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CN111943996B - Trachelospermi analogue, genetic engineering bacterium for producing same and application thereof - Google Patents

Trachelospermi analogue, genetic engineering bacterium for producing same and application thereof Download PDF

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CN111943996B
CN111943996B CN201910403327.6A CN201910403327A CN111943996B CN 111943996 B CN111943996 B CN 111943996B CN 201910403327 A CN201910403327 A CN 201910403327A CN 111943996 B CN111943996 B CN 111943996B
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triandrin
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刘涛
毕慧萍
王帅
庄以彬
马延和
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Tianjin National Synthetic Biotechnology Innovation Center Co ltd
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Abstract

The invention relates to the field of genetic engineering, in particular to a rosavin analogue, a genetic engineering bacterium for producing the rosavin analogue and application thereof. The rosavin analogue has a structure shown in a formula I, a formula II, a formula III or a formula IV, and the genetic engineering bacteria contain genes for coding phenylalanine ammonia lyase or tyrosine ammonia lyase, 4-coumaric acid coenzyme A ligase 4CL, cinnamoyl coenzyme A reductase CCR and UDP glucosyltransferase UGT. The genetic engineering bacteria can ferment to produce Triandrin and Triandrin B (formula IV); or Triandrin, Triandrin B, Triandrin C (formula II) and Triandrin D (formula III), wherein the yield of Triandrin and Triandrin B can reach 1.7g/L and 1.6g/L at most; or the torsavide B (formula I) with the yield of 1.6g/L at most.

Description

Trachelospermi analogue, genetic engineering bacterium for producing same and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to a rosavin analogue, a genetic engineering bacterium for producing the rosavin analogue, an application of the genetic engineering bacterium in producing the rosavin analogue and a method for producing the rosavin analogue.
Background
Cinnamyl alcohol glycoside compounds (cannelyl alcohol glycosides, CAGs) are important active ingredients in endangered plant rhodiola rosea and have the functions of resisting oxidation, protecting heart, enhancing learning and memory and the like. Such compounds include losslem, loservin, losslem glycoside, Triandrin, and the like. The rosa roxburghii, rosavin and rosalin are collectively called rosavin compounds, wherein rosavin is the main active ingredient and is also an important marker commonly used for evaluating the quality of the rhodiola rosea extract. Research finds that the composition has various beneficial pharmacological effects, such as intelligence development, cancer resistance, immunity enhancement, depression resistance, ultraviolet radiation resistance and the like.
Currently, the production of the rosavin is mainly from plant extraction. Rhodiola rosea grows in high and cold areas, and wild resources are rare. In recent years, rhodiola rosea has been listed as an endangered species by several countries including china due to excessive mining. The rhodiola rosea is not artificially planted in a large scale until now under the restriction of growth conditions, plant diseases and insect pests and the like; the accumulation period of the active ingredients in the plant rhodiola rosea is very long, and the sufficient active ingredients can be obtained only by growing for about 5 to 7 years. In 2006, Patov et al reported the chemical synthesis of Trachelosin (Kishida, M., Akita, H. (2005) Simple synthesis of phenyl propenoid β -D-glucopyranoside derivatives based on microwave-Heck type reaction of organoboron reagents, tetrahedron Lett.46, 4123-4125.). But the chemical synthesis steps are more, the yield is low, the cost is high, and the environmental pollution is serious. In 2014, Grech-Baran et al used a hairy root culture method, cinnamyl alcohol was added to a culture medium, and we obtained Lucelawerver and its derivative Lucelawerver by Biotransformation, and the maximum yield of Lucelawerver after 14 days of culture could reach 505mg/L (Grech-Baran, M., Syklowska-Baranek, K., Krajewska-Patan, A., Wyrwal, A., Pietrosiuk, A. (2014) Biotransformation of cinnamyl alcohol to Rosans by non-transformed wire type and hair ot rocuures of Rhodiola kirilowii.Biotechnol. Lett.36, 649-656.).
The existing compound of the rosavin class has single species and low yield.
Disclosure of Invention
The present invention aims to overcome the above-mentioned defects of the prior art and provide a novel rosavin analogue and a genetically engineered bacterium capable of producing the novel rosavin analogue, wherein the genetically engineered bacterium is capable of producing the novel rosavin analogue in high yield.
