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WO2023076976A1 - Methods for improving pyrroloquinoline quinone production in methylopila - Google Patents

Methods for improving pyrroloquinoline quinone production in methylopila Download PDF

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Publication number
WO2023076976A1
WO2023076976A1 PCT/US2022/078752 US2022078752W WO2023076976A1 WO 2023076976 A1 WO2023076976 A1 WO 2023076976A1 US 2022078752 W US2022078752 W US 2022078752W WO 2023076976 A1 WO2023076976 A1 WO 2023076976A1
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gene
seq
bacterium
pqqa
methylopila
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PCT/US2022/078752
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French (fr)
Inventor
Wanli Lu
Yisheng WU
David Nunn
Oliver YU
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Conagen Inc.
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Publication of WO2023076976A1 publication Critical patent/WO2023076976A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the field of the invention relates to transgenic bacteria and methods for increasing the biosynthetic production of pyrroloquinoline quinone (PQQ). More specifically, the present methods and processes make use of genetically engineered bacteria Methylopila sp. YHT-1 that have been transformed to include nucleic acids encoding a set of PQQ biosynthetic enzymes. The copy numbers and expression level of the PQQ biosynthetic genes have been increased to improve the PQQ production in the genetic engineered Methylopila sp. YHT-1 strains.
  • Pyrroloquinoline quinone (4,5-dihydro-4,5-dioxo-lH-pyrrolo-[2,3-/]quinoline-2,7,9- tricarboxylic acid; PQQ; FIG. 1), also named as methoxatin, is a water-soluble, aromatic, tricyclic 0-quinone that was discovered as a redox cofactor of bacterial glucose dehydrogenase in 1964.
  • PQQ is found in soil, fruits, vegetables, and meats as well as human breast milk, currently PQQ has only been reported being synthesized by microorganisms. PQQ enters plants from soil bacteria, and subsequently enters human diets.
  • PQQ has been demonstrated to act as a redox cofactor of methanol/alcohol dehydrogenase and glucose dehydrogenase enzymes.
  • PQQ has been recognized as the third type of redox cofactors discovered after pyridine nucleotide- and flavin-dependent cofactors.
  • PQQ possesses potent antioxidant activity, much stronger than other quinones and enediols including vitamin C (ascorbic acid). In terms of redox recycling, it has been estimated to have about 20,000 potential redox cycles and is therefore much more stable than ascorbic acid which only has about 4.
  • PQQ has provoked significant interest because of its benefits for human health, such as for diabetes, anti-aging, neuroprotection, cognition, antioxidant activity, and lowering the level of c- reactive protein in response to inflammation. Also, PQQ supplementation has been proved to promote mitochondrial efficiency and induce mitochondrial biogenesis. PQQ was suggested as a newcomer to the B groups of vitamins in a research article published In Nature In 2003 1 , although controversy regarding its role as a vitamin has persisted ever since.
  • PQQ polystyrene resin
  • PQQ polystyrene resin
  • known chemical PQQ synthetic processes are not ideal for safe and sustainable production due to a number of disadvantages such as costly key starting materials, the use of toxic precursors, complicated purification processes, generation of undesirable byproducts, and potential cause of environmental pollution.
  • consumers generally prefer natural derivatives and are usually willing to pay higher prices for natural ingredients.
  • MP688 and genetically engineered Methylobacterium extorquens AMI are the most promising disclosed strains which may be cultivated in a medium containing methanol as a carbon source and reportedly produce 125 mg/L and 114 mg/L PQQ in test tube fermentations, respectively.
  • PQQ yields are not ideal for production on an industrial scale. PQQ is still both expensive and difficult to obtain by biological production. Accordingly, there is a need for improved microbial fermentation methods to produce PQQ economically, reliably, and in an environmentally friendly manner.
  • Methylopila is a genus of bacteria from the Methylocystaceae, a family of bacteria that are capable of obtaining carbon and energy from methane. Such bacteria are known as methanotroplis, and the Methylocystaceae belong to the type II methanotroplis, which are structurally and biochemically distinct from the Methylococcaceae or type I methanotroplis .
  • methane is oxidized to form formaldehyde, which is assimilated by the serine pathway. This involves combining formaldehyde and glycine to form serine, which may be converted into glyceraldehyde and thus into other organic molecules.
  • This family of bacteria can also fix nitrogen, similarly to many other members of the order Rhizobiales.
  • Methylopila sp. YHT-1 A novel PQQ-producing Methylopila strain named Methylopila sp. YHT-1 was isolated from the soil in Wuxi, Jiangsu, China and was found to use methanol as its single carbon source. The strain was reported to produce 30 mg/L PQQ in a test tube fermentation setting and 113.6 mg/L PQQ in a 3-liter fermenter. Genes associated with the PQQ biosynthesis have been discovered in several species of bacteria including Methylobacterium extorquens AMI, Methylovorus sp. MP688, Acinetobacter calcoaceticus, Enterobacter intermedium 60-2G, Klebsiella pneumoniae, and Gluconobacter oxydans.
  • Methylobacterium extorquens AMI Methylovorus sp. MP688, Acinetobacter calcoaceticus, Enterobacter intermedium 60-2G, Klebsiella pneumoniae
  • the core PQQ biosynthetic genes are mostly arranged in one or two clusters pqqA-B-C-D-E-F or pqqA-B-C-D-E (also known as pqqABCDEF or pqqABCDE, respectively) and pqqF-G (also known as ppqFG).
  • pqq gene cluster can mean a gene cluster or operon that encodes proteins involved in the biosynthesis of PQQ.
  • PQQ biosynthesis is believed to start from a ribosomal translated precursor peptide, PqqA.
  • PqqA ribosomal translated precursor peptide
  • the PqqA sequence in Methylopila sp. YHT-l strain is MAIWTAPIVEETPVGLEVTSYSPAEL (SEQ ID NO: 1).
  • the first step is catalyzed by the radical SAM (S-adenosyl-L- methionine) enzyme PqqE, in complex with PqqD, the small chaperone protein, binding with the substrate PqqA.
  • SAM S-adenosyl-L- methionine
  • the modified PqqA peptide is recognized by protease M16B family enzymes PqqF and/or PqqG, which catalyze cutting of the generated glutamic acid-tyrosine pair out of the PqqA peptide to release the suitable substrate for the downstream enzyme PqqB.
  • PqqB was recently characterized as an iron-dependent hydroxylase catalyzing a series of complicated oxygeninsertion reactions to produce the quinone moiety 10 .
  • AHQQ the immediate precursor of PQQ
  • PqqC a cofactor-less oxidase.
  • AHQQ is thus converted into PQQ in a reaction that involves overall eight-electron oxidations, leading to pyrrole- and pyridine-ring formation.
  • PQQ can be reliably produced at a high yield by genetic engineering and fermentation technology using recombinant Methylopila sp. YHT-l cell cultures. These genetically engineered Methylopila sp. YHT-l cell cultures can synthesize PQQ in commercially significant yields.
  • provided herein are genetic engineering methods which contribute to increase the copy numbers and enhance the expression level of the precursor peptide pqqA gene and the entire PQQ biosynthetic gene clusters, which in turn create a way to sustainably and economically produce unprecedented amounts of PQQ product.
  • a transgenic bacterial cell or population thereof wherein the transgenic bacterial cell can produce PQQ at a high yield and the bacterium is Methylopila sp. YHT-1.
  • a transgenic Methylopila sp. YHT-1 cell or population thereof, the transgenic Methylopila sp. YHT-1 cell including nucleotide sequences capable of expressing a PQQ precursor peptide PqqA, an iron-dependent hydroxylase PqqB, a cofactorless oxidase PqqC, a PqqA precursor binding chaperone protein PqqD, a radical SAM enzyme PqqE, protease M16B family enzymes PqqF and/or PqqG, a redox-sensitive transcriptional activator SoxR, wherein the nucleotide sequences and amino acids sequences are from Methylopila sp. YHT-1.
  • a transgenic Methylopila sp. YHT-1 cell or population thereof.
  • the transgenic Methylopila sp. YHT-1 cell comprises a disrupted gene encoding a putative PQQ periplasmic binding protein PqqT, wherein the pqqT and its flanking region nucleotide sequences are from Methylopila sp. YHT-1 .
  • a transgenic Methylopila sp. YHT-1 cell or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising one or more replicative broad host range vectors for use in overexpression of PQQ biosynthetic genes, wherein the replicative broad host range vectors include but not are not limited to Bordetella bronchiseptica-derived pBBRIMCS series vectors, or RK2 -based expression vectors like pJB656, or IncP type vectors such as pRK310 and pCM series vectors.
  • the replicative broad host range vectors include but not are not limited to Bordetella bronchiseptica-derived pBBRIMCS series vectors, or RK2 -based expression vectors like pJB656, or IncP type vectors such as pRK310 and pCM series vectors.
  • YHT-1 cell or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising native or synthetic promoters for use in overexpression of PQQ biosynthetic genes, wherein the promoters include but are not limited to the native promoter located upstream of the pqqA-B-C -D-E operon, the native promoter located upstream of pqqE gene, the native promoter located upstream of the mdhl operon, the native promoter located upstream of the mdh.2 operon, the native promoter located upstream of the pqqF-Q operon, the native promoter located upstream of the pqqT gene, PmxaF promoter of the PQQ-dependent methanol dehydrogenase gene of Methylobacterium extorquens AML PfumC promoter of the fumarase gene of Methylobacterium extorquens AML PcoxB promoter of the cytochrome
  • a transgenic Methylopila sp. YHT-1 cell or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising one or more gene cassettes comprising native or strong synthetic promoters, nucleotide sequences capable of expressing the precursor peptide pqqA gene, and the whole PQQ biosynthetic gene cluster that are integrated into the chromosome of Methylopila sp. YHT-1 strain at the locus upstream of the pqqA-B-C-D- E operon or upstream of the pqqA2 gene.
  • a method for the production of the PQQ comprising culturing genetically engineered Methylopila sp. YHT-1 cells in culture and fermentation media.
  • the culture and fermentation media for genetic engineered Methylopila sp. YHT-1 cells contain methanol as a carbon source.
  • the vast majority of the PQQ is secreted into the growth media.
  • amino acid sequences and nucleotide sequences of proteins, deoxyribonucleic acids, and promoters utilized for genetic engineering of Methylopila sp. YHT-1 cells which are listed in the Sequence Listing section hereinbelow.
  • Some aspects of the present disclosure provide for producing pyrroloquinoline quinone (PQQ) comprising: cultivating in a culture medium a bacterium belonging to the genus Methylopila, and collecting PQQ from the culture medium, wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
  • the pqqABCDE gene cluster comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.
  • the pqqABCDE gene cluster comprises the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.
  • the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 70%', at least 75%, at least 80%, at least 85%', at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof;
  • the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof;
  • the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at
  • the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51 , 53, 55, 57, or variants thereof: the pqqABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1 , 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1 , 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium.
  • the pqqABCDE gene cluster is inserted into the chromosome of the bacterium. In some embodiments, pqqABCDE gene cluster is introduced into the bacterium via a plasmid. In some embodiments, a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
  • the potent promoter is selected from the group consisting of PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47), or variants thereof.
  • the potent promoter comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46. or SEQ ID NO: 47.
  • the potent promoter comprises the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.
  • the potent promoter is Ptac promoter (SEQ ID NO: 47), or valiants thereof.
  • the bacterium has been further modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
  • the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and variants thereof.
  • the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. and SEQ ID NO: 11.
  • the bacterium has been further modified to delete or knock out a pqqT gene.
  • the pqqT is identified by SEQ ID NO: 31.
  • the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, and uncultured Methylopila sp. In some embodiments, the bacterium is Methylopila sp. YH-1.
  • any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp, Yamaguchi, Methylopila sp. Ml 07, Methylopila sp. 73B, or uncultured Methylopila sp.
  • any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1.
  • the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
  • the culture medium includes methanol at a concentration of not less than 3 grams (e.g., 10, 9, 8, 7, 6, 5, 4, 3 grams) of methanol per liter of water solvent to at most 15 grams (e.g,, 5, 6, 9, 8, 9, 10, 11, 12, 13, 14, 15 grams) of methanol per liter water solvent.
  • bacterium belonging to the genus Methylopila wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
  • the pqqABCDE gene cluster comprises a nucleotide sequence that is at least 70%, at least 15%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.
  • the pqqABCDE gene cluster comprises the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22,
  • the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 70%', at least 75%, at least 80%, at least 85%', at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or valiants thereof;
  • the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof;
  • the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%,
  • the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof: the pqq ABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71 , 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene comprising the nucle
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1 , 2, or 3 copies) of the pqq A gene is inserted into the chromosome of the bacterium, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
  • one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium.
  • the pqqABCDE gene cluster is inserted into the chromosome of the bacterium.
  • pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
  • a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
  • the potent promoter is selected from the group consisting of PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Plac promoter (SEQ ID NO: 47), or variants thereof.
  • PMDH1 promoter SEQ ID NO: 34
  • PMDH2 promoter SEQ ID NO: 35
  • PmxaF promoter SEQ ID NO: 42
  • PfumC promoter SEQ ID NO: 43
  • PcoxB promoter SEQ ID NO: 44
  • Ptuf promoter SEQ ID NO: 45
  • PMP688A2 promoter SEQ ID NO: 46
  • Plac promoter SEQ ID NO: 47
  • the potent promoter comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.
  • the potent promoter comprises the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.
  • the potent promoter is Ptac promoter (SEQ ID NO: 47), or variants thereof.
  • the bacterium has been further modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
  • the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof.
  • the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.
  • bacterium belonging to the genus Methylopila wherein the bacterium has been modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
  • the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof.
  • the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.
  • the bacterium has been further modified to delete or knock out a pqqT gene.
  • the pqqT is identified by SEQ ID NO: 31 .
  • the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. Ml 07, Methylopila sp. 73B, and uncultured Methylopila sp.
  • the bacterium is Methylopila sp. YH-1.
  • any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, or uncultured Methylopila sp.
  • any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1.
  • the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
  • Methods for producing pyrroloquinoline quinone are also provided, the method comprising cultivating in a culture medium a bacterium described here, and collecting PQQ from the culture medium.
  • SEQ ID NO: 1 is the amino acid sequence of PqqA of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 2.
  • PqqA is a PQQ precursor peptide.
  • SEQ ID NO: 3 is the nucleotide sequence of the second copy of pqqA (pqqA2) of Methylopila sp. YHT-1.
  • SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11 are codon-randomized nucleotide sequence of pqqA.
  • SEQ ID NO: 12 is the amino acid sequence of PqqB ofi Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 13 and codon-optimized nucleotide SEQ ID NO: 14.
  • PqqB is an iron-dependent hydroxylase.
  • SEQ ID NO: 15 is the amino acid sequence of PqqC of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 16 and codon-optimized nucleotide SEQ ID NO: 17.
  • PqqC is a cofactorless oxidase.
  • SEQ ID NO: 18 is the amino acid sequence of PqqD of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 19 and codon-optimized nucleotide SEQ ID NO: 20.
  • PqqD is a PqqA precursor binding chaperone protein.
  • SEQ ID NO: 21 is the amino acid sequence of PqqE of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 22 and codon-optimized nucleotide SEQ ID NO: 23,
  • PqqE is a radical SAM enzyme
  • SEQ ID NO: 24 is the amino acid sequence of SoxR of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 25.
  • SoxR is a putative redox-sensitive transcriptional activator.
  • SEQ ID NO: 26 is the amino acid sequence of PqqF of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 27.
  • PqqF is a protease M16B family enzyme.
  • SEQ ID NO: 28 is the amino acid sequence of PqqG of Methylopila sp, YHT-1, corresponding to nucleotide SEQ ID NO: 29.
  • PqqG is a protease M16B family enzyme.
  • SEQ ID NO: 30 is the amino acid sequence of PqqT of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 31.
  • PqqT is a putative PQQ periplasmic binding protein.
  • SEQ ID NO: 32 is the amino acid sequence of MDH1 of Methylopila sp. YHT-1 , corresponding to nucleotide SEQ ID NO: 33.
  • SEQ ID NO: 34 is the nucleotide sequence of the promoter region located upstream of the mdhl operon.
  • MDH1 is a putative PQQ-dependent methanol dehydrogenase.
  • SEQ ID NO: 35 is the amino acid sequence of MDH2 of Methylopila sp. YHT-1 , corresponding to nucleotide SEQ ID NO: 36.
  • SEQ ID NO: 37 is the nucleotide sequence of the promoter region located upstream of the mdh2 operon.
  • MDH2 is another putative PQQ- dependent methanol dehydrogenase.
  • SEQ ID NO: 38 is the nucleotide sequence of the promoter region located upstream of the pqqA-B-C-D-E operon of Methylopila sp. YHT-1.
  • SEQ ID NO: 39 is the nucleotide sequence of the promoter region located upstream of the pqqE gene of Methylopila sp. YHT-1.