Through a large number of researches, the inventor constructs a gene containing a gene capable of coding tyrosine ammonia lyase TAL, a gene capable of coding 4-coumaric acid coenzyme A ligase 4CL, a gene capable of coding cinnamoyl coenzyme A reductase CCR and a gene capable of coding UDP glucosyltransferase UGT, and realizes the microbial de novo synthesis of coumaryl alcohol hydroxyl monoglucoside Triandrin and coumaryl alcohol phenol hydroxyl monoglucoside (a new compound shown in a formula IV and named as Triandrin B); meanwhile, a coding gene of glycosyltransferase for extending sugar chains is introduced into the genetically engineered bacteria, and the microbial de novo synthesis of coumaryl alcohol hydroxy diglucoside (a new compound, shown in formula II and named as Triandrin C) and coumaryl alcohol hydroxy diglucoside (a new compound, shown in formula III and named as Trandrin D) is realized in the presence of glucose. On the basis of a genetically engineered bacterium into which a gene encoding a glycosyltransferase which extends a sugar chain is introduced, a gene encoding tyrosine ammonia lyase TAL is substituted with a gene encoding phenylalanine ammonia lyase PAL, and in the presence of glucose, another novel compound, sucroside (cinnamyl alcohol diglucoside, a novel compound represented by formula I, and named as "Lussevir B" or "Rosavin B") is obtained. The yield of the product obtained by the genetic engineering bacteria provided by the invention is high and can reach gram level at most.
Based on the above findings, in one aspect, the present invention provides a voseville analog having a structure represented by formula I, formula II, formula III, or formula IV below;
Figure BDA0002060464820000021
in a second aspect, the present invention provides a genetically engineered bacterium that produces a rosavin analogue as described above, comprising a gene capable of encoding phenylalanine ammonia lyase PAL or a gene encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, and a gene capable of encoding UDP glucosyltransferase UGT.
Preferably, the genetically engineered bacterium further contains a gene capable of encoding a glycosyltransferase that extends a sugar chain.
In a third aspect, the invention provides the application of the genetically engineered bacteria in the production of the rosavin analogue.
In a fourth aspect, the present invention provides a method for producing a voseville analog, comprising culturing the genetically engineered bacterium as described above in a medium containing glucose, and inducing expression of a foreign gene to produce the voseville analog.
The gene engineering bacteria for producing the coumaryl alcohol glucoside can be used for producing the coumaryl alcohol, the coumaryl alcohol hydroxyl monoglucoside Triandrin and the novel compound coumaryl alcohol hydroxyl monoglucoside Triandrin B (formula IV) by fermentation, and the yields of the Triandrin and the Triandrin B can reach 1.7g/L and 1.6g/L at most; it is also possible to produce a novel compound, coumaryl alcohol hydroxydiglucoside Triandrin C (formula II) and coumaryl alcohol hydroxydiglucoside Triandrin D (formula III), by further introducing a gene encoding a glycosyltransferase that extends a sugar chain and in the presence of glucose. In addition, preferably on the basis of introducing the genetic engineering bacteria of the gene of glycosyltransferase making the sugar chain extend, the gene of coding tyrosine ammonia lyase TAL is replaced by the gene of coding phenylalanine ammonia lyase PAL, in the presence of glucose, the recombinant Escherichia coli for producing the cinnamyl alcohol glucoside can be obtained, and cinnamyl alcohol, cinnamyl alcohol monoglucoside complex and cinnamyl alcohol biglucoside complex B can be produced by fermentation, wherein the complex B is a novel compound (formula I), and the yield can reach 1.6 g/L. Therefore, the invention provides a new biosynthesis way for high yield of cinnamyl alcohol or coumaryl alcohol glucoside, lays a foundation for large-scale industrial production of cinnamyl alcohol and/or coumaryl alcohol glucoside, and has important scientific research value and social benefit.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of the biosynthesis pathway of cinnamyl alcohol and/or coumaryl alcohol mono-glucoside (Triandrin B) and di-glucoside (Triandrin C and Triandrin D) in E.coli expression strains according to the present invention.
FIG. 2 is an HPLC result of fermentation products of Escherichia coli strains BWT-PDG, BPHE-PDG, BWT-TDG and BTAL-TDG, wherein a is an HPLC detection chart of the fermentation product of the strain BWT-PDG, B is an HPLC detection chart of the fermentation product of the strain BPHE-PDG, c is an HPLC detection chart of the fermentation product of the strain BWT-TDG, d is an HPLC detection chart of the fermentation product of the strain BTAL-TDG, peak 1 is cinnamyl alcohol, peak 2 is a net plug, and peak 3 is a tramadol B; peak 4 is coumaryl alcohol, peak 5 is Triandrin, peak 6 is Triandrin B, peak 7 is Triandrin C, and peak 8 is Triandrin D.