  • SEQ ID NO: 40 is the nucleotide sequence of the promoter region located upstream of the pqqF-Q operon of Methylopila sp. YHT-1.
  • SEQ ID NO: 41 is the nucleotide sequence of the promoter region located upstream of the pqqT gene of Methylopila sp. YHT-1.
  • SEQ ID NO: 42 is the nucleotide sequence of the PmxaF promoter of the PQQ-dependent methanol dehydrogenase gene of Methylobacterium extorquens AMI.
  • SEQ ID NO: 43 is the nucleotide sequence of the PfumC promoter of the fumarase gene of Methylobacterium. extorquens AMI.
  • SEQ ID NO: 44 is the nucleotide sequence of the PcoxB promoter of the cytochrome c oxidase subunit II gene of Methylobacterium extorquens AMI.
  • SEQ ID NO: 45 is the nucleotide sequence of the Pluf promoter of the EF-Tu gene coding for the translation elongation factor thermo-unstable of Methylobacterium extorquens AMI.
  • SEQ ID NO: 46 is the nucleotide sequence of the PqqA2 promoter (MP688A2) of the pqqA2 gene of Methylovorus sp. MP688.
  • SEQ ID NO: 47 is the nucleotide sequence of the hybrid Ptac promoter.
  • SEQ ID NO: 48 is the amino acid sequence of PqqA of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 49.
  • SEQ ID NO: 50 is the amino acid sequence of PqqA of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 51.
  • SEQ ID NO: 52 is the amino acid sequence of PqqA of Methylopila sp. M107, corresponding to nucleotide SEQ ID NO: 53.
  • SEQ ID NO: 54 is the amino acid sequence of PqqA of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 55.
  • SEQ ID NO: 56 is the amino acid sequence of PqqA of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 57.
  • SEQ ID NO: 58 is the amino acid sequence of PqqB of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 59.
  • SEQ ID NO: 60 is the amino acid sequence of PqqB of Methylopila. sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 61.
  • SEQ ID NO: 62 is the amino acid sequence of PqqB of Methylopila sp. M107, corresponding to nucleotide SEQ ID NO: 63.
  • SEQ ID NO: 64 is the amino acid sequence of PqqB of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 65.
  • SEQ ID NO: 66 is the amino acid sequence of PqqB of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 67.
  • SEQ ID NO: 68 is the amino acid sequence of PqqC of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 69.
  • SEQ ID NO: 70 is the amino acid sequence of PqqC of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 71.
  • SEQ ID NO: 72 is the amino acid sequence of PqqC of Methylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 73.
  • SEQ ID NO: 74 is the amino acid sequence of PqqC of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 75.
  • SEQ ID NO: 76 is the amino acid sequence of PqqC of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 77.
  • SEQ ID NO: 78 is the amino acid sequence of PqqD of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 79.
  • SEQ ID NO: 80 is the amino acid sequence of PqqD of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 81.
  • SEQ ID NO: 82 is the amino acid sequence of PqqD of Methylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 83.
  • SEQ ID NO: 84 is the amino acid sequence of PqqD of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 85.
  • SEQ ID NO: 86 is the amino acid sequence of PqqD of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 87.
  • SEQ ID NO: 88 is the amino acid sequence of PqqE of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 89.
  • SEQ ID NO: 90 is the amino acid sequence of PqqE of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 91.
  • SEQ ID NO: 92 is the amino acid sequence of PqqE of MePhylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 93.
  • SEQ ID NO: 94 is the amino acid sequence of PqqE of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 95.
  • SEQ ID NO: 96 is the amino acid sequence of PqqE of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 97.
  • FIG. 1 shows the chemical structure of PQQ.
  • FIG. 2 shows the comparison of PQQ biosynthetic genes (gene clusters) in Methylobacterium extorquens AMI strain (panel A) and Methylopila sp. YHT-1 strain (panel B).
  • FIG. 3 illustrates the proposed biosynthetic pathway to PQQ in Methylopila sp. YHT-1 strain.
  • FIG. 4 represents a schematic diagram of the biparental conjugation system for Methylopila sp. YHT-1 strain.
  • FIG. 5 includes the maps of plasmids the inventors have generated to screen strong promoters tax Methylopila sp. YHT-1 strain.
  • FIG. 6 includes the maps of plasmids the inventors have generated for a deletion analysis of the promoter region located upstream of the pqqA-B-C-D-E operon of Methylopila sp. YHT-1 strain.
  • FIG. 7 show's the real-time GFP signal and biomass results when Methylopila sp.
  • YHT-1 strains hosting pBBRlMCS2-Ptac-GFP plasmid or pBBRlMCS2-IGRpqqA351-GFP plasmid were cultured in methanol minimal B medium.
  • FIG. 8 includes the map of a plasmid the inventors have generated for the analysis of the promoter region located upstream of the pqqE gene of Methylopila sp.
  • YHT-1 strain panel A
  • YHT-1 clones transformed with pBBRlMCS2-IGRpqqE-GFP under blue light panel B).
  • FIG. 9 includes the maps of plasmids the inventors have generated for overexpression of the precursor peptide pqqA gene, pqqA-B-C-D-E operon, and the whole PQQ biosynthetic gene cluster using both the native promoter of the pqqA-B-C-D-E operon and the Ptac promoter.
  • FIG. 10 includes the maps of plasmids the inventors have generated for overexpression of the redox-sensitive transcriptional activator soxR gene located upstream of the pqqF-G operon of Methylopila sp. YHT-1 strain.
  • FIG. 11 includes the maps of plasmids the inventors have generated for the insertion of the Ptac promoter, Ptac-pqqAs cassette, pqqAs cassette into the upstream region of the pqqA-B- C-D-E operon or the pqqA2 gene in the chromosome of Methylopila sp. YHT-1 strain.
  • FIG. 12 includes the map of plasmid the inventors have generated for in-frame deletion of the pqqT gene encoding a putative PQQ periplasmic binding protein PqqT from the chromosome of Methylopila sp. YHT-1 strain.
  • FIG. 13 illustrates an example of the 630 nm absorbance curve of the GDH assay (panel A) and reports PQQ GDH assay results of representative bacterial strains (panel B).
  • FIG. 14 includes HPLC profiles illustrating PQQ production in test tube cultures of the Methylopila sp. YHT-1 wildtype strain and of genetically engineered strains.
  • FIG. 15 includes a HPLC profile and UV spectra showing PQQ production in the fermentation culture of an engineered Methylopila sp. YHT-1 strain.
  • the present disclosure provides new and improved bacterial strains enhancing the expression of certain PQQ-related genes and/or gene clusters.
  • a series of recombinant, genetically engineered Methylopila sp. YHT- 1 strains were generated to increase gene copy numbers and enhance the expression of the precursor peptide pqqA gene and of the entire PQQ biosynthetic gene clusters.
  • these new strains may be used in novel methods for the biosynthetic production of PQQ by in cellular systems at previously unattained yields.
  • PQQ-producing bacterium can mean a bacterium which has an ability to produce and excrete a PQQ into a medium when the bacterium is cultured in the medium.
  • PQQ-producing bacterium also can mean a bacterium which is able to produce and cause accumulation of PQQ in a culture medium in an amount larger than a wild-type or parental strain, for example Methylopila sp. YH-1 (China Center for Type Culture Collection (CCT'CC) Deposit No. M 2014016) or other Methylocistaceae.
  • bacterium belonging to the genus Methylopila can mean that the bacterium is classified into the genus Methylopila according to the classification known to a person skilled in the art of microbiology, although the bacterium is not particularly limited. Specifically, those classified into the group Methylopila according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) may be used.
  • NCBI National Center for Biotechnology Information
  • PQQ-producing bacterium can also mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.3 mg/L, not less than 0.5 mg/L, or not less than 1 mg/L of PQQ.
  • enhancing the expression of gene(s) and/or gene cluster(s) can mean that the expression of the gene(s) and/or cluster(s) in an improved strain is higher than that of a nonmodified strain, for example, a wild-type strain such as Methylopila sp. YH-1.
  • modifications can include increasing the copy number of the expressed gene or gene cluster per cell, increasing the expression level of the gene cluster or gene, and so forth.
  • the copy number of an expressed gene cluster or gene is measured, for example, by restricting the chromosomal and/or plasmid DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like.
  • the level of gene cluster or gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like.
  • pqqA-like gene can mean a gene that is located in a bacterial genome and encodes a precursor for PQQ biosynthesis.
  • the presence of a pqqA-like gene in a bacterial genome can be determined by analysis of the genome sequence, in addition to databases containing information about annotated pqqA genes from various microorganisms.
  • pqqA-like genes from a wide range of microorganisms can be used. Examples of the bacterium containing in-genome pqqA-like gene are not limited to the genus Methylopila.
  • Examples of the bacterium containing in-genome pqqA-like genes can also include, Methylococcus capsulatus Bath, Colwellia psychrerythraea 34H, Gluconobacter oxydans 621H, Methylobacdllus flagellatus KT, Dinoroseobacter shibae DFL 12, Leptothrix cholodnii SP-6, Erwinia amylovora ATCC 49946, Acinetobacter sp. RUH2624, Saccharopolyspora erythraea NRRL23338, Brady rhizobium sp. ORS278, Brady rhizobium sp.
  • Some of the bacteria can have multiple copies of pqqA-like gene in-genome, for instance, Met hy lot e> t era mobilis JLW8, Methylovorus sp. SIP3-4, Methylobacterium extorquens DM4, and Methyloba.cterium sp. 4-46.
  • the gene cluster and the gene(s) of which expression is/are enhanced are not limited to the genes shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9.
  • SEQ ID NO: 10 SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63.
  • SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81.
  • the protein variants encoded by the genes may have exemplary homology of not less than 80%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, or not less than 99.9% with respect to the entire amino acid sequence shown in SEQ ID NO:1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24; SEQ ID NO:26.
  • SEQ ID NO: 74 SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, as long as the activity or function of the corresponding protein is maintained.
  • protein variant means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids.
  • the number of changes in the variant proteins can depend on the position in the three- dimensional structure of the proteins or the type of amino acid residues. Exemplary embodiments can be 1 to 30, 1 to 15, 1 to 5, or 1 to 3 in SEQ ID NO:1 , SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24; SEQ ID NO:26.
  • a protein having the function of PQQ precursor can mean that the protein can be involved in PQQ biosynthesis as a precursor for PQQ, specifically, the protein can have a three-dimensional structure which is sufficient to be recognized and used as a substrate by PQQ biosynthesis enzymes with subsequent conversion into PQQ.
  • substitution, deletion, insertion or addition of one or several amino acid residues should typically be conservative mutation(s) so that the activity or the function is maintained.
  • the representative conservative mutation give rise to a conservative substitution.
  • conservative substitutions can include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Vai or Phe for He, substitution of He, Met, Vai or Phe for Leu, substitution of Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Vai or Phe for Met, substitution of Trp, Tyr, Met, He or Le
  • the gene cluster and gene(s) may be a variant(s) which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ
  • SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97 but can include genes homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29.
  • SEQ ID NO: 49 SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional protein.
  • Stringent conditions can include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed.
  • Other exemplary homologies can include not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, or not less than 99.9%.
  • stringent conditions are exemplified by washing one time or more, such as two or three times, at a salt concentration of IxSSC, 0.1% SDS.
  • Another exemplary salt concentration can include O.lxSSC, 0.1% SDS at 60° C.
  • Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. By way of example, washing can be performed 2 to 3 times.
  • the length of the probe can be suitably selected depending on the hybridization conditions, and can be 100 bp to 1 kbp, for example.
  • codons in the gene sequences may be replaced with other equivalent codons which are easily used in the host into which the genes are introduced.
  • a cellular system may include any cell or cells that can be used to ectopically express PQQ biosynthetic genes from the genus Methylopila.
  • Methylopila sp. YHT-1 strain e.g. Methylopila sp. YHT-1 strain, Methylopila capsulata strain, Methylopila sp. Yamaguchi strain, Methylopila sp. M107 strain, Methylopila sp. 73B strain, and uncultured Methylopila sp. strain
  • It includes bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes.
  • a cell culture refers to any cell or cells that are in a culture. Culturing or incubating is the process in which cells are grown under controlled conditions, typically outside of their natural environment. For example, cells, such as bacterial or yeast cells, may be grown as a cell suspension in liquid nutrient broth.
  • a cell culture includes, but is not limited to, a bacterial cell culture, a yeast cell culture, a plant cell culture, and an animal cell culture. In some embodiments, the cell culture comprises bacterial cells, yeast cells, or a combination thereof.
  • a bacterial cell culture comprises bacterial cells including, but not limited to, Escherichia, Streptomyces, Zymomonas, Acetobacter, Citrobacter, Synechocystis, Rhizobium, Clostridium, Corynebacterium, Streptococcus, Xanlhomonas, Lactobacillus, Lactococcus, Bacillus, Alcaligenes, Pseudomonas, Aeromonas, Azotobacter, Comamonas, Mycobacterium, Rhodococcus, Gluconobacter, Ralstonia, Acidlthiobacillus, Microlunatus, Geobacter, Geobacillus, Arlhrobacter, Flavobacterium, Serratia, Saccharopolyspora, Thermus, Stenotrophomonas, Chromobacterium, Sino rhizobium, Saccharopolyspora, Agrobacterium., Panto
  • a cell culture as described herein may be in an aqueous medium including one or more nutrient substances as known in the art.
  • the medium may be either a synthetic or natural medium, so long as the medium includes a carbon source, a nitrogen source, minerals and, if necessary, appropriate amounts of nutrients which the bacterium may require for growth.
  • the carbon source can include various carbohydrates such as glucose and sucrose, various organic acids, alcohol including methanol, ethanol and glycerol. Methanol is usually preferable.
  • the nitrogen source can include various ammonium salts such as ammonia and ammonium sulphate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism.
  • the sulfur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like.
  • Minerals can include potassium monophosphate, sodium chloride, calcium chloride, and the like.
  • Vitamins can include thiamine, yeast extract, and the like.
  • cells are cultured at a temperature of 16°C to 40°C.
  • cells may be cultured at a temperature of 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23 °C, 24°C, 25°C, 26°C, 27"C, 28°C. 29°C, 30°C, 3 VC, 32“C, 33°C, 34°C, 35"C, 36°C. 37°C, 38°C, 39°C or 40°C.
  • cells are cultured at a pH range from about 3 to about 9, for example in the range of from about 4 to about 8.
  • the pH can be regulated by the addition of an inorganic or organic acid or base such as hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate, ammonia, or by the addition of a buffer such as phosphate, phthalate or Tris'®.
  • cells are cultured for a period of 0.5 hours to 96 hours, or more.
  • cells may be cultured for a period of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
  • cells such as bacterial cells, are cultured for a period of 12 to 24 hours.
  • cells are cultured for 12 to 24 hours at a temperature of 37”C.
  • cells are cultured for 12 to 24 hours at a temperature of 16°C.
  • cells are cultured to a density of 1 x 10 s (OD600 ⁇ 1) to 2 x 10 11 (ODfjoo ⁇ 200) viable cells/ml cell culture medium.
  • cells are cultured to a density of 1 x 10 s , 2 x 10 s , 3 x 10 s , 4 x 10 8 , 5 x 10 8 , 6 x 10 s , 7 x 10 s , 8 x 10 s , 9 x 10 s , 1 x 10 9 , 2 x 10 9 , 3 x 10 9 , 4 x 10 9 , 5 x 10 9 , 6 x 10 9 , 7 x 10 9 , 8 x 10 9 , 9 x 10 9 , 1 x 10 10 , 2 x 10 10 , 3 x 10 i0 , 4 x l() i0 , 5 x l() i0 , 6 x 10 i0 , 7 x 10 10 9 , 10 9 x
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such vectors are within the scope of the present disclosure.
  • the expression vector includes those genetic elements for expression of the recombinant polypeptide in bacterial cells.
  • the elements for transcription and translation in the bacterial cell may include a promoter, a coding region for the protein complex, and a transcriptional terminator.
  • polynucleotide used for incorporation into the expression vector of the subject technology can be prepared by routine techniques such as polymerase chain reaction (PGR).
  • PGR polymerase chain reaction
  • complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA.
  • the vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary' homopolymeric tads to form recombinant DNA molecules.
  • synthetic linkers containing one or more restriction sites are used to operably link the polynucleotide of the subject technology to the expression vector.
  • the polynucleotide is generated by restriction endonuclease digestion.
  • the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single- stranded termini with their 3'-5'-exonucleolytic activities and fill-in recessed 3 '-ends with their polymerizing activities, thereby generating blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends.
  • These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the polynucleotide.
  • LIC ligation-independent cloning
  • PCR in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR.
  • Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.
  • a polynucleotide for incorporation into an expression vector of the subject technology is prepared by the use of PCR using appropriate oligonucleotide primers.