FIG. 3 is an MS spectrum of peaks 1-8. a is cinnamyl alcohol, B is tamponade, c is tamponade B; d is coumaryl alcohol, e is Triandrin, f is Triandrin B, g is Triandrin C, and h is Triandrin D.
FIG. 4 is an NMR chart of Aservin B, and a is1H NMR, b is13C NMR。
FIG. 5 shows Triandrin1H NMR chart.
FIG. 6 is an NMR chart of Triandrin B, a is1H NMR, b is13C NMR。
FIG. 7 shows Triandrin C1H NMR chart.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the invention discloses a voseville analogue which has a structure shown as formula I, formula II, formula III or formula IV;
Figure BDA0002060464820000031
Figure BDA0002060464820000041
according to the invention, the compound with the structure shown in the formula I is cinnamyl alcohol diglucoside, is named as Trachelin B, is named as Rosavin B in English, has a chemical name of trans-cinnamyl- (6' -O-beta-D-glucopyranosyl) -O-beta-D-glucopyranoside, and has a molecular formula C21H30O11Molecular weight 458.18.
According to the invention, the compound with the structure shown in the formula II is coumaryl alcohol hydroxy diglucoside, which is named as Triandrin C in English in the invention, has the chemical name of 4-hydroxy-cinnamyl- (6' -O-beta-D-glucopyranosyl) -O-beta-D-glucopyranoside and the molecular formula of C21H30O12Molecular weight 474.17.
According to the invention, the compound with the structure shown in the formula III is coumaryl alcohol hydroxy diglucoside, which is named as Triandrin D in English in the invention, has the chemical name of 4-hydroxy-cinnamyl alcohol- (6' -O-beta-D-glucopyranosyl) -O-beta-D-glucopyranoside and the molecular formula of C21H30O12Molecular weight 474.17.
According to the invention, the compound with the structure shown in the formula IV is coumaryl alcohol hydroxyl mono-glucoside, the invention is named as Triandrin B in English, the chemical name is 4-hydroxy-cinnamyl alcohol-4-O-beta-D-glucopyranoside,molecular formula C15H20O7Molecular weight 312.12.
In a second aspect, the present invention provides a genetically engineered bacterium that produces a rosavin analogue as described above, comprising a gene capable of encoding phenylalanine ammonia lyase PAL or a gene encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, and a gene capable of encoding UDP glucosyltransferase UGT.
According to the invention, when the genetically engineered bacteria contain a gene capable of coding tyrosine ammonia lyase TAL, the genetically engineered bacteria can biosynthesize the novel compound Triandrin B shown as the formula IV and can biosynthesize coumaryl alcohol and coumaryl alcohol hydroxyl monoglucoside Triandrin.
Wherein, the coumarol has the following characteristics: the English name is p-coumaryl alcohol, the chemical name is 4- [ (E) -3-hydroxyprop-1-enyl]Phenol, formula C9H10O2Molecular weight 150.18, CAS number 3690-05-9, structural formula
Figure BDA0002060464820000042
Coumarol alcohol hydroxyl mono-glucoside Triandrin has the following characteristics: the English name is Triandrin (Sachaliside), the chemical name is 4-hydroxy-cinnamyl-O-beta-D-glucopyranoside, and the molecular formula is C15H20O7Molecular weight 312.12, CAS number 132294-76-9, structural formula
Figure BDA0002060464820000043
According to the invention, when the genetically engineered bacteria contain a gene capable of encoding phenylalanine ammonia lyase PAL, the genetically engineered bacteria can biosynthesize cinnamyl alcohol and obstruction;
wherein, cinnamyl alcohol has the following characteristics: english name is Cinnamic alcohol, chemical name is 3-phenyl-2-propene-1-ol, molecular formula is C9H10O, molecular weight of 134.18, CAS number of 104-54-1, structural formula
Figure BDA0002060464820000051
The vein plug has the following characteristics: english name Rosin, chemical name
(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(E)-3-phenylprop-2-enoxy]oxide-3, 4,5-triol or trans-cinnamyl-O-beta-D-glucopyranoside, molecular formula C15H20O6Molecular weight of 296.32, CAS number of 85026-55-7, and structural formula
Figure BDA0002060464820000052
The inventors of the present invention have further introduced a gene encoding glycosyltransferase for extending a sugar chain into the above genetically engineered bacteria, and have incorporated a gene encoding tyrosine ammonia lyase TAL in the presence of glucose, thereby biologically synthesizing the novel compounds Triandrin C and Triandrin D of the present invention; the genetically engineered bacteria containing the gene capable of encoding phenylalanine ammonia lyase PAL can be further biosynthesized into the novel compound Rosavin B.