  • the coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product.
  • the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector.
  • the expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of appropriate cells with an expression vector of the subject technology Is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation.
  • Successfully transformed cells that is, those cells containing the expression vector, can be identified by techniques well known in the art.
  • cells transfected with an expression vector of the subject technology can be cultured to produce polypeptides described herein.
  • Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.
  • the host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.
  • the transformed cell can be a bacterial cell, a yeast cell, an algal cell, a fungal cell, a plant cell, an insect cell or an animal cell.
  • the cell is a plant cell selected from the group consisting of: canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.
  • Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct vectors for expression of the recombinant polypeptide of the subjection technology in a microbial host cell. These vectors may then be introduced into appropriate microorganisms via transformation to allow for high level expression of the recombinant polypeptide of the subject technology.
  • Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art.
  • the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3’ of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.
  • Initiation control regions or promoters which are useful to drive expression of the recombinant polypeptide in the desired microbial host cell are numerous and familiar to those skilled in the art.
  • the term “potent promoter” refers to a promoter that increases expression of a nucleic acid sequence encoding a polypeptide compared to the parent, constitutive promoter.
  • Non-limiting examples of potent promoters which may provide a high level of gene expression in a bacterium belonging to the genus Methylopila may be chosen from among PMDH1 (SEQ ID NO: 34), PMDH2 (SEQ ID NO: 35), PmxaF (SEQ ID NO: 42), PfumC (SEQ ID NO: 43), PcoxB (SEQ ID NO: 44), /hn/(SEQ ID NO: 45), PMP688A2 (SEQ ID NO: 46) and Ptac (SEQ ID NO: 47), or variants thereof.
  • Termination control regions may also be derived from various genes native to the microbial hosts.
  • a termination site optionally may be included for the microbial hosts described herein.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window' of alignment of components, e.g., nucleotides or amino acids.
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the w'indow of comparison).
  • Optimal alignment of sequences for aligning a comparison window' are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part, of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA).
  • identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • the percent of sequence identity is preferably determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software PackageTM (Version 10; Genetics Computer Group, Inc., Madison, WI). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, JOURNAL OF MOLECULAR BIOLOGY 48:443-53, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, ADVANCES IN APPLIED MATHEMATICS, 2:482-489, 1981, Smith et al., NUCLEIC ACIDS RESEARCH 11 :2205- 2220, 1983). The percent identity is most preferably determined using the “Best Fit” program.
  • BLAST Basic Local Alignment Search Tool
  • the term “substantial percent sequence identity” refers to a percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity.
  • one embodiment of the disclosure is a polynucleotide molecule that has at least about ?()% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity with a polynucleotide sequence described herein. Identity and Similarity
  • Identity is the fraction of amino acids that are the same between a pair of sequences after an alignment of the sequences (which can be done using only sequence information or structural information or some other information, but usually it is based on sequence information alone), and similarity is the score assigned based on an alignment using some similarity matrix.
  • the similarity index may be any one of the following BLOSUM62, PAM250, or GONNET, or any matrix used by one skilled in the art for the sequence alignment of proteins.
  • Identity is the degree of correspondence between two sub-sequences (no gaps between the sequences). An identity of 25% or higher typically implies similarity of function, while 18- 25% implies similarity of structure or function. It should be kept in mind that two completely unrelated or random sequences (that are greater than 100 residues) may have higher than 20% identity. Similarity is the degree of resemblance between two sequences when they are compared. This is dependent on their identity.
  • exemplary is used herein to mean “serving as an example, instance, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.
  • complementary is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary' to guanine. Accordingly, the subjection technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
  • nucleic acid and “nucleotide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • Coding sequence is to be given its ordinary and customary meaning to a person of ordinary'- skill in the art, and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.
  • isolated is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
  • incubating and “incubation” as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing the desired product.
  • degenerate variant refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions.
  • Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues.
  • a nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.
  • polypeptide refers to peptides, polypeptides, and proteins, unless otherwise noted.
  • polypeptide and “peptide” are used interchangeably herein when referring to a polynucleotide product.
  • exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • polypeptide fragment and “fragment,” when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
  • polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).
  • amino acid position “corresponding to” a reference position refers to a position that aligns with a reference sequence, as identified by aligning the amino acid sequences. Such alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.
  • Suitable regulatory sequences is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3’ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3’ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the ait that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • Promoters which cause a gene to be expressed in most cell types at most times, are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • expression is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the subject technology.
  • mRNA sense
  • antisense RNA derived from the nucleic acid fragment of the subject technology.
  • Over-expression refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non- tran sformed organ! sms .
  • Transformation is to be given its ordinary and customary meaning to a person of reasonable skill in the field, and is used without limitation to refer to the transfer of a polynucleotide into a target cell for further expression by that cell.
  • the transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal.
  • Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic” or “recombinant” or “transformed” organisms.
  • transformed when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • heterologous when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.
  • recombinant when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Titus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3’ untranslated sequence into a cell.
  • Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.
  • “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
  • Example 1 Development of a genetic toolkit for Methylopila sp. YHT-1 strain
  • Methylopila sp. YHT-1 was a newly discovered PQQ- producing bacterium, and toolkits for its genetic manipulation were not available. Moreover, constitutive promoters utilized for gene overexpression in Methylopila sp. YHT-1 were yet to be identified and quantified. Methylopila is a genus of bacteria from the family of Methylocystaceae . No well- developed protocols for this family of microorganisms had been reported at the time of the invention. As such, the inventors developed an efficient biparental conjugation system for Methylopila sp. YHT-1 strain (FIG. 4).
  • Rifampicin-resistant clones were generated through plating > IO 8 Methylopila sp. YHT-1 cells onto multiple nutrient agar plates with rifampicin (50 pg/mL).
  • the Bordetella bronchiseptica-derived broad host range conjugative plasmid pBBRlMCS2 was used to develop the conjugation system. Briefly, pBBRlMCS2 DNA was transformed into E. coll S 17- 1, a strain having chromosomally integrated conjugal transfer functions (RP4 transfer functions). Rifampicin-resistant Methylopila sp. YHT-1 cells and E.
  • coli S 17- 1 cells hosting pBBRlMCS2 plasmid were cultured in methanol minimal B medium and Luria-Bertani, respectively.
  • the mid -exponential phase recipient and donor cell cultures were washed with Luria-Bertani liquid medium, mixed in a 6:1 recipient-to-donor ratio, and plated onto nutrient agar plates.
  • the cells were stripped from nutrient agar plates with a disposable cell spreader and plated onto the selective medium (Succinate Minimal Salts medium plates with 25 pg/mL kanamycin and 50 pg/mL rifampicin). After incubation at 30°C for a week, transconjugant colonies were picked and streaked onto methanol minimal B medium plates with 25 pg/mL kanamycin to ensure the absence of donor cells.
  • the selective medium Succinate Minimal Salts medium plates with 25 pg/mL kanamycin and 50 pg/mL rifampicin.
  • Replicative plasmids are useful tools for gene overexpression or promoter screening.
  • a polynucleotide sequence may be genetically inserted without modification in a plasmid vector; however, a DNA sequence having a promoter activity may be added. Furthermore, mutations may be introduced at random and polynucleotides with improved promoter activity may be used. Usually, a coding region of enzyme protein or the like is positioned on the 3’ side of the promoter sequence.
  • Commercially available plasmid vectors can be applicable if they already have a promoter sequence, and the promoter functions in one or more Methylopila species. As the plasmid vector, any vectors can be used insofar as they are present, stable, and replicable in transformed cells.
  • plasmid vector pUC series, pBR series and the like that are used for transformation of E. coli
  • shuttle vectors linked with a plasmid vector which is replicable in the target cells can be exemplified.
  • a desired DNA sequence may be inserted into an appropriate plasmid vector with the use of a replication region of a broad-host-range vector, and used as a shuttle vector.
  • a shuttle vector may be prepared by inserting a replication region of RK2 vector into an appropriate position of a pUC series vector, and the shuttle vector is able to utilize E. coli.
  • pBBR series plasmids which have relatively small DN A sizes and are replicable in a broad range of hosts.
  • pBBR series plasmids include pBBR122, pBBRIMCS, pBBRlMCS2, pBBRlMCS3, pBBR!MCS4, and pBBRlMCS5. These plasmid vectors are characterized, for example, by different antibiotic markers, and may be selected for use after evaluation of antibiotic resistance of a transformed cell. Furthermore, a plasmid retained by a cell to be transformed may be used. Replicative broad host range vectors including pBBRlMCS2, RK2-based expression vectors, and IncP type vectors have been tested and utilized in the Methylopila sp. YHT-1 strain 12 . pBBRl MCS2-derived plasmids can be stably maintained for many generations in the presence of a low concentration of kanamycin.
  • a broad host range vector pK18mobsacB was used in the Methylopila sp. YHT-1 strain.
  • pK18mobsacB -derived plasmids can be efficiently transformed into Methylopila sp. YHT-1 cells using the biparental conjugation system.
  • Single crossover transconjugant colonies were selected with kanamycin, and then second crossover events were selected using the sucrose counterselection strategy to remove the plasmid backbone from the chromosome and produce the colonies with the gene insertion or gene deletion. Colony PCR and Sanger sequencing methods were used for genotyping.
  • YHT-1 and a number of well-characterized promoters used in other methanolutilizing microorganisms, as well as the widely used Ptac promoter, were tested and quantified in the Methylopila sp. YHT-1 strain (FIG. 5).
  • the PMDHI promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47) were each cloned into the upstream region of RBS site of the GFP gene to generate the replicative plasmids shown in FIG. 5, panels B-I.
  • the native promoter located upstream of the pqqA-B-C-D-E operon (SEQ ID NO: 38) was also amplified and inserted into the same location to serve as a control (FIG. 6, panel A). All of the aforementioned plasmids were each transformed into Methylopila sp. YHT-1 cells. The confirmed transconjugant colonies were inoculated into 1 ml, of either methanol minimal B medium or succinate minimal salts (SMS) medium with methanol or succinate as carbon source, respectively, then grown in a 48-well MTP, flower plate at 30°C and a shaking frequency of 1000 rpm in a BioLector microbioreactor.
  • SMS succinate minimal salts
  • Example 2 Deletion analysis of the native promoter of the pqqA-B-C ⁇ D-E operon
  • the 351 bp intergenic region (IGR) sequence upstream from the pqqA gene start codon was searched with BProm software and a putative -35 region and -10 region were identified.
  • IGRpqqA351 meaning 351 bp upstream from the pqqA gene start codon
  • IGRpqqA300, IGRpqqA270, IGRpqqA200, IGRpqqA152 and IGRpqqA-del97 meaning 97 bp upstream of the pqqA gene start, codon was removed
  • GFP signals and biomass of each well were measured in a time-course manner. Based on all GFP and biomass measurement data, and biomass-normalized GFP data, there was no significant difference among strains hosting plasmids of all these promoters. The combined data supported the conclusion that the 152 bp region upstream of the pqqA gene contains the complete promoter sequence, and that the 97 bp region upstream of the pqqA gene is essential for the transcription of the pqqA-B-C-D- E operon.
  • Example 3 Promoter identification for the IGR sequence upstream of the pqqE gene
  • a number of pBBR1MCS2-derived replicative plasmids were constructed for the overexpression of the pqqA-B-C-D-E operon, six additional copies of synthetic pqqA genes (SEQ ID NOs: 4-9) and/or the pqqA-B-C-D-E operon combined with the pqqF-G operon.
  • the native promoter, the putative pqqA-B-C-D-E operon, and a terminator were amplified and inserted into the pBBR!MCS2 vector using the Gibson Assembly cloning method, thereby generating the pBBRlMCS2-PQQ plasmid (FIG. 9, panel A).
  • the pqqE promoter was removed to create a plasmid named pBBRlMCS2-pqqAs-PQQ-delPpqqE- GFP (FIG. 9, panel E).
  • the pqqF-G operon was amplified and then inserted in between the pqqE gene and the terminator of the pBBRlMCS2-pqqAs-PQQ-GFP plasmid to generate the pB BRI MCS2-pqqAs-PQQ-FG-GFP plasmid (FIG. 9, panel F).
  • the Ptac promoter was validated as a stronger promoter than the native promoter, so the native promoter in pBBRlMCS2-pqqAs-PQQ-GFP, pBBRlMCS2-pqqAs-PQQ-delPpqqE-GFP, and pBBRlMCS2-pqqAs-PQQ-FG-GFP were replaced with Ptac promoter to build the constructs of pBBRlMCS2-Ptac-pqqAs-PQQ-GFP, pBBRlMCS2-Ptac-pqqAs-PQQ-delPpqqqE-GFP, and pBBRlMCS2-Ptac-pqqAs-PQQ-FG-GFP, respectively (FIG. 9, panels G-I).
  • SoxR gene (SEQ ID NO: 25) encoding a putative redox- sensitive transcriptional activator SoxR is located immediately upstream of the pqqF gene on the chromosome of Methylopila sp. YHT-1.
  • SoxR a MerR-family homodimeric transcription factor with a 2Fe-2S cluster in each monomer, is known to function as a sensor and a transcriptional activator for a superoxide response regulon.
  • PQQ plays an important antioxidant role in the cells.
  • the Ptac promoter or methanolinducible PmxaF promoter or PMDHI promoter, an RBS sequence, and the soxR gene were cloned into the pBBR!MCS2 vector to create the pBBR!MCS2-Ptac-SoxR, pBBR!MCS2- PmxaF-SoxR and pBBR!MCS2-PMDHl-SoxR plasmids, respectively (FIG. 10, panels A-C).
  • soxR-pqqF-pqqG gene fragment was amplified and inserted in between the pqqE gene and the terminator of the pBBR1MCS2-pqqAs-PQQ-GFP plasmid and pBBRIMCS2-Ptac- pqqAs-PQQ-GFP plasmid to generate the pBBRlMCS2-pqqAs-PQQ-SoxR-FG-GFP plasmid and pBBR!MCS2-Ptac-pqqAs-PQQ-SoxR-FG-GFP plasmid, respectively (FIG. 10, panel D, panel E).
  • Example 6 Chromosomal site-directed insertion of the strong promoter, multiple pqqA genes, and an additional copy of PQQ biosynthetic gene cluster into the Methylopila sp. YHT-1 strain
  • site-directed chromosomal integration strains were built based on pK18mobSacB derived constructs and sucrose counterselection strategy.
  • the strong Ptac promoter, two ⁇ l-kb fragments of 5’ and 3’ DNA regions flanking the start codon of pqqA gene were cloned into pKl 8mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB-Ptac-PQQ-Arms (FIG. 11, panel A).
  • the strong Ptac promoter with six additional copies of synthetic pqqA genes including pqqA_r6, pqqA_ r ⁇ 5, pqqA_r]., pqqA_xAA, pqqA_y ⁇ 0, and pqqA_rA%, along with their own RBS sites, were inserted immediately upstream of the Ptac promoter of the pKl 8mobSacB-Ptac-PQQ-Arms plasmid to create the pK18mobSacB-PtacPqqAs-PtacPQQ-Arms plasmid (FIG. 11, panel B).
  • the native promoter was then replaced with methanol-inducible PMDHI promoter using the Gibson Assembly cloning method to build the plasmid pK18mobSacB-PMDHl-PqqAs-PQQ-Arms (FIG. 11, panel E). Similar to pK18mobSacB-Ptac-PqqAs-PQQ-Arms and pK18mobSacB- NativePromoter-PqqAs-PQQ-Arms plasmids, pKl 8mobSacB-Ptac-PqqAs-pqqA2-Arms (FIG.
  • plasmids were cloned, their insertion sites being immediately upstream of the pqqA2 gene in the Methyiopila sp. YHT-1 chromosome. All of the above plasmids were transformed into Methylopila sp. YHT-1 cells. The single crossover transconjugant colonies were confirmed by colony PCR followed with Sanger sequencing.
  • the confirmed single crossover transconjugant colonies were picked and cultured in methanol minimal B medium without kanamycin, and then streaked onto counterselection nutrient agar plates containing 10% sucrose. Single colonies from these counterselection plates were then picked and streaked once more to obtain sufficient biomass for genotype screening (colony PCR and Sanger sequencing) of the correct double crossover colonies.
  • the confirmed engineered colonies were cultured in methanol minimal B medium and an assay measuring the amount of product PQQ was conducted as described in Example 8.
  • PqqT a PQQ periplasmic (solute) binding protein
  • Methylobacterium extorquens AMI Methylobacterium extorquens AMI. It was proposed that PqqT is involved in the uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis 16 .
  • the pqqT gene was also discovered from the genome of the Methylopila sp. YHT-1 strain (SEQ ID NO: 31) and located in an operon containing three genes, wherein the other two genes encode a transport system permease protein and an ABC transporter ATP-binding protein. The inventors hypothesized that the deletion of pqqT gene from the chromosome of Methylopila sp.
  • YHT-1 strain might block the import of exogenous PQQ into the cytosol, thereby improving endogenous PQQ production.
  • Two ⁇ l-kb fragments of 5’ and 3’ DNA regions flanking the pqqT gene were cloned into pK18mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB -pqqT -KO- Arms.
  • the pqqT gene was in-frame deleted from the chromosome of Methylopila sp.
  • the confirmed engineered colonies were cultured in methanol minimal B medium and an assay measuring the amount of product PQQ was conducted as described in Example 8.
  • Example 8 Quantification of PQQ produced by genetically engineered Methylopila sp. YHT-1 strains
  • the wildtype and genetically engineered Methylopila sp. YHT-1 strains were streaked onto methanol minimal B medium plate and grown at 30 °C.
  • a solution was formed by combining 2 g (NHahSOr, 1-4 g KH2PO4, 3 g Na2HPO4, 0.2 g MgSO4, 30 mg ferric citrate, 30 mg CaCh, 5 mg MnCh, 5 mg ZnSCh, 0.5 mg C11SO4, and 6 g methanol per liter of water solvent, to form methanol minimal B medium at pH 7.0, then 20 g/L agar were added to yield methanol minimal B plate medium.
  • PQQ titer in test tube cultures using minimal medium was frequently below the detection limit of HPLC and, when the nutrition rich medium was used, the HPLC peak area corresponding to PQQ might be overestimated because of the background noise.
  • the PQQ GDH assay was modified from the method published in The Journal of Archives of Biochemistry and Biophysics in 1996 (doi.org/10.1006/abbi.1996.0530). The assay made use of the activity of the glucose dehydrogenase enzyme, using PQQ as a cofactor, to measure PQQ concentration.
  • GDH was combined with glucose, a dye (2,6-Dichlorophenolindophenol, DCPIP), and an electron acceptor (phenazine methosulfate).
  • PQQ glucose a dye
  • DCPIP 2,6-Dichlorophenolindophenol
  • an electron acceptor phenazine methosulfate
  • a flow rate of 1.0 ml/min was applied, and the mobile phase was composed of (A) 10 mM ammonium acetate (pH 5.0) and (B) methanol.
  • the elution program was 0-2 min 15% B: 2-7 min 15%-50% B; 7-7.1 min 50%-15% B; 7.1-10 min 15% B.
  • the detection wavelength was 254 nm for PQQ.
  • PQQ standard showed up at around 1.9 minutes (FIG. 14, panel A).
  • YHT-1 strain hosting pBBR!MCS2-Ptac-pqqAs-PQQ-GFP plasmid (FIG. 14, panel C) and the engineered strain hosting pBBRlMCS2-pqqAs-PQQ-GFP plasmid (FIG. 14, panel D) showed a 20- or 14-fold increase in PQQ production, respectively.
  • representative genetically engineered Methylopila sp. YHT-1 strains were inoculated into 3-Liter fermenters for fed-batch culture, resulting in a consistent production of 550 mg/L of PQQ or more in about 7 days. As shown in FIG.
  • PQQ could be produced as the dominant product (FIG. 15, panel B).
  • the retention time and UV spectrum (FIG. 15, panel D) of the PQQ peak were consistent with those of the PQQ standard (FIG. 15, panels A and C).

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Abstract

Provided herein are methods for improving the biosynthetic production of PQQ in Methylopila sp, YHT-1 strain using genetic engineering approaches to increase the copy numbers and the expression levels of the PQQ biosynthetic genes.

Description

METHODS FOR IMPROVING PYRROLOQUINOLINE QUINONE PRODUCTION IN
METHYLOPILA
RELATED APPLICATION
This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/272,440, filed on October 27, 2021, entitled “METHODS FOR IMPROVING PYRROLOQUINOLINE QUINONE PRODUCTION IN METHYLOPILA,” the entire contents of which are incorporated herein by reference.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing (C149770064WQ00-SEQ-ZJG.xml; Size: 139,105 bytes; and Date of Creation: October 1 1, 2022) is herein incorporated by reference in its entirety.
FIELD OF THE IN VENTION
The field of the invention relates to transgenic bacteria and methods for increasing the biosynthetic production of pyrroloquinoline quinone (PQQ). More specifically, the present methods and processes make use of genetically engineered bacteria Methylopila sp. YHT-1 that have been transformed to include nucleic acids encoding a set of PQQ biosynthetic enzymes. The copy numbers and expression level of the PQQ biosynthetic genes have been increased to improve the PQQ production in the genetic engineered Methylopila sp. YHT-1 strains.
BACKGROUND
Pyrroloquinoline quinone (4,5-dihydro-4,5-dioxo-lH-pyrrolo-[2,3-/]quinoline-2,7,9- tricarboxylic acid; PQQ; FIG. 1), also named as methoxatin, is a water-soluble, aromatic, tricyclic 0-quinone that was discovered as a redox cofactor of bacterial glucose dehydrogenase in 1964. Although PQQ is found in soil, fruits, vegetables, and meats as well as human breast milk, currently PQQ has only been reported being synthesized by microorganisms. PQQ enters plants from soil bacteria, and subsequently enters human diets. PQQ has been demonstrated to act as a redox cofactor of methanol/alcohol dehydrogenase and glucose dehydrogenase enzymes. PQQ has been recognized as the third type of redox cofactors discovered after pyridine nucleotide- and flavin-dependent cofactors. PQQ possesses potent antioxidant activity, much stronger than other quinones and enediols including vitamin C (ascorbic acid). In terms of redox recycling, it has been estimated to have about 20,000 potential redox cycles and is therefore much more stable than ascorbic acid which only has about 4.
As an important nutrient involved in numerous physiological and biochemical processes, PQQ has provoked significant interest because of its benefits for human health, such as for diabetes, anti-aging, neuroprotection, cognition, antioxidant activity, and lowering the level of c- reactive protein in response to inflammation. Also, PQQ supplementation has been proved to promote mitochondrial efficiency and induce mitochondrial biogenesis. PQQ was suggested as a newcomer to the B groups of vitamins in a research article published In Nature In 20031, although controversy regarding its role as a vitamin has persisted ever since. A recent study on five mouse PQQ-binding proteins confirmed the conversion of lactate to pyruvate by rabbit L- lactate dehydrogenase is regulated by the cofactor PQQ, which strongly supports the hypothesis that PQQ may function as a vitamin in mammals. Numerous experiments conducted on PQQ in rodents and cats also support the theory that PQQ plays a critical role in mitochondrial health. In 2018, in a perspective article published in the Proceedings of the National Academy of Sciences2, the authors propose PQQ as a “longevity vitamin” which Is not essential for immediate survival, but necessary for long-term health.
The worldwide market for PQQ has been rapidly increasing in size in recent years. Currently, PQQ can be prepared by chemical synthesis methods on a multi-gram scale in high yield and high purity. However, known chemical PQQ synthetic processes are not ideal for safe and sustainable production due to a number of disadvantages such as costly key starting materials, the use of toxic precursors, complicated purification processes, generation of undesirable byproducts, and potential cause of environmental pollution. Moreover, consumers generally prefer natural derivatives and are usually willing to pay higher prices for natural ingredients. Methods for the production of PQQ using wildtype bacteria strains belonging to the genera of Achronobacter, Alteromonas, Ancyclobacter, Hyphomicrobium, Methanomonas, Methylobacillus, Methylomonas, Methy lophilus, Methy lophaga, Methylobac ■terium, Microcyclus, Mycoplana, Pseudomonas, P rotammobacter, Protomonas, Thiobacillus, Xanthobacter, Myxococcus and Paracoccus have been disclosed3, 4’ 5. Among all of these different genera of bacteria, Methylovorus sp. MP688 and genetically engineered Methylobacterium extorquens AMI are the most promising disclosed strains which may be cultivated in a medium containing methanol as a carbon source and reportedly produce 125 mg/L and 114 mg/L PQQ in test tube fermentations, respectively. However, currently reported PQQ yields are not ideal for production on an industrial scale. PQQ is still both expensive and difficult to obtain by biological production. Accordingly, there is a need for improved microbial fermentation methods to produce PQQ economically, reliably, and in an environmentally friendly manner.
SUMMARY OF THE INVENTION
Methylopila is a genus of bacteria from the Methylocystaceae, a family of bacteria that are capable of obtaining carbon and energy from methane. Such bacteria are known as methanotroplis, and the Methylocystaceae belong to the type II methanotroplis, which are structurally and biochemically distinct from the Methylococcaceae or type I methanotroplis . In this family methane is oxidized to form formaldehyde, which is assimilated by the serine pathway. This involves combining formaldehyde and glycine to form serine, which may be converted into glyceraldehyde and thus into other organic molecules. This family of bacteria can also fix nitrogen, similarly to many other members of the order Rhizobiales.
A novel PQQ-producing Methylopila strain named Methylopila sp. YHT-1 was isolated from the soil in Wuxi, Jiangsu, China and was found to use methanol as its single carbon source. The strain was reported to produce 30 mg/L PQQ in a test tube fermentation setting and 113.6 mg/L PQQ in a 3-liter fermenter. Genes associated with the PQQ biosynthesis have been discovered in several species of bacteria including Methylobacterium extorquens AMI, Methylovorus sp. MP688, Acinetobacter calcoaceticus, Enterobacter intermedium 60-2G, Klebsiella pneumoniae, and Gluconobacter oxydans. The core PQQ biosynthetic genes are mostly arranged in one or two clusters pqqA-B-C-D-E-F or pqqA-B-C-D-E (also known as pqqABCDEF or pqqABCDE, respectively) and pqqF-G (also known as ppqFG). In this context, the term “pqq gene cluster” can mean a gene cluster or operon that encodes proteins involved in the biosynthesis of PQQ.
Decades of studies on the PQQ biosynthesis in the Methylobacterium extorquens AMI strain, particularly by the Klinman laboratory at the University of California, Berkeley, have elucidated the complete PQQ biosynthetic pathway and characterized most of the key enzymes at each step. The structure of PQQ biosynthetic gene clusters in the Methylobacterium extorquens AMI strain is schematically illustrated in FIG. 2, panel A. The genome of the Methylopila sp. YHT-l strain was sequenced using the Illumina Next-Generation Sequencing method. PQQ biosynthetic genes in the Methylopila sp. YHT-l strain are schematically illustrated in FIG. 2, panel B. Similar to clusters in Methylobacterium extorquens AMI strain, the core PQQ biosynthetic genes in Methylopila sp. YHT-l strain were found to be arranged in two clusters pqqABC DE and pqqFG. In addition, a second copy of the pqqA gene (pqqA2), a pqqT gene encoding a putative PQQ binding protein, and a soxR gene encoding a redox- sensitive transcriptional activator located in the pqqFG operon have been annotated.
As shown in FIG. 3, PQQ biosynthesis is believed to start from a ribosomal translated precursor peptide, PqqA. Typically, the peptide PqqA sequence of each microorganism is relatively conserved, although the length varies from 23 to 39 amino acids among different known microorganisms. The PqqA sequence in Methylopila sp. YHT-l strain is MAIWTAPIVEETPVGLEVTSYSPAEL (SEQ ID NO: 1). There are believed to be five steps to PQQ biosynthesis6, z’ 8’ 9’ !0, 11. The first step is catalyzed by the radical SAM (S-adenosyl-L- methionine) enzyme PqqE, in complex with PqqD, the small chaperone protein, binding with the substrate PqqA. The glutamic acid and the tyrosine in PqqA are then cross-linked.
Subsequently, the modified PqqA peptide is recognized by protease M16B family enzymes PqqF and/or PqqG, which catalyze cutting of the generated glutamic acid-tyrosine pair out of the PqqA peptide to release the suitable substrate for the downstream enzyme PqqB. PqqB was recently characterized as an iron-dependent hydroxylase catalyzing a series of complicated oxygeninsertion reactions to produce the quinone moiety10. After a spontaneous cyclization, AHQQ, the immediate precursor of PQQ, is generated, followed by the last step of PQQ formation, which is believed to be catalyzed by PqqC, a cofactor-less oxidase. AHQQ is thus converted into PQQ in a reaction that involves overall eight-electron oxidations, leading to pyrrole- and pyridine-ring formation.
As described herein, PQQ can be reliably produced at a high yield by genetic engineering and fermentation technology using recombinant Methylopila sp. YHT-l cell cultures. These genetically engineered Methylopila sp. YHT-l cell cultures can synthesize PQQ in commercially significant yields. To this end, provided herein are genetic engineering methods which contribute to increase the copy numbers and enhance the expression level of the precursor peptide pqqA gene and the entire PQQ biosynthetic gene clusters, which in turn create a way to sustainably and economically produce unprecedented amounts of PQQ product. hi embodiments, disclosed herein is a transgenic bacterial cell or population thereof, wherein the transgenic bacterial cell can produce PQQ at a high yield and the bacterium is Methylopila sp. YHT-1.
In embodiments, disclosed is a transgenic Methylopila sp. YHT-1 cell, or population thereof, the transgenic Methylopila sp. YHT-1 cell including nucleotide sequences capable of expressing a PQQ precursor peptide PqqA, an iron-dependent hydroxylase PqqB, a cofactorless oxidase PqqC, a PqqA precursor binding chaperone protein PqqD, a radical SAM enzyme PqqE, protease M16B family enzymes PqqF and/or PqqG, a redox-sensitive transcriptional activator SoxR, wherein the nucleotide sequences and amino acids sequences are from Methylopila sp. YHT-1.
In embodiments, disclosed herein is a transgenic Methylopila sp. YHT-1 cell, or population thereof. The transgenic Methylopila sp. YHT-1 cell comprises a disrupted gene encoding a putative PQQ periplasmic binding protein PqqT, wherein the pqqT and its flanking region nucleotide sequences are from Methylopila sp. YHT-1 . hi embodiments, disclosed is a transgenic Methylopila sp. YHT-1 cell, or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising multiple copies of synthetic genes encoding a PQQ precursor peptide PqqA.
In embodiments, disclosed is a transgenic Methylopila sp. YHT-1 cell, or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising one or more replicative broad host range vectors for use in overexpression of PQQ biosynthetic genes, wherein the replicative broad host range vectors include but not are not limited to Bordetella bronchiseptica-derived pBBRIMCS series vectors, or RK2 -based expression vectors like pJB656, or IncP type vectors such as pRK310 and pCM series vectors. hi embodiments, disclosed is a transgenic Methylopila sp. YHT-1 cell, or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising native or synthetic promoters for use in overexpression of PQQ biosynthetic genes, wherein the promoters include but are not limited to the native promoter located upstream of the pqqA-B-C -D-E operon, the native promoter located upstream of pqqE gene, the native promoter located upstream of the mdhl operon, the native promoter located upstream of the mdh.2 operon, the native promoter located upstream of the pqqF-Q operon, the native promoter located upstream of the pqqT gene, PmxaF promoter of the PQQ-dependent methanol dehydrogenase gene of Methylobacterium extorquens AML PfumC promoter of the fumarase gene of Methylobacterium extorquens AML PcoxB promoter of the cytochrome c oxidase subunit II gene of Methylobacterium extorquens AMI, Ptuf promoter of the EF-Tu gene coding for the translation elongation factor thermo-unstable of Methylobacterium extorquens AMI, Pqq-M promoter of the pqqA2 gene of Methylovorus sp. MP688 and the hybrid Ptac promoter.
In embodiments, disclosed is a transgenic Methylopila sp. YHT-1 cell, or population thereof, the transgenic Methylopila sp. YHT-1 cell comprising one or more gene cassettes comprising native or strong synthetic promoters, nucleotide sequences capable of expressing the precursor peptide pqqA gene, and the whole PQQ biosynthetic gene cluster that are integrated into the chromosome of Methylopila sp. YHT-1 strain at the locus upstream of the pqqA-B-C-D- E operon or upstream of the pqqA2 gene.
In embodiments, disclosed is a method for the production of the PQQ, the method comprising culturing genetically engineered Methylopila sp. YHT-1 cells in culture and fermentation media.
In embodiments, the culture and fermentation media for genetic engineered Methylopila sp. YHT-1 cells contain methanol as a carbon source.
In embodiments, the vast majority of the PQQ is secreted into the growth media.
In embodiments, disclosed are amino acid sequences and nucleotide sequences of proteins, deoxyribonucleic acids, and promoters utilized for genetic engineering of Methylopila sp. YHT-1 cells, which are listed in the Sequence Listing section hereinbelow.