According to a specific embodiment of the invention, the exogenous genes of the genetically engineered bacteria are a gene capable of encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, and a gene capable of encoding UDP glucosyltransferase UGT, and can biosynthesize a novel compound Triandrin B as shown in formula IV above, and can biosynthesize coumaryl alcohol and coumaryl alcohol hydroxyl mono glucoside Triandrin.
According to a specific embodiment of the invention, the exogenous genes of the genetically engineered bacteria are a gene capable of encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, a gene capable of encoding UDP glucosyltransferase UGT, and a gene capable of encoding glycosyltransferase for extending sugar chains, and can biosynthesize a novel compound Triandrin B represented by formula IV above, a novel compound Triandrin C represented by formula II, and a novel compound Triandrin D represented by formula III above, and biosynthesize coumaryl alcohol and coumaryl alcohol hydroxyl monoglucoside Triandrin.
According to a specific embodiment of the present invention, the exogenous genes of the genetically engineered bacteria are a gene capable of encoding phenylalanine ammonia lyase PAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, and a gene capable of encoding UDP glucosyltransferase UGT and a gene capable of encoding glycosyltransferase extending a sugar chain, which can biosynthesize Rosavin B as shown in formula I above, and can biosynthesize cinnamyl alcohol and clovir.
According to the present invention, the term "phenylalanine ammonia lyase PAL" is phenylalanine ammonia lyase ammonia-lyase, abbreviated PAL, which is an enzyme that catalyzes the direct removal of ammonia from phenylalanine to produce trans-cinnamic acid, and can be of various origins, e.g., microbial origin, plant origin, and the corresponding amino acid sequences thereof are well known to those skilled in the art, and can be obtained by conventional technical means in the art, e.g., by searching in databases (e.g., NCBI), or by searching in related publications, and the present invention is not described in detail herein.
According to the present invention, the term "tyrosine ammonia lyase TAL" is tyrosine ammonia-lyase, abbreviated TAL, which is an enzyme that catalyzes the direct removal of ammonia from tyrosine, and can be of various origins, e.g., microbial origin, plant origin, and the corresponding amino acid sequences thereof are well known to those skilled in the art, and can be obtained by means of techniques conventional in the art, e.g., by searching in databases (e.g., NCBI), or by searching in related publications, and the present invention is not further described herein.
According to the present invention, the term "4-coumaric acid-coenzyme A ligase 4 CL" is hydroxycinnamate-CoA lyase, abbreviated as 4CL, which is a key enzyme in the phenylpropanoid metabolic pathway in monolignol and flavonoid biosynthesis. They may be of various origins, e.g. of microbial origin, of plant origin, the corresponding amino acid sequences of which are known to those skilled in the art, and may be obtained by means of techniques conventional in the art, e.g. by searching in databases (e.g. NCBI), or by searching in relevant publications, and will not be described in any further detail herein.
According to the present invention, the term "cinnamoyl-CoA reductase CCR" is cinnamyl-CoA reductase, abbreviated CCR, responsible for catalyzing the most important metabolic reactions in monolignol biosynthesis, which transfers phenylpropanoid metabolites into the lignin synthesis pathway. They may be of various origins, e.g. of microbial origin, of plant origin, the corresponding amino acid sequences of which are known to those skilled in the art, and may be obtained by means of techniques conventional in the art, e.g. by searching in databases (e.g. NCBI), or by searching in relevant publications, and will not be described in any further detail herein.
According to the present invention, the term "UDP glucosyltransferase UGT" is UDP-glycosyltransferase, abbreviated UGT, which catalytically activates glucosyl groups in vivo to different acceptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules, thus conferring a number of biological functions to the glycosylated product. They may be of various origins, e.g. of microbial origin, of plant origin, the corresponding amino acid sequences of which are known to those skilled in the art, and may be obtained by means of techniques conventional in the art, e.g. by searching in databases (e.g. NCBI), or by searching in relevant publications, and will not be described in any further detail herein.