Some aspects of the present disclosure provide for producing pyrroloquinoline quinone (PQQ) comprising: cultivating in a culture medium a bacterium belonging to the genus Methylopila, and collecting PQQ from the culture medium, wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
(i) increasing the copy number of the gene cluster,
(ii) introducing at least one copy of the gene cluster into the chromosome of the bacterium,
(iii) placing at least one copy of the gene cluster under the control of a potent promoter, (iv) introducing at least one copy of the gene cluster into the bacterium by inserting one or mor genes of the gene cluster into the chromosome, and placing the remaining genes of the gene cluster under the control of a potent promoter, and
(v) combinations thereof. in some embodiments, the pqqABCDE gene cluster comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22. In some embodiments, the pqqABCDE gene cluster comprises the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.
In some embodiments, the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 70%', at least 75%, at least 80%, at least 85%', at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof; the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71 , 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising a nucleotide sequence at least 70%', at least 75%, at least 80%, at least 85%', at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof.
In some embodiments, the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51 , 53, 55, 57, or variants thereof: the pqqABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene comprising the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof. in some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
In some embedments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid. hi some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid. in some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
In some embodiments, one or more copies (e.g., 1 , 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
In some embodiments, one or more copies (e.g., 1 , 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium.
In some embodiments, the pqqABCDE gene cluster is inserted into the chromosome of the bacterium. In some embodiments, pqqABCDE gene cluster is introduced into the bacterium via a plasmid. In some embodiments, a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
In some embodiments, the potent promoter is selected from the group consisting of PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47), or variants thereof. In some embodiments, the potent promoter comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46. or SEQ ID NO: 47. In some embodiments, the potent promoter comprises the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the potent promoter is Ptac promoter (SEQ ID NO: 47), or valiants thereof.
In some embodiments, the bacterium has been further modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium,
(iii) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
In some embodiments, the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and variants thereof. In some embodiments, the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. and SEQ ID NO: 11.
In some embodiments, the bacterium has been further modified to delete or knock out a pqqT gene. In some embodiments, the pqqT is identified by SEQ ID NO: 31.
In some embodiments, the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, and uncultured Methylopila sp. In some embodiments, the bacterium is Methylopila sp. YH-1. Ill some embodiments, any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp, Yamaguchi, Methylopila sp. Ml 07, Methylopila sp. 73B, or uncultured Methylopila sp. In some embodiments, any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1. In some embodiments, the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
In some embodiments, the culture medium includes methanol at a concentration of not less than 3 grams (e.g., 10, 9, 8, 7, 6, 5, 4, 3 grams) of methanol per liter of water solvent to at most 15 grams (e.g,, 5, 6, 9, 8, 9, 10, 11, 12, 13, 14, 15 grams) of methanol per liter water solvent.
Further provided herein are recombinant bacterium belonging to the genus Methylopila, wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
(i) increasing the copy number of the gene cluster,
(ii) introducing at least one copy of the gene cluster into the chromosome of the bacterium,
(iii) placing at least one copy of the gene cluster under the control of a potent promoter,
(iv) introducing at least one copy of the gene cluster into the bacterium by inserting one or mor genes of the gene cluster into the chromosome, and placing the remaining genes of the gene cluster under the control of a potent promoter, and
(v) combinations thereof.
In some embodiments, the pqqABCDE gene cluster comprises a nucleotide sequence that is at least 70%, at least 15%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22. In some embodiments, the pqqABCDE gene cluster comprises the nucleotide sequence of any one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22,
In some embodiments, the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 70%', at least 75%, at least 80%, at least 85%', at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or valiants thereof; the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81 , 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene comprising a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%' identical to the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof. in some embodiments, the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof: the pqq ABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71 , 73, 75, 77, or variants thereof; the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and the pqqABCDE gene cluster comprises a pqqE gene comprising the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof.
In some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid. In some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid. In some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid. In some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid. In some embodiments, one or more copies (e.g., 1 , 2, or 3 copies) of the pqq A gene is inserted into the chromosome of the bacterium, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid. In some embodiments, one or more copies (e.g., 1, 2, or 3 copies) of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium. In some embodiments, the pqqABCDE gene cluster is inserted into the chromosome of the bacterium. In some embodiments, pqqABCDE gene cluster is introduced into the bacterium via a plasmid. In some embodiments, a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid. hi some embodiments, the potent promoter is selected from the group consisting of PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Plac promoter (SEQ ID NO: 47), or variants thereof. In some embodiments, the potent promoter comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the potent promoter comprises the nucleotide sequence of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the potent promoter is Ptac promoter (SEQ ID NO: 47), or variants thereof.
In some embodiments, the bacterium has been further modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium, (iii) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
In some embodiments, the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof. In some embodiments, the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.
Further provided herein are recombinant bacterium belonging to the genus Methylopila, wherein the bacterium has been modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium,
(iii) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
In some embodiments, the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof. In some embodiments, the nucleotide sequence of each pqqA-like gene independently comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.
In some embodiments, the bacterium has been further modified to delete or knock out a pqqT gene. In some embodiments, the pqqT is identified by SEQ ID NO: 31 . In some embodiments, the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. Ml 07, Methylopila sp. 73B, and uncultured Methylopila sp. In some embodiments, the bacterium is Methylopila sp. YH-1. In some embodiments, any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, or uncultured Methylopila sp. In some embodiments, any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1. In some embodiments, the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
Methods for producing pyrroloquinoline quinone (PQQ) are also provided, the method comprising cultivating in a culture medium a bacterium described here, and collecting PQQ from the culture medium.
SEQUENCE LISTING
The amino acids and nucleotide sequences listed in the accompanying sequence listing are shown using standard 1 -letter abbreviations for both amino acid and nucleotide base. Only one strand of the nucleotide sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NO: 1 is the amino acid sequence of PqqA of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 2. PqqA is a PQQ precursor peptide. SEQ ID NO: 3 is the nucleotide sequence of the second copy of pqqA (pqqA2) of Methylopila sp. YHT-1. SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11 are codon-randomized nucleotide sequence of pqqA.
SEQ ID NO: 12 is the amino acid sequence of PqqB ofi Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 13 and codon-optimized nucleotide SEQ ID NO: 14. PqqB is an iron-dependent hydroxylase.
SEQ ID NO: 15 is the amino acid sequence of PqqC of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 16 and codon-optimized nucleotide SEQ ID NO: 17. PqqC is a cofactorless oxidase.
SEQ ID NO: 18 is the amino acid sequence of PqqD of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 19 and codon-optimized nucleotide SEQ ID NO: 20. PqqD is a PqqA precursor binding chaperone protein. SEQ ID NO: 21 is the amino acid sequence of PqqE of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 22 and codon-optimized nucleotide SEQ ID NO: 23, PqqE is a radical SAM enzyme,
SEQ ID NO: 24 is the amino acid sequence of SoxR of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 25. SoxR is a putative redox-sensitive transcriptional activator.
SEQ ID NO: 26 is the amino acid sequence of PqqF of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 27. PqqF is a protease M16B family enzyme.
SEQ ID NO: 28 is the amino acid sequence of PqqG of Methylopila sp, YHT-1, corresponding to nucleotide SEQ ID NO: 29. PqqG is a protease M16B family enzyme.
SEQ ID NO: 30 is the amino acid sequence of PqqT of Methylopila sp. YHT-1, corresponding to nucleotide SEQ ID NO: 31. PqqT is a putative PQQ periplasmic binding protein.
SEQ ID NO: 32 is the amino acid sequence of MDH1 of Methylopila sp. YHT-1 , corresponding to nucleotide SEQ ID NO: 33. SEQ ID NO: 34 is the nucleotide sequence of the promoter region located upstream of the mdhl operon. MDH1 is a putative PQQ-dependent methanol dehydrogenase.
SEQ ID NO: 35 is the amino acid sequence of MDH2 of Methylopila sp. YHT-1 , corresponding to nucleotide SEQ ID NO: 36. SEQ ID NO: 37 is the nucleotide sequence of the promoter region located upstream of the mdh2 operon. MDH2 is another putative PQQ- dependent methanol dehydrogenase.
SEQ ID NO: 38 is the nucleotide sequence of the promoter region located upstream of the pqqA-B-C-D-E operon of Methylopila sp. YHT-1.
SEQ ID NO: 39 is the nucleotide sequence of the promoter region located upstream of the pqqE gene of Methylopila sp. YHT-1.
SEQ ID NO: 40 is the nucleotide sequence of the promoter region located upstream of the pqqF-Q operon of Methylopila sp. YHT-1.
SEQ ID NO: 41 is the nucleotide sequence of the promoter region located upstream of the pqqT gene of Methylopila sp. YHT-1.
SEQ ID NO: 42 is the nucleotide sequence of the PmxaF promoter of the PQQ-dependent methanol dehydrogenase gene of Methylobacterium extorquens AMI. SEQ ID NO: 43 is the nucleotide sequence of the PfumC promoter of the fumarase gene of Methylobacterium. extorquens AMI.
SEQ ID NO: 44 is the nucleotide sequence of the PcoxB promoter of the cytochrome c oxidase subunit II gene of Methylobacterium extorquens AMI.
SEQ ID NO: 45 is the nucleotide sequence of the Pluf promoter of the EF-Tu gene coding for the translation elongation factor thermo-unstable of Methylobacterium extorquens AMI.
SEQ ID NO: 46 is the nucleotide sequence of the PqqA2 promoter (MP688A2) of the pqqA2 gene of Methylovorus sp. MP688.
SEQ ID NO: 47 is the nucleotide sequence of the hybrid Ptac promoter.
SEQ ID NO: 48 is the amino acid sequence of PqqA of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 49.
SEQ ID NO: 50 is the amino acid sequence of PqqA of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 51.
SEQ ID NO: 52 is the amino acid sequence of PqqA of Methylopila sp. M107, corresponding to nucleotide SEQ ID NO: 53.
SEQ ID NO: 54 is the amino acid sequence of PqqA of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 55.
SEQ ID NO: 56 is the amino acid sequence of PqqA of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 57.
SEQ ID NO: 58 is the amino acid sequence of PqqB of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 59.
SEQ ID NO: 60 is the amino acid sequence of PqqB of Methylopila. sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 61.
SEQ ID NO: 62 is the amino acid sequence of PqqB of Methylopila sp. M107, corresponding to nucleotide SEQ ID NO: 63.
SEQ ID NO: 64 is the amino acid sequence of PqqB of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 65.
SEQ ID NO: 66 is the amino acid sequence of PqqB of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 67. SEQ ID NO: 68 is the amino acid sequence of PqqC of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 69.
SEQ ID NO: 70 is the amino acid sequence of PqqC of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 71.
SEQ ID NO: 72 is the amino acid sequence of PqqC of Methylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 73.
SEQ ID NO: 74 is the amino acid sequence of PqqC of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 75.
SEQ ID NO: 76 is the amino acid sequence of PqqC of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 77.
SEQ ID NO: 78 is the amino acid sequence of PqqD of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 79.
SEQ ID NO: 80 is the amino acid sequence of PqqD of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 81.
SEQ ID NO: 82 is the amino acid sequence of PqqD of Methylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 83.
SEQ ID NO: 84 is the amino acid sequence of PqqD of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 85.
SEQ ID NO: 86 is the amino acid sequence of PqqD of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 87.
SEQ ID NO: 88 is the amino acid sequence of PqqE of Methylopila capsulata, corresponding to nucleotide SEQ ID NO: 89.
SEQ ID NO: 90 is the amino acid sequence of PqqE of Methylopila sp. Yamaguchi, corresponding to nucleotide SEQ ID NO: 91.
SEQ ID NO: 92 is the amino acid sequence of PqqE of MePhylopila sp. Ml 07, corresponding to nucleotide SEQ ID NO: 93.
SEQ ID NO: 94 is the amino acid sequence of PqqE of Methylopila sp. 73B, corresponding to nucleotide SEQ ID NO: 95.
SEQ ID NO: 96 is the amino acid sequence of PqqE of uncultured Methylopila sp., corresponding to nucleotide SEQ ID NO: 97. BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows the chemical structure of PQQ.
FIG. 2 shows the comparison of PQQ biosynthetic genes (gene clusters) in Methylobacterium extorquens AMI strain (panel A) and Methylopila sp. YHT-1 strain (panel B).
FIG. 3 illustrates the proposed biosynthetic pathway to PQQ in Methylopila sp. YHT-1 strain.
FIG. 4 represents a schematic diagram of the biparental conjugation system for Methylopila sp. YHT-1 strain.
FIG. 5 includes the maps of plasmids the inventors have generated to screen strong promoters tax Methylopila sp. YHT-1 strain.
FIG. 6 includes the maps of plasmids the inventors have generated for a deletion analysis of the promoter region located upstream of the pqqA-B-C-D-E operon of Methylopila sp. YHT-1 strain.
FIG. 7 show's the real-time GFP signal and biomass results when Methylopila sp. YHT-1 strains hosting pBBRlMCS2-Ptac-GFP plasmid or pBBRlMCS2-IGRpqqA351-GFP plasmid were cultured in methanol minimal B medium.
FIG. 8 includes the map of a plasmid the inventors have generated for the analysis of the promoter region located upstream of the pqqE gene of Methylopila sp. YHT-1 strain (panel A) and an image of Methylopila sp. YHT-1 clones transformed with pBBRlMCS2-IGRpqqE-GFP under blue light (panel B).
FIG. 9 includes the maps of plasmids the inventors have generated for overexpression of the precursor peptide pqqA gene, pqqA-B-C-D-E operon, and the whole PQQ biosynthetic gene cluster using both the native promoter of the pqqA-B-C-D-E operon and the Ptac promoter.
FIG. 10 includes the maps of plasmids the inventors have generated for overexpression of the redox-sensitive transcriptional activator soxR gene located upstream of the pqqF-G operon of Methylopila sp. YHT-1 strain. FIG. 11 includes the maps of plasmids the inventors have generated for the insertion of the Ptac promoter, Ptac-pqqAs cassette, pqqAs cassette into the upstream region of the pqqA-B- C-D-E operon or the pqqA2 gene in the chromosome of Methylopila sp. YHT-1 strain.
FIG. 12 includes the map of plasmid the inventors have generated for in-frame deletion of the pqqT gene encoding a putative PQQ periplasmic binding protein PqqT from the chromosome of Methylopila sp. YHT-1 strain.
FIG. 13 illustrates an example of the 630 nm absorbance curve of the GDH assay (panel A) and reports PQQ GDH assay results of representative bacterial strains (panel B).
FIG. 14 includes HPLC profiles illustrating PQQ production in test tube cultures of the Methylopila sp. YHT-1 wildtype strain and of genetically engineered strains.
FIG. 15 includes a HPLC profile and UV spectra showing PQQ production in the fermentation culture of an engineered Methylopila sp. YHT-1 strain.
DETAILED DESCRIPTION hi a first aspect, the present disclosure provides new and improved bacterial strains enhancing the expression of certain PQQ-related genes and/or gene clusters. In one exemplary embodiment, a series of recombinant, genetically engineered Methylopila sp. YHT- 1 strains were generated to increase gene copy numbers and enhance the expression of the precursor peptide pqqA gene and of the entire PQQ biosynthetic gene clusters. In a second aspect, these new strains may be used in novel methods for the biosynthetic production of PQQ by in cellular systems at previously unattained yields.
The term “PQQ-producing bacterium” can mean a bacterium which has an ability to produce and excrete a PQQ into a medium when the bacterium is cultured in the medium. The term “PQQ-producing bacterium” also can mean a bacterium which is able to produce and cause accumulation of PQQ in a culture medium in an amount larger than a wild-type or parental strain, for example Methylopila sp. YH-1 (China Center for Type Culture Collection (CCT'CC) Deposit No. M 2014016) or other Methylocistaceae. The phrase “bacterium belonging to the genus Methylopila can mean that the bacterium is classified into the genus Methylopila according to the classification known to a person skilled in the art of microbiology, although the bacterium is not particularly limited. Specifically, those classified into the group Methylopila according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) may be used.
The term “PQQ-producing bacterium” can also mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.3 mg/L, not less than 0.5 mg/L, or not less than 1 mg/L of PQQ.
The phrase “enhancing the expression of gene(s) and/or gene cluster(s)” can mean that the expression of the gene(s) and/or cluster(s) in an improved strain is higher than that of a nonmodified strain, for example, a wild-type strain such as Methylopila sp. YH-1. Examples of such modifications can include increasing the copy number of the expressed gene or gene cluster per cell, increasing the expression level of the gene cluster or gene, and so forth. The copy number of an expressed gene cluster or gene is measured, for example, by restricting the chromosomal and/or plasmid DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene cluster or gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like.
The term “pqqA-like gene” can mean a gene that is located in a bacterial genome and encodes a precursor for PQQ biosynthesis. The presence of a pqqA-like gene in a bacterial genome can be determined by analysis of the genome sequence, in addition to databases containing information about annotated pqqA genes from various microorganisms. pqqA-like genes from a wide range of microorganisms can be used. Examples of the bacterium containing in-genome pqqA-like gene are not limited to the genus Methylopila. Examples of the bacterium containing in-genome pqqA-like genes can also include, Methylococcus capsulatus Bath, Colwellia psychrerythraea 34H, Gluconobacter oxydans 621H, Methylobacdllus flagellatus KT, Dinoroseobacter shibae DFL 12, Leptothrix cholodnii SP-6, Erwinia amylovora ATCC 49946, Acinetobacter sp. RUH2624, Saccharopolyspora erythraea NRRL23338, Brady rhizobium sp. ORS278, Brady rhizobium sp. BTAil/ATCC BAA-1182, Ralstonia pickettii 12J, Pseudomonas fluorescens, etc. Some of the bacteria can have multiple copies of pqqA-like gene in-genome, for instance, Met hy lot e> t era mobilis JLW8, Methylovorus sp. SIP3-4, Methylobacterium extorquens DM4, and Methyloba.cterium sp. 4-46.
Since there may be some differences in DNA sequences between the genera, species or strains, the gene cluster and the gene(s) of which expression is/are enhanced are not limited to the genes shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63. SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77. SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, but can include genes homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11. SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81. SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97. Therefore, the protein variants encoded by the genes may have exemplary homology of not less than 80%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, or not less than 99.9% with respect to the entire amino acid sequence shown in SEQ ID NO:1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24; SEQ ID NO:26. SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32. or SEQ ID NO:35, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54. SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72. SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, as long as the activity or function of the corresponding protein is maintained.
The term “homology” may also be used to refer to “identity”. The phrase “protein variant”, as used in the presently disclosed subject matter, means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids. The number of changes in the variant proteins can depend on the position in the three- dimensional structure of the proteins or the type of amino acid residues. Exemplary embodiments can be 1 to 30, 1 to 15, 1 to 5, or 1 to 3 in SEQ ID NO:1 , SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24; SEQ ID NO:26. SEQ ID NO:28, SEQ ID NO:3(), SEQ ID NO:32, SEQ ID NO:35, or SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62. SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70. SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86. SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96. These changes in the variants can typically occur in regions of the protein which are not critical for the three-dimensional structure of the protein. This is because some amino acids have high homology to one another so the three-dimensional structure is not affected by such a change. By way of example, the term “a protein having the function of PQQ precursor” can mean that the protein can be involved in PQQ biosynthesis as a precursor for PQQ, specifically, the protein can have a three-dimensional structure which is sufficient to be recognized and used as a substrate by PQQ biosynthesis enzymes with subsequent conversion into PQQ.
The substitution, deletion, insertion or addition of one or several amino acid residues should typically be conservative mutation(s) so that the activity or the function is maintained. The representative conservative mutation give rise to a conservative substitution. Examples of conservative substitutions can include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Vai or Phe for He, substitution of He, Met, Vai or Phe for Leu, substitution of Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Vai or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Tip, substitution of His, Phe or Trp for Tyr, and substitution of Met, Be or Leu for Vai.
Therefore, the gene cluster and gene(s) may be a variant(s) which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87. SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, but can include genes homologous to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29. SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional protein. “Stringent conditions” can include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. Other exemplary homologies can include not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, or not less than 99.9%. For example, stringent conditions are exemplified by washing one time or more, such as two or three times, at a salt concentration of IxSSC, 0.1% SDS. Another exemplary salt concentration can include O.lxSSC, 0.1% SDS at 60° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. By way of example, washing can be performed 2 to 3 times. The length of the probe can be suitably selected depending on the hybridization conditions, and can be 100 bp to 1 kbp, for example. Moreover, codons in the gene sequences may be replaced with other equivalent codons which are easily used in the host into which the genes are introduced. Cellular Systems
As referred to herein, a cellular system may include any cell or cells that can be used to ectopically express PQQ biosynthetic genes from the genus Methylopila. (e.g. Methylopila sp. YHT-1 strain, Methylopila capsulata strain, Methylopila sp. Yamaguchi strain, Methylopila sp. M107 strain, Methylopila sp. 73B strain, and uncultured Methylopila sp. strain). It includes bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes.
Cell Culture
A cell culture refers to any cell or cells that are in a culture. Culturing or incubating is the process in which cells are grown under controlled conditions, typically outside of their natural environment. For example, cells, such as bacterial or yeast cells, may be grown as a cell suspension in liquid nutrient broth. A cell culture includes, but is not limited to, a bacterial cell culture, a yeast cell culture, a plant cell culture, and an animal cell culture. In some embodiments, the cell culture comprises bacterial cells, yeast cells, or a combination thereof.
In some embodiments, a bacterial cell culture according to the present disclosure comprises bacterial cells including, but not limited to, Escherichia, Streptomyces, Zymomonas, Acetobacter, Citrobacter, Synechocystis, Rhizobium, Clostridium, Corynebacterium, Streptococcus, Xanlhomonas, Lactobacillus, Lactococcus, Bacillus, Alcaligenes, Pseudomonas, Aeromonas, Azotobacter, Comamonas, Mycobacterium, Rhodococcus, Gluconobacter, Ralstonia, Acidlthiobacillus, Microlunatus, Geobacter, Geobacillus, Arlhrobacter, Flavobacterium, Serratia, Saccharopolyspora, Thermus, Stenotrophomonas, Chromobacterium, Sino rhizobium, Saccharopolyspora, Agrobacterium., Pantoea, Vibrio natriegens, Achronobacter, Alteromonas, Ancyclobacter, Hyphomicrobium, Methanomonas, Methylobacillus, Methylomonas, Me thy lophilus, Me.lhylophagci, Methylopila, Aiethylobacterium, Microc yclus, Mycoplana, Pseudomonas, Protaminobacter, Protomonas, Thiobacillus, Xanthobacter, Myxococcus and Paracoccus. in some embodiments, a cell culture as described herein may be in an aqueous medium including one or more nutrient substances as known in the art. The medium may be either a synthetic or natural medium, so long as the medium includes a carbon source, a nitrogen source, minerals and, if necessary, appropriate amounts of nutrients which the bacterium may require for growth. The carbon source can include various carbohydrates such as glucose and sucrose, various organic acids, alcohol including methanol, ethanol and glycerol. Methanol is usually preferable. The nitrogen source can include various ammonium salts such as ammonia and ammonium sulphate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism. The sulfur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like. Minerals can include potassium monophosphate, sodium chloride, calcium chloride, and the like. Vitamins can include thiamine, yeast extract, and the like.
In some embodiments, cells are cultured at a temperature of 16°C to 40°C. For example, cells may be cultured at a temperature of 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23 °C, 24°C, 25°C, 26°C, 27"C, 28°C. 29°C, 30°C, 3 VC, 32“C, 33°C, 34°C, 35"C, 36°C. 37°C, 38°C, 39°C or 40°C.
In some embodiments, cells are cultured at a pH range from about 3 to about 9, for example in the range of from about 4 to about 8. The pH can be regulated by the addition of an inorganic or organic acid or base such as hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate, ammonia, or by the addition of a buffer such as phosphate, phthalate or Tris'®.
In some embodiments, cells are cultured for a period of 0.5 hours to 96 hours, or more. For example, cells may be cultured for a period of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours. Typically, cells, such as bacterial cells, are cultured for a period of 12 to 24 hours. In some embodiments, cells are cultured for 12 to 24 hours at a temperature of 37”C. In some embodiments, cells are cultured for 12 to 24 hours at a temperature of 16°C.
In some embodiments, cells are cultured to a density of 1 x 10s (OD600< 1) to 2 x 1011 (ODfjoo ~ 200) viable cells/ml cell culture medium. In some embodiments, cells are cultured to a density of 1 x 10s, 2 x 10s, 3 x 10s, 4 x 108, 5 x 108, 6 x 10s, 7 x 10s, 8 x 10s, 9 x 10s, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x 1010, 2 x 1010, 3 x 10i0, 4 x l()i0, 5 x l()i0, 6 x 10i0, 7 x 10i0, 8 x 10J0, 9 x 10J0, 1 x 10r! , or 2 x 1011 viable cells/ml. (Conversion factor: OD 1 = 8 x 10s cells/ml).
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and the PQQ can be collected and purified by methods such as ion-exchange, concentration, and crystallization. Synthetic Biology
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook, J., Ekitsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"): and by Silhavy, T, J., Bennan, M. L. and Enquist, L. W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and Ausubel, F, M. et al., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, published by GREENE PUBLISHING AND WILEY-INTERSCIENCE, 1987; the entirety of each of which is hereby incorporated herein by reference.
Microbial Production Systems
Expression of proteins in transformed host cells is most often carried out in a bacterial or yeast host cell with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such vectors are within the scope of the present disclosure.
In an embodiment, the expression vector includes those genetic elements for expression of the recombinant polypeptide in bacterial cells. The elements for transcription and translation in the bacterial cell may include a promoter, a coding region for the protein complex, and a transcriptional terminator.
Persons of ordinary skill in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. The polynucleotide used for incorporation into the expression vector of the subject technology, as described herein, can be prepared by routine techniques such as polymerase chain reaction (PGR).
A number of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary' homopolymeric tads to form recombinant DNA molecules.
In alternative embodiments, synthetic linkers containing one or more restriction sites provide are used to operably link the polynucleotide of the subject technology to the expression vector. In an embodiment, the polynucleotide is generated by restriction endonuclease digestion. In an embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single- stranded termini with their 3'-5'-exonucleolytic activities and fill-in recessed 3 '-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the polynucleotide.
Alternatively, a vector having ligation-independent cloning (LIC) sites can be employed. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18 6069-74, (1990), Haun, et al, BIOTECHNIQUES 13, 515-18 (1992), which is incorporated herein by reference to the extent it is consistent herewith).
In an embodiment, in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.
In an embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared by the use of PCR using appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In an embodiment, the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector.
The expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of appropriate cells with an expression vector of the subject technology Is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation.
Successfully transformed cells, that is, those cells containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce polypeptides described herein. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.
The host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.
In some embodiments, the transformed cell can be a bacterial cell, a yeast cell, an algal cell, a fungal cell, a plant cell, an insect cell or an animal cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.
Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct vectors for expression of the recombinant polypeptide of the subjection technology in a microbial host cell. These vectors may then be introduced into appropriate microorganisms via transformation to allow for high level expression of the recombinant polypeptide of the subject technology.
Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art. Typically, the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3’ of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.
Initiation control regions or promoters, which are useful to drive expression of the recombinant polypeptide in the desired microbial host cell are numerous and familiar to those skilled in the art. In this context, the term “potent promoter” refers to a promoter that increases expression of a nucleic acid sequence encoding a polypeptide compared to the parent, constitutive promoter. Non-limiting examples of potent promoters which may provide a high level of gene expression in a bacterium belonging to the genus Methylopila may be chosen from among PMDH1 (SEQ ID NO: 34), PMDH2 (SEQ ID NO: 35), PmxaF (SEQ ID NO: 42), PfumC (SEQ ID NO: 43), PcoxB (SEQ ID NO: 44), /hn/(SEQ ID NO: 45), PMP688A2 (SEQ ID NO: 46) and Ptac (SEQ ID NO: 47), or variants thereof.
Termination control regions may also be derived from various genes native to the microbial hosts. A termination site optionally may be included for the microbial hosts described herein.
Analysis of Sequence Similarity Using Identity Scoring
As used herein, “sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window' of alignment of components, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the w'indow of comparison). Optimal alignment of sequences for aligning a comparison window' are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part, of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this disclosure "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
The percent of sequence identity is preferably determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, WI). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, JOURNAL OF MOLECULAR BIOLOGY 48:443-53, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, ADVANCES IN APPLIED MATHEMATICS, 2:482-489, 1981, Smith et al., NUCLEIC ACIDS RESEARCH 11 :2205- 2220, 1983). The percent identity is most preferably determined using the “Best Fit” program.
Useful methods for determining sequence identity are also disclosed in the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. MOL. BIOL. 215:403-10 (1990); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity. As used herein, the term “substantial percent sequence identity” refers to a percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the disclosure is a polynucleotide molecule that has at least about ?()% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity with a polynucleotide sequence described herein. Identity and Similarity
Identity is the fraction of amino acids that are the same between a pair of sequences after an alignment of the sequences (which can be done using only sequence information or structural information or some other information, but usually it is based on sequence information alone), and similarity is the score assigned based on an alignment using some similarity matrix. The similarity index may be any one of the following BLOSUM62, PAM250, or GONNET, or any matrix used by one skilled in the art for the sequence alignment of proteins.
Identity is the degree of correspondence between two sub-sequences (no gaps between the sequences). An identity of 25% or higher typically implies similarity of function, while 18- 25% implies similarity of structure or function. It should be kept in mind that two completely unrelated or random sequences (that are greater than 100 residues) may have higher than 20% identity. Similarity is the degree of resemblance between two sequences when they are compared. This is dependent on their identity.
Explanation of Terms Used Herein:
As used herein, the singular forms “a, an” and “the” include plural references unless the content clearly dictates otherwise.
To the extent that the term “include,” “have,” or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term “comprise” as "comprise" is interpreted when employed as a transitional word in a claim.
The word “exemplary” is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. The term “complementary” is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary' to guanine. Accordingly, the subjection technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
The terms “nucleic acid” and “nucleotide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
“Coding sequence” is to be given its ordinary and customary meaning to a person of ordinary'- skill in the art, and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.
The term “isolated” is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
The terms “incubating” and “incubation” as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing the desired product.
The term “degenerate variant” refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.
The terms “polypeptide,” “protein,” and “peptide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art; the three terms are sometimes used interchangeably, and are used without limitation to refer to a polymer of amino acids, or amino acid analogs, regardless of its size or function. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms In the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms "protein," "polypeptide," and "peptide” are used interchangeably herein when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
The terms "polypeptide fragment" and "fragment," when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).
An amino acid position “corresponding to” a reference position refers to a position that aligns with a reference sequence, as identified by aligning the amino acid sequences. Such alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.
"Suitable regulatory sequences" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3’ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
"Promoter” is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3’ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the ait that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters, which cause a gene to be expressed in most cell types at most times, are commonly referred to as "constitutive promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term "expression" as used herein, is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the subject technology. "Over-expression” refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non- tran sformed organ! sms .
"Transformation” is to be given its ordinary and customary meaning to a person of reasonable skill in the field, and is used without limitation to refer to the transfer of a polynucleotide into a target cell for further expression by that cell. The transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant” or "transformed" organisms.
The terms "transformed," "transgenic," and "recombinant," when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
The terms "recombinant," "heterologous," and "exogenous," when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.
Similarly, the terms "recombinant," "heterologous," and "exogenous," when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Titus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.
The terms "plasmid," "vector," and "cassette" are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3’ untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials andmethods are described below.
The disclosure will be more fully understood upon consideration of the following nonlimiting Examples. It should be understood that these Examples, while indicating exemplary embodiments of the subject technology, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various uses and conditions.
EXAMPLES
Example 1: Development of a genetic toolkit for Methylopila sp. YHT-1 strain
At the time of the invention, Methylopila sp. YHT-1 was a newly discovered PQQ- producing bacterium, and toolkits for its genetic manipulation were not available. Moreover, constitutive promoters utilized for gene overexpression in Methylopila sp. YHT-1 were yet to be identified and quantified. Methylopila is a genus of bacteria from the family of Methylocystaceae . No well- developed protocols for this family of microorganisms had been reported at the time of the invention. As such, the inventors developed an efficient biparental conjugation system for Methylopila sp. YHT-1 strain (FIG. 4). Rifampicin-resistant clones were generated through plating > IO8 Methylopila sp. YHT-1 cells onto multiple nutrient agar plates with rifampicin (50 pg/mL). The Bordetella bronchiseptica-derived broad host range conjugative plasmid pBBRlMCS2 was used to develop the conjugation system. Briefly, pBBRlMCS2 DNA was transformed into E. coll S 17- 1, a strain having chromosomally integrated conjugal transfer functions (RP4 transfer functions). Rifampicin-resistant Methylopila sp. YHT-1 cells and E. coli S 17- 1 cells hosting pBBRlMCS2 plasmid were cultured in methanol minimal B medium and Luria-Bertani, respectively. The mid -exponential phase recipient and donor cell cultures were washed with Luria-Bertani liquid medium, mixed in a 6:1 recipient-to-donor ratio, and plated onto nutrient agar plates.
Following incubation at 30°C for about 20 to 24 hours, the cells were stripped from nutrient agar plates with a disposable cell spreader and plated onto the selective medium (Succinate Minimal Salts medium plates with 25 pg/mL kanamycin and 50 pg/mL rifampicin). After incubation at 30°C for a week, transconjugant colonies were picked and streaked onto methanol minimal B medium plates with 25 pg/mL kanamycin to ensure the absence of donor cells.