According to the present invention, the term "glycosyltransferase that extends a sugar chain" is any enzyme that can extend a sugar chain of an existing sugar compound, and can be of various origins, for example, microbial origins, plant origins, and corresponding amino acid sequences thereof are well known to those skilled in the art, and can be obtained by means of ordinary techniques in the art, for example, by searching in a database (e.g., NCBI), or by searching in related publications, and the present invention will not be described in detail.
According to the present invention, preferably, said UDP glucosyltransferase UGT is UDP glucosyltransferase UGT73C 5.
According to the present invention, preferably, the glycosyltransferase extending a sugar chain is a sugar-glycosyltransferase GGT, more preferably a sugar-glycosyltransferase CaUGT 3.
It is known that 18 amino acids, except Met (ATG) or Trp (TGG), among the 20 different amino acids that make up a protein, are each encoded by 2-6 codons (Sambrook et al, molecular cloning, Cold spring harbor laboratory Press, New York, USA, second edition, 1989, see appendix D page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in the triplet codon will not change the composition of the amino acid, so that the nucleotide sequences of genes encoding the same protein may differ. The nucleotide sequences of the genes encoding them, which are obtained by biological methods (e.g., PCR methods, mutation methods) or chemical synthesis methods, can be completely deduced by those skilled in the art from the amino acid sequences of the corresponding enzymes according to well-known codon tables.
According to a preferred embodiment of the invention, the gene encoding phenylalanine ammonia lyase PAL is the AtPAL gene from Arabidopsis thaliana, GenBank: AY 133595.1.
According to a preferred embodiment of the invention, the gene coding for tyrosine ammonia lyase TAL is represented by SEQ ID No.1, or the RgTAL gene from rhodotorula glutinis, GeneBank: AAA33883.1, preferably as shown in SEQ ID No. 1.
According to a preferred embodiment of the invention, the gene encoding 4-coumarate-CoA ligase 4CL is shown in SEQ ID No.2 or is the Pc4CL gene from parsley, GenBank: X13325.1, preferably shown in SEQ ID No. 2.
According to a preferred embodiment of the invention, the gene encoding cinnamoyl-CoA reductase CCR is the AtCCR gene from Arabidopsis thaliana, GenBank: AF 332459.1.
According to a preferred embodiment of the invention, the gene encoding the UDP glucosyltransferase UGT73C5 is shown in SEQ ID No.3 or the AtUGT73C5 gene from Arabidopsis thaliana, GenBank: KJ138865.1, preferably shown in SEQ ID No. 3.
According to a preferred embodiment of the invention, the gene encoding the sugar-glycosyltransferase CaUGT3 is represented by SEQ ID No.4 or CaUGT3 gene from Catharanthus roseus, GenBank: AB443870, preferably as represented by SEQ ID No. 4.
The strain used for constructing the genetically engineered bacterium according to the present invention may be various strains conventionally used in the art, for example, a fungus such as yeast, or a bacterium such as bacillus. According to a preferred embodiment of the present invention, the strain is Escherichia coli, and the species of Escherichia coli used for constructing the Escherichia coli expression strain is not particularly limited, and may be various Escherichia coli commonly used in the art capable of expressing a desired gene. In order to enable better expression of the gene of interest, the E.coli is preferably BL21(DE3) and E.coli DH5 α, more preferably E.coli strain BL21(DE 3).
The inventors of the present invention have found in their studies that the efficiency of obtaining each target product can be further improved when constructing escherichia coli into high phenylalanine-producing escherichia coli or high tyrosine-producing escherichia coli, compared to when using wild-type escherichia coli, after introducing each gene of the present invention as above.
Wherein, the escherichia coli for highly producing phenylalanine may be escherichia coli not expressing tyrR (DNA-binding transcriptional dual regulator tyrR), tyrA (fused chloride mutase/preprenate dehydrogenase) and trpe (anthralinanate synthase subunit) genes, and the construction thereof may be realized by various methods, for example, may include a λ Red recombination system, a CRISPR-Cas9 recombination system, RNAi, and the like. Preferably, the high-phenylalanine-producing Escherichia coli construction adopts a lambda Red recombination system. The escherichia coli with high phenylalanine yield constructed by the invention is more suitable for synthesizing Rosavin B, cinnamyl alcohol and vein plug.