Replicative plasmids are useful tools for gene overexpression or promoter screening. A polynucleotide sequence may be genetically inserted without modification in a plasmid vector; however, a DNA sequence having a promoter activity may be added. Furthermore, mutations may be introduced at random and polynucleotides with improved promoter activity may be used. Usually, a coding region of enzyme protein or the like is positioned on the 3’ side of the promoter sequence. Commercially available plasmid vectors can be applicable if they already have a promoter sequence, and the promoter functions in one or more Methylopila species. As the plasmid vector, any vectors can be used insofar as they are present, stable, and replicable in transformed cells. Furthermore, as the plasmid vector, pUC series, pBR series and the like that are used for transformation of E. coli, as well as shuttle vectors linked with a plasmid vector which is replicable in the target cells can be exemplified. A desired DNA sequence may be inserted into an appropriate plasmid vector with the use of a replication region of a broad-host-range vector, and used as a shuttle vector. For example, a shuttle vector may be prepared by inserting a replication region of RK2 vector into an appropriate position of a pUC series vector, and the shuttle vector is able to utilize E. coli. Another example is provided by pBBR series plasmids, which have relatively small DN A sizes and are replicable in a broad range of hosts. Popular pBBR series plasmids include pBBR122, pBBRIMCS, pBBRlMCS2, pBBRlMCS3, pBBR!MCS4, and pBBRlMCS5. These plasmid vectors are characterized, for example, by different antibiotic markers, and may be selected for use after evaluation of antibiotic resistance of a transformed cell. Furthermore, a plasmid retained by a cell to be transformed may be used. Replicative broad host range vectors including pBBRlMCS2, RK2-based expression vectors, and IncP type vectors have been tested and utilized in the Methylopila sp. YHT-1 strain12. pBBRl MCS2-derived plasmids can be stably maintained for many generations in the presence of a low concentration of kanamycin.
For chromosomal site-directed gene insertion or gene deletion, a broad host range vector pK18mobsacB was used in the Methylopila sp. YHT-1 strain. pK18mobsacB -derived plasmids can be efficiently transformed into Methylopila sp. YHT-1 cells using the biparental conjugation system. Single crossover transconjugant colonies were selected with kanamycin, and then second crossover events were selected using the sucrose counterselection strategy to remove the plasmid backbone from the chromosome and produce the colonies with the gene insertion or gene deletion. Colony PCR and Sanger sequencing methods were used for genotyping.
Overexpression of individual PQQ biosynthetic genes and the construction of PQQ biosynthetic pathways in Methylopila sp. YHT-1 were found to work best with a set of constitutive strong promoters. Previous studies in Methylobacterium extorquens AMI demonstrated that the large subunit of methanol dehydrogenase represents up to 9% of the total soluble protein under methylotrophic growth condition and the promoter region of the mxaF gene (PmxaF) has been validated as one of the strongest promoters in methylotrophs13, !4,
Figure imgf000039_0001
Two native promoters that are located upstream of the putative methanol dehydrogenase genes of Methylopila sp. YHT-1, and a number of well-characterized promoters used in other methanolutilizing microorganisms, as well as the widely used Ptac promoter, were tested and quantified in the Methylopila sp. YHT-1 strain (FIG. 5). A promoterless GFP reporter vector, pBBRl MCS2- promoterlessGFP, was cloned using the Gibson Assembly cloning method. The RBS sequence, GFP gene, and a terminator were cloned into the pBBR!MCS2 vector. The PMDHI promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47) were each cloned into the upstream region of RBS site of the GFP gene to generate the replicative plasmids shown in FIG. 5, panels B-I. The native promoter located upstream of the pqqA-B-C-D-E operon (SEQ ID NO: 38) was also amplified and inserted into the same location to serve as a control (FIG. 6, panel A). All of the aforementioned plasmids were each transformed into Methylopila sp. YHT-1 cells. The confirmed transconjugant colonies were inoculated into 1 ml, of either methanol minimal B medium or succinate minimal salts (SMS) medium with methanol or succinate as carbon source, respectively, then grown in a 48-well MTP, flower plate at 30°C and a shaking frequency of 1000 rpm in a BioLector microbioreactor. GFP signals and biomass of each well were measured in a time-course manner. Based on all GFP and biomass measurement data, and biomass normalized GFP data, it was concluded that among all promoters screened, Ptac was the strongest one in Methylopila sp. YHT-1 in both methanol minimal B medium and SMS medium. As shown in FIG. 7, panel A, in the methanol minimal B medium Ptac started to induce GFP gene expression and the GFP signal reached the exponential phase around 8 hours earlier than in the instance of the native promoter of the pqqA -B-C-D-E operon. There was no significant difference in the growth rate of the cells (FIG. 7, panel B). Thus, the Ptac promoter and the native promoter were selected for genes and PQQ biosynthetic cluster overexpression.
Example 2: Deletion analysis of the native promoter of the pqqA-B-C~D-E operon
It has been reported that a region within the pqqA2 promoter in Methylovorus sp. MP688 (SEQ ID NO: 46) inhibits transcription. Deletion of a 137 bp sequence upstream of the PMP688A2 promoter caused a four- to five-fold increase in the activity of the reporter enzyme, suggesting the existence of negative transcriptional regulators binding to the promoter region in the course of PQQ biosynthetic genes transcription. To characterize the transcriptional regulator binding region of the native promoter of the pqqA-B-C-D-E operon (SEQ ID NO: 38), the 351 bp intergenic region (IGR) sequence upstream from the pqqA gene start codon was searched with BProm software and a putative -35 region and -10 region were identified. Based on the BProm prediction, different lengths of native promoter region covered the putative -35 region and -10 region upstream of pqqA gene designated IGRpqqA351 (meaning 351 bp upstream from the pqqA gene start codon), IGRpqqA300, IGRpqqA270, IGRpqqA200, IGRpqqA152 and IGRpqqA-del97 (meaning 97 bp upstream of the pqqA gene start, codon was removed) were cloned into the upstream region of RBS site of GFP gene in the pBBRlMCS2-promoterlessGFP vector (FIG. 6, panels A-F). All of the above plasmids were transformed into Methylopila sp. YHT-1 cells. There was no GFP signal detected after pBBRlMCS2-IGRpqqA-de!97-GFP plasmid was transformed into Methylopila sp. YHT-1 cell, indicating that this 97 bp region is essential for the transcription of the pqqA-B-C-D-E operon and that there may be a positive transcriptional regulator binding to this region. All of the other confirmed transconjugant colonies were inoculated into 1 mL of methanol minimal B medium, then grown in a 48 well MTP, flower plate at 30cC and 1000 rpm in a BioLector microbioreactor. GFP signals and biomass of each well were measured in a time-course manner. Based on all GFP and biomass measurement data, and biomass-normalized GFP data, there was no significant difference among strains hosting plasmids of all these promoters. The combined data supported the conclusion that the 152 bp region upstream of the pqqA gene contains the complete promoter sequence, and that the 97 bp region upstream of the pqqA gene is essential for the transcription of the pqqA-B-C-D- E operon.
Example 3: Promoter identification for the IGR sequence upstream of the pqqE gene
Sequence analysis of the PQQ biosynthetic gene clusters revealed a 194 bp IGR sequence upstream of the pqqE gene. PqqE, along with PqqD, is the first group of enzymes catalyze the reactions on the precursor peptide PqqA. Without being bound to any particular theory, the inventors proposed that the IGR sequence might contain a complete promoter being regulated to control the transcription of PQQ biosynthetic genes. The 194 bp IGR DNA was amplified and inserted into the Xhol site upstream of the RBS site of the GFP gene in the pBBR!MCS2- promoterlessGFP vector (FIG. 8, panel A). The newly cloned plasmid was then transformed into Methylopila sp. YHT-1 cells. Relatively weak GFP signal from the transconjugant colonies could be visualized under blue light (FIG. 8, panel B), which confirmed that the transcription of pqqE gene is initiated from its own promoter. Example 4: Construction of replicative plasmids containing multiple pqqA genes and PQQ biosynthetic gene clusters to enhance their expression in Methytopila sp. YHT-1
A number of pBBR1MCS2-derived replicative plasmids were constructed for the overexpression of the pqqA-B-C-D-E operon, six additional copies of synthetic pqqA genes (SEQ ID NOs: 4-9) and/or the pqqA-B-C-D-E operon combined with the pqqF-G operon. The native promoter, the putative pqqA-B-C-D-E operon, and a terminator were amplified and inserted into the pBBR!MCS2 vector using the Gibson Assembly cloning method, thereby generating the pBBRlMCS2-PQQ plasmid (FIG. 9, panel A). For ease of identification of the positive Iran sconjugant colonies, a GFP gene along with its RBS site was inserted in between the
Figure imgf000042_0001
gene and the terminator to create the pBBRlMCS2-PQQ-GFP plasmid (FIG. 9, panel B). Since there was an independent promoter upstream of the pqqE gene, to coordinate the expression of all five PQQ biosynthetic genes, this IGR sequence was removed to generate the pBBR1MCS2- PQQ-delPpqqE-GFP plasmid (FIG. 9, panel C). Six additional copies of synthetic pqqA genes, including pqqAjth, pqqA_j\5, pqqA_x\ , pqqAjtl l, pqqA_GQ, and pqqAjclS, along with their own RBS sites, were inserted in between the native promoter and the pqqA gene of the pBBRlMCS2-PQQ-GFP plasmid. The resulting plasmid was named pBBR!MCS2-pqqAs- PQQ-GFP (FIG. 9, panel D). Similar to the pBBRlMCS2-PQQ-delPpqqE-GFP plasmid, the pqqE promoter was removed to create a plasmid named pBBRlMCS2-pqqAs-PQQ-delPpqqE- GFP (FIG. 9, panel E). In addition, the pqqF-G operon was amplified and then inserted in between the pqqE gene and the terminator of the pBBRlMCS2-pqqAs-PQQ-GFP plasmid to generate the pB BRI MCS2-pqqAs-PQQ-FG-GFP plasmid (FIG. 9, panel F). As disclosed above, the Ptac promoter was validated as a stronger promoter than the native promoter, so the native promoter in pBBRlMCS2-pqqAs-PQQ-GFP, pBBRlMCS2-pqqAs-PQQ-delPpqqE-GFP, and pBBRlMCS2-pqqAs-PQQ-FG-GFP were replaced with Ptac promoter to build the constructs of pBBRlMCS2-Ptac-pqqAs-PQQ-GFP, pBBRlMCS2-Ptac-pqqAs-PQQ-delPpqqE-GFP, and pBBRlMCS2-Ptac-pqqAs-PQQ-FG-GFP, respectively (FIG. 9, panels G-I).
All of the above plasmids were transformed into Methylopila sp. YHT-1 cells. The tran sconjugant colonies were confirmed by the presence of GFP signal and/or colony PCR followed with Sanger sequencing. The confirmed transconjugant colonies were cultured in methanol minimal B medium and the assay of the amount of produced PQQ was conducted as described in Example 8. Example 5: Investigation of the impact of a putative redox-sensitive transcriptional activator SoxR on PQQ biosynthesis
A soxR gene (SEQ ID NO: 25) encoding a putative redox- sensitive transcriptional activator SoxR is located immediately upstream of the pqqF gene on the chromosome of Methylopila sp. YHT-1. SoxR, a MerR-family homodimeric transcription factor with a 2Fe-2S cluster in each monomer, is known to function as a sensor and a transcriptional activator for a superoxide response regulon. PQQ) plays an important antioxidant role in the cells. To check whether the soxR gene overexpression affects PQQ production, the Ptac promoter or methanolinducible PmxaF promoter or PMDHI promoter, an RBS sequence, and the soxR gene were cloned into the pBBR!MCS2 vector to create the pBBR!MCS2-Ptac-SoxR, pBBR!MCS2- PmxaF-SoxR and pBBR!MCS2-PMDHl-SoxR plasmids, respectively (FIG. 10, panels A-C). In addition, the soxR-pqqF-pqqG gene fragment was amplified and inserted in between the pqqE gene and the terminator of the pBBR1MCS2-pqqAs-PQQ-GFP plasmid and pBBRIMCS2-Ptac- pqqAs-PQQ-GFP plasmid to generate the pBBRlMCS2-pqqAs-PQQ-SoxR-FG-GFP plasmid and pBBR!MCS2-Ptac-pqqAs-PQQ-SoxR-FG-GFP plasmid, respectively (FIG. 10, panel D, panel E).
All of the above plasmids were transformed into Methylopila sp. YHT-1 cells. The tran sconjugant colonies were confirmed by colony PCR followed by Sanger sequencing. The confirmed transconjugant colonies were cultured in methanol minimal B medium and an assay measuring the amount of product PQQ was conducted as described in Example 8.
Example 6: Chromosomal site-directed insertion of the strong promoter, multiple pqqA genes, and an additional copy of PQQ biosynthetic gene cluster into the Methylopila sp. YHT-1 strain
To eliminate the antibiotic resistance gene in the genetically engineered strains and to develop an antibiotic-free fermentation process, site-directed chromosomal integration strains were built based on pK18mobSacB derived constructs and sucrose counterselection strategy. The strong Ptac promoter, two ~l-kb fragments of 5’ and 3’ DNA regions flanking the start codon of pqqA gene were cloned into pKl 8mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB-Ptac-PQQ-Arms (FIG. 11, panel A). The strong Ptac promoter with six additional copies of synthetic pqqA genes, including pqqA_r6, pqqA_ r\5, pqqA_r]., pqqA_xAA, pqqA_y\0, and pqqA_rA%, along with their own RBS sites, were inserted immediately upstream of the Ptac promoter of the pKl 8mobSacB-Ptac-PQQ-Arms plasmid to create the pK18mobSacB-PtacPqqAs-PtacPQQ-Arms plasmid (FIG. 11, panel B). The Ptac promoter immediately upstream of the pqqA gene was then removed to create pK18mobSacB-Ptac-PqqAs-PQQ-Arms plasmid (FIG. 11, panel C). Six additional copies of synthetic pqqA genes, a ~l-kb fragment of the 5’ flanking region of the pqqA gene (containing the native promoter) and a ~l-kb fragment of the 3' flanking region of the start codon of pqqA gene were cloned into pK18mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB-NativePromoter-PqqAs~PQQ-Arms (FIG. 11, panel D). The native promoter was then replaced with methanol-inducible PMDHI promoter using the Gibson Assembly cloning method to build the plasmid pK18mobSacB-PMDHl-PqqAs-PQQ-Arms (FIG. 11, panel E). Similar to pK18mobSacB-Ptac-PqqAs-PQQ-Arms and pK18mobSacB- NativePromoter-PqqAs-PQQ-Arms plasmids, pKl 8mobSacB-Ptac-PqqAs-pqqA2-Arms (FIG.
11, panel F) and pK18mobSacB-NativePromoter-PqqAs-pqqA2-Arms (FIG. 11, panel G) plasmids were cloned, their insertion sites being immediately upstream of the pqqA2 gene in the Methyiopila sp. YHT-1 chromosome. All of the above plasmids were transformed into Methylopila sp. YHT-1 cells. The single crossover transconjugant colonies were confirmed by colony PCR followed with Sanger sequencing. The confirmed single crossover transconjugant colonies were picked and cultured in methanol minimal B medium without kanamycin, and then streaked onto counterselection nutrient agar plates containing 10% sucrose. Single colonies from these counterselection plates were then picked and streaked once more to obtain sufficient biomass for genotype screening (colony PCR and Sanger sequencing) of the correct double crossover colonies.
To further improve the copy numbers of PqqA precursor gene and PQQ biosynthetic genes in the antibiotic resistance gene-free strains, the Ptac or native promoter, two additional copies of synthetic pqqA genes (pqqA_x3 and pr?<jA..iT3; SEQ ID NOs: 10, 11), a synthetic PQQ operon (p^B_opt, pqqC_opt, pqqD_opt, pqqEjypV, SEQ ID NOs: 14, 17, 20 and 23), and two ~l-kb fragments of 5’ and 3’ DNA regions flanking the start codon of pqqA2 gene were cloned into the pK18mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB-Ptac-PqqAs-PQQ-PqqA2-Arms and pK18mobSacB-NativePromoter- PqqAs-PQQ-PqqA2-Arms plasmids, respectively. Using the engineered Methylopila sp. YHT-1 strains that promoter and six copies of pqqAs were inserted at the pqqA site as the host strains and followed the same procedure described above, the synthetic pqqAs -pqqB-pqqC-pqqD-pqqE cassettes were then inserted into the pqqA2 site of the engineered Methylopila sp. YHT-1 strains. Overall, eight copies of pqqA genes and one copy of each of the pqqB, pqqC, pqqD, and pqqE genes were inserted into the Methylopila sp. YHT-1 strain.
The confirmed engineered colonies were cultured in methanol minimal B medium and an assay measuring the amount of product PQQ was conducted as described in Example 8.
Example 7: Investigation of the impact of a putative PQQ periplasmic binding protein PqqT on PQQ biosynthesis
PqqT, a PQQ periplasmic (solute) binding protein, was recently characterized from Methylobacterium extorquens AMI. It was proposed that PqqT is involved in the uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis16. The pqqT gene was also discovered from the genome of the Methylopila sp. YHT-1 strain (SEQ ID NO: 31) and located in an operon containing three genes, wherein the other two genes encode a transport system permease protein and an ABC transporter ATP-binding protein. The inventors hypothesized that the deletion of pqqT gene from the chromosome of Methylopila sp. YHT-1 strain might block the import of exogenous PQQ into the cytosol, thereby improving endogenous PQQ production. Two ~l-kb fragments of 5’ and 3’ DNA regions flanking the pqqT gene were cloned into pK18mobSacB vector using the Golden Gate Assembly cloning method to build the plasmid pK18mobSacB -pqqT -KO- Arms. Following the procedure described in Example 6, the pqqT gene was in-frame deleted from the chromosome of Methylopila sp. YHT-1 strain with a truncated scar sequence left. The confirmed engineered colonies were cultured in methanol minimal B medium and an assay measuring the amount of product PQQ was conducted as described in Example 8.
Example 8: Quantification of PQQ produced by genetically engineered Methylopila sp. YHT-1 strains
The wildtype and genetically engineered Methylopila sp. YHT-1 strains were streaked onto methanol minimal B medium plate and grown at 30 °C. A solution was formed by combining 2 g (NHahSOr, 1-4 g KH2PO4, 3 g Na2HPO4, 0.2 g MgSO4, 30 mg ferric citrate, 30 mg CaCh, 5 mg MnCh, 5 mg ZnSCh, 0.5 mg C11SO4, and 6 g methanol per liter of water solvent, to form methanol minimal B medium at pH 7.0, then 20 g/L agar were added to yield methanol minimal B plate medium. Following 6-7 days of incubation, the large round colonies, or colonies showing strong GFP signals for certain genetically engineered strains, were picked and grown in a test tube containing 5 ml of liquid methanol minimal B medium at 250 rpm and 30°C for 4 days. One ml of cell culture was harvested by centrifugation at maximum speed for 10 minutes and the supernatant was filtered through a 0.22 Micron syringe filters for PQQ glucose dehydrogenase (GDH) assay and HPLC analysis.
PQQ titer in test tube cultures using minimal medium was frequently below the detection limit of HPLC and, when the nutrition rich medium was used, the HPLC peak area corresponding to PQQ might be overestimated because of the background noise. Thus, for the purpose of PQQ quantification, the inventors adopted a PQQ GDH assay for the screening of high titer PQQ producing strains followed by HPLC analysis of the methanol minimal B medium cell cultures. The PQQ GDH assay was modified from the method published in The Journal of Archives of Biochemistry and Biophysics in 1996 (doi.org/10.1006/abbi.1996.0530). The assay made use of the activity of the glucose dehydrogenase enzyme, using PQQ as a cofactor, to measure PQQ concentration. GDH was combined with glucose, a dye (2,6-Dichlorophenolindophenol, DCPIP), and an electron acceptor (phenazine methosulfate). When PQQ was present, GDH would reduce the dye, and the DCPIP dye’s absorbance at 630 nm would decrease. A Biomek Automated Liquid Handlers Workstation was used to quickly mix all the components into the 96 well plates, and then a SpectraMax i3 Platform from Molecular Devices was used to measure the absorbance of DCPIP at 630 nm every 30 seconds for 10 minutes. Based on the time-course 630 nm absorbance data, the Vmax value was calculated (FIG. 13, panel A). The absolute numbers of Vmax were correlated with the PQQ titer, which was used to rank the titers of the tested strains. Four colonies from the Methylopila sp. YHT-1 wildtype strain and each of the genetically engineered Methylopila sp. YHT-1 strains were cultured for the PQQ GDH assay. PQQ GDH assay results from representative strains are shown in FIG. 13, panel B. Genetically engineered Methylopila sp. YHT-1 strain hosting the pBBRlMCS2-Ptac-pqqAs-PQQ-GFP plasmid exhibited the highest Vmax value among all tested strains. Removal of the pqqE promoter, o verexpression of the soxR gene and deletion of the pqqT gene could not further improve PQQ titers, whereas overexpression of multiple pqqA. genes and PQQ biosynthetic clusters were found capable of contributing to the PQQ titer improvement. Antibiotic resistance gene-free strains could produce more PQQ than the wildtype strain using methanol minimal B medium, but the strains hosting replicative vectors were better PQQ producers than the chromosomal integrated PQQ-producing strains.
Samples from the Methylopila sp. YHT-1 wildtype strain and a number of genetically engineered Methylopila sp. YHT- 1 strains were then utilized for HPLC analysis. HPLC analysis of PQQ was performed using an Ultimate 3000 HPLC System (Dionex, Sunnyvale, CA) that included a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorbance detector. A SunFireIM C18 HPLC column (150mm x 4.6mm 5 pm) with a guard column was used for the characterization of PQQ. A flow rate of 1.0 ml/min was applied, and the mobile phase was composed of (A) 10 mM ammonium acetate (pH 5.0) and (B) methanol. The elution program was 0-2 min 15% B: 2-7 min 15%-50% B; 7-7.1 min 50%-15% B; 7.1-10 min 15% B. The detection wavelength was 254 nm for PQQ. As shown in FIG. 14, PQQ standard showed up at around 1.9 minutes (FIG. 14, panel A). As compared to samples from the wildtype strain (FIG. 14, panel B), the genetically engineered Methylopila sp. YHT-1 strain hosting pBBR!MCS2-Ptac-pqqAs-PQQ-GFP plasmid (FIG. 14, panel C) and the engineered strain hosting pBBRlMCS2-pqqAs-PQQ-GFP plasmid (FIG. 14, panel D) showed a 20- or 14-fold increase in PQQ production, respectively. To mass-produce PQQ in industrial applications, representative genetically engineered Methylopila sp. YHT-1 strains were inoculated into 3-Liter fermenters for fed-batch culture, resulting in a consistent production of 550 mg/L of PQQ or more in about 7 days. As shown in FIG. 15, in a 3-Liter fermenter culture of a genetically engineered Methylopila sp. YHT-1 strain, PQQ could be produced as the dominant product (FIG. 15, panel B). The retention time and UV spectrum (FIG. 15, panel D) of the PQQ peak were consistent with those of the PQQ standard (FIG. 15, panels A and C).
SEQUENCES OF INTEREST
Amino acid sequence of PqqA of Methylopila sp. YHT-1 (SEQ ID NO: 1):
MA1WTAPIVEETPVGLEVTSYSPAEL
Nucleotide sequence of pqqA of Methylopila sp. YHT-1 (SEQ ID NO: 2):
ATGGCCATTTGGACCGCCCCGATCGTTGAAGAGACCCCCGTCGGCCTCGAAGTCACG
TCCTACAGCCCGGCTGAGCTCTGA Nucleotide sequence of the second copy of pqqA (pqqA2) of Methylopila sp. YHT-1 (SEQ ID NO: 3):
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
REFERENCES 2, 832. 2018, 115. 43, 10826-10844. 68. 02,061,278. ,334. 0, 142, 29, 12620-12634. oc. 2004, 126, 17, 5342-5343. 007, 46, 7174-7186. 9, 294, 15025-15036. 019, 141, 10, 4398-4405. 011, 50, 1556-1566. ng) 2001, 147, 8, 2065-2075.nthetic Biology 2015, 4, 4, 430-443. 5, 55, 312-323. Sci. USA 1983, 80, 21-25. 2665-2669.

Claims

1. A method for producing pyrroloquinoline quinone (PQQ) comprising: culti vating in a culture medium a bacterium belonging to the genus Methylopila, and collecting PQQ from the culture medium, wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
(i) increasing the copy number of the gene cluster,
(ii) introducing at least one copy of the gene cluster into the chromosome of the bacterium,
(iii) placing at least one copy of the gene cluster under the control of a potent promoter,
(iv) introducing at least one copy of the gene cluster into the bacterium by inserting one or mor genes of the gene cluster into the chromosome, and placing the remaining genes of the gene cluster under the control of a potent promoter, and
(v) combinations thereof.
2. The method according to claim 1, wherein the pqqABCDE gene cluster comprises nucleotide sequences at least 80% identical to the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, or variants thereof.
3. The method according to claim 1 or claim 2, wherein the pqqABCDE gene cluster comprises the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, or variants thereof.
4. The method according to claim 1, wherein the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51 , 53, 55, 57, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqD gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81 , 83, 85, 87, or variants thereof; and/or wherein the pqqABCDE gene cluster comprises a pqqE gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof,
5. The method according to claim 1, wherein the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof: and/or wherein the pqqABCDE, gene cluster comprises a pqqE gene comprising the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof.
6. The method according to any one of claims 1 to 5, wherein one or more copies of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
7. The method according to any one of claims 1 to 5, wherein one or more copies of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
8. The method according to any one of claims 1 to 5, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
9. The method according to any one of claims 1 to 5, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
10. The method of according to any one of claims 1-5, wherein one or more copies of the pqqA gene is inserted into the chromosome of the bacterium, one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
11. The method of according to any one of claims 1 -5, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium.
12. The method according to any one of claims 1 to 5, wherein the pqqABCDE gene cluster is inserted into the chromosome of the bacterium.
13. The method according to any one of claims 1 to 5, wherein pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
14. The method according to any one of claims 1 to 5, wherein a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
15. The method according to any of claims 1 to 14, wherein the potent promoter is selected from the group consisting of PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47), or variants thereof.
16. The method of any of claims 1 to 15, wherein the potent promoter is Ptac promoter (SEQ ID NO: 47), or variants thereof.
17. The method of any of claims 1 to 16, wherein the bacterium has been further modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium,
(iii) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
18. The method of claim 17, wherein the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof.
19. The method of any of claims 1 to 18, wherein the bacterium has been further modified to delete or knock out a pqqT gene.
20. The method of claim 19, wherein the pqqT is identified by SEQ ID NO: 31
21. The method of any of claims I to 20, wherein the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi,
Methylopila sp. M107, Methylopila sp. 73B, and uncultured Methylopila sp.
22. The method of claim 21, wherein the bacterium is Methylopila sp. YH-1.
23. The method of any of claims 1 to 22, wherein any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, or uncultured Methylopila sp.
24. The method of claim 23, wherein any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1.
25. The method of claim 23 or claim 24, wherein the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
26. The method according to any of claims 1 to 25, wherein the culture medium includes methanol at a concentration of not less than 3 grams of methanol per liter of water solvent to at most 15 grams of methanol per liter water solvent.
27. A recombinant bacterium belonging to the genus Methylopila, wherein the bacterium has been modified to enhance expression of a pqqABCDE gene cluster from a Methylopila sp. by a method selected from the group consisting of:
(i) increasing the copy number of the gene cluster,
(ii) introducing at least one copy of the gene cluster into the chromosome of the bacterium,
(Hi) placing at least one copy of the gene cluster under the control of a potent promoter, (iv) introducing at least one copy of the gene cluster into the bacterium by inserting one or mor genes of the gene cluster into the chromosome, and placing the remaining genes of the gene cluster under the control of a potent promoter, and
(v) combinations thereof.
28. The recombinant bacterium according to claim 27, wherein the pqqABCDE cluster comprises nucleotide sequences at least 80% identical to the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22. or variants thereof.
29. The recombinant bacterium according to claim 27 or claim 28, wherein the pqqABCDE gene cluster comprises the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 13,
SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, or variants thereof.
30. The recombinant bacterium according to claim 27, wherein the pqqABCDE gene cluster comprises a pqqA gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqB gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61 , 63, 65, 67, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqC gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; wherein the pqqABCDE. gene cluster comprises a pqqD gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof; and wherein the pqqABCDE gene cluster comprises a pqqE gene comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91 , 93, 95, 97, or variants thereof.
31. The recombinant bacterium according to claim 27, wherein the pqqABCDE gene cluster comprises a pqqA gene comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 49, 51, 53, 55, 57, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqB gene comprising the nucleotide sequence of any one of SEQ ID NOs: 13, 59, 61, 63, 65, 67, or variants thereof: wherein the pqqABCDE gene cluster comprises a pqqC gene comprising the nucleotide sequence of any one of SEQ ID NOs: 16, 69, 71, 73, 75, 77, or variants thereof; wherein the pqqABCDE gene cluster comprises a pqqD gene comprising the nucleotide sequence of any one of SEQ ID NOs: 19, 79, 81, 83, 85, 87, or variants thereof: and wherein the pqqABCDE. gene cluster comprises a pqqE gene comprising the nucleotide sequence of any one of SEQ ID NOs: 22, 89, 91, 93, 95, 97, or variants thereof.
32. The recombinant bacterium according to any one of claims 14-28, wherein one or more copies of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
33. The recombinant bacterium according to any one of claims 27-32, wherein one or more copies of the pqqA gene is inserted into the chromosome of the bacterium, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a plasmid.
34. The recombinant bacterium according to any one of claims 27-32, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
35. The recombinant bacterium according to any one of claims 27-32, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
36. The recombinant bacterium of according to any one of claims 27-32, wherein one ormore copies of the pqqA gene is inserted into the chromosome of the bacterium, one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are introduced into the bacterium via a second plasmid.
37. The recombinant bacterium of according to any one of claims 27-32, wherein one or more copies of the pqqA gene is introduced into the bacterium via a first plasmid, and the pqqB gene, the pqqC gene, and the pqqD gene, are introduced into the bacterium via a second plasmid, and the pqqE gene is inserted into the chromosome of the bacterium.
38. The recombinant bacterium according to any one of claims 27-32, wherein the pqqABCDE gene cluster is inserted into the chromosome of the bacterium.
39. The recombinant bacterium according to any one of claims 27-32, wherein pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
40. The recombinant bacterium according to any one of claims 27-32, wherein a first copy of the pqqABCDE gene cluster is inserted into the chromosome of the bacterium and a second copy of the pqqABCDE gene cluster is introduced into the bacterium via a plasmid.
41. The recombinant bacterium according to any of claims 27-40, wherein the potent promoter is selected from the group consisting of ' PMDH1 promoter (SEQ ID NO: 34), PMDH2 promoter (SEQ ID NO: 35), PmxaF promoter (SEQ ID NO: 42), PfumC promoter (SEQ ID NO: 43), PcoxB promoter (SEQ ID NO: 44), Ptuf promoter (SEQ ID NO: 45), PMP688A2 promoter (SEQ ID NO: 46) and Ptac promoter (SEQ ID NO: 47), or variants thereof.
42. The recombinant bacterium according to any of claims 27-40, wherein the potent promoter is Ptac promoter (SEQ ID NO: 47), or variants thereof.
43. The recombinant bacterium according to any of claims 27-42, wherein the recombinant bacterium has been further modified to enhance expression of at least one pqqA- like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium,
(Hi) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
44. The recombinant bacterium according to claim 43, wherein the nucleotide sequence of each pqqA-like gene is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof.
45. A recombinant bacterium belonging to the genus Methylopila, wherein the bacterium has been modified to enhance expression of at least one pqqA-like gene by a method selected from the group consisting of:
(i) increasing the copy number of the pqqA-like gene,
(ii) introducing multiple copies of the pqqA-like gene into the chromosome of the bacterium,
(Hi) placing the pqqA-like gene under the control of a potent promoter, and
(iv) combinations thereof.
46. The recombinant bacterium according to claim 45, wherein the nucleotide sequence of each pqqA-like gene Is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and variants thereof.
47. The recombinant bacterium according to claim 45 or claim 46, wherein the bacterium has been further modified to delete or knock out a pqqT gene.
48. The recombinant bacterium according to claim 47, wherein the pqqT is identified by SEQ ID NO: 31.
49. The recombinant bacterium according to any of claims 27-48, wherein the bacterium is selected from the group consisting of Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. M107, Methylopila sp. 73B, and uncultured Methylopila sp.
50. The recombinant bacterium according to claim 49, wherein the bacterium is Methylopila sp. YH-1.
51. The recombinant bacterium according to any of claims 27-50, wherein any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1, Methylopila capsulata, Methylopila sp. Yamaguchi, Methylopila sp. Ml 07, Methylopila sp. 73B, or uncultured Methylopila sp.
52. The recombinant bacterium according to claim 50, wherein any one of the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene is from Methylopila sp. YH-1 .
53. The method of claim 51 or claim 52, wherein the pqqABCDE gene cluster, the pqqA gene, the pqqB gene, the pqqC gene, the pqqD gene, and the pqqE gene are from Methylopila sp. YH-1.
54. A method for producing pyrroloquinoline quinone (PQQ) comprising: cultivating in a culture medium a bacterium according to any one of claims 27-53, and collecting PQQ from the culture medium.
PCT/US2022/078752 2021-10-27 2022-10-27 Methods for improving pyrroloquinoline quinone production in methylopila WO2023076976A1 (en)

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