Wherein, the escherichia coli with high tyrosine yield can be escherichia coli which does not express tyrR (DNA-binding transcriptional dual regulator tyrR), pheA (fused chloride mutase/preprenase) and trpE (anthracycline TrpE) genes, and the construction can be realized by adopting various methods, for example, a lambda Red recombination system, a CRISPR-Cas9 recombination system, RNAi and the like. Preferably, the construction of the high tyrosine-producing Escherichia coli of the present invention employs a lambda Red recombination system. The escherichia coli with high tyrosine yield constructed by the invention is more suitable for biosynthesis of Triandrin B, Triandrin C and Triandrin D, and coumarol and Triandrin.
According to the present invention, the construction method of the genetically engineered bacterium can be accomplished by a conventional technical means in the art, for example, by introducing a vector into a corresponding strain, into which a corresponding gene is inserted, and the present invention has no particular requirement on the type of expression vector, and can be various expression vectors commonly used in the art, such as plasmids, which can express a target gene in a strain. It will be understood by those skilled in the art that the construction method of the expression vector may adopt various methods commonly used in the art, such as the enzyme digestion treatment of the target gene and the ligation into the vector, which are not described herein again.
Wherein, the gene for constructing the genetically engineered bacterium of the invention can be cloned in a vector and introduced into escherichia coli, and preferably, the gene for constructing the genetically engineered bacterium of the invention is cloned into 2-3 vectors and introduced into escherichia coli for the bearing capacity of the vector and the bearing capacity of the number of exogenous vectors of escherichia coli.
The sequence of the gene in the vector for constructing the genetically engineered bacterium of the present invention is not particularly limited, as long as it can be expressed efficiently.
In a third aspect, the invention provides the application of the genetically engineered bacteria in the production of the rosavin analogue.
In a fourth aspect, the present invention provides a method for producing a voseville analog, comprising culturing the genetically engineered bacterium as described above in a medium containing glucose, and inducing expression of a foreign gene to produce the voseville analog.
According to the present invention, the culture conditions are conventional culture conditions, such as culture at 35-40 ℃ to OD using a medium containing an antibiotic as a selection marker600Is 0.5-0.7, thenAdding isopropyl-beta-D-thiogalactoside (IPTG) to induce expression at 14-20 ℃. In the fermentation culture, the above gene can be translated into the corresponding protein and the corresponding protein can exert its effect.
Wherein, taking the genetically engineered bacterium as an example, the culture medium can be LB culture medium (solvent is water, solute and final concentration thereof are 10g/L of Tryptone, 5g/L of yeast extract and 10g/L of NaCl respectively), and can also be M9 (Na)2HPO4·12H2O 15.12g/L，KH2PO4 3.0g/L，NaCl 0.5g/L，MgSO4·7H2O 0.5g/L，CaCl20.011g/L，NH4Cl 1.0g/L, glucose 20g/L) liquid medium, and the selection can be carried out by a person skilled in the art according to actual conditions.
According to the present invention, when the obtained genetically engineered bacterium is cultured to produce a vomerosal analog, it is also preferable to prepare a seed solution by subjecting the constructed genetically engineered bacterium to an activation treatment, for example, in a medium containing an antibiotic as a selection marker, and culturing at 35 to 40 ℃ for 12 to 20 hours.
According to a preferred embodiment of the present invention, the above LB medium may be used for activation of genetically engineered bacteria, i.e., the above M9 medium may be used for production of a vomerosal analog, for example, for preparation of seed solutions. The amount of the seed solution to be inoculated can be selected conventionally in the art, for example, the amount of the seed solution to be inoculated is 1 vol%, that is, 1mL of the seed solution is added per 100mL of M9 liquid medium.
Further, the concentration of IPTG in the present invention is also not particularly limited, and it is preferably added in such an amount that the final concentration thereof in the M9 liquid medium becomes 0.08 to 0.12 mM.
According to the invention, the antibiotic is used as a screening marker on an expression vector, taking a common transformation vector of escherichia coli as an example, and the antibiotic can be streptomycin and kanamycin; the concentration of the antibiotic is a matter of routine choice in the art, for example, the streptomycin is added in an amount such that the final concentration is 80-120 mg/L; the kanamycin is added in an amount to make the final concentration 40-60 mg/L.
According to the present invention, it is understood that a person skilled in the art can select a corresponding genetically engineered bacterium according to the kind of a compound to be produced.
Specifically, when only Triandrin B needs to be produced, genetically engineered bacteria of which the exogenous gene can encode tyrosine ammonia lyase TAL, 4-coumarate-CoA ligase 4CL, cinnamoyl-CoA reductase CCR and UDP glucosyltransferase UGT can be selected and cultured under the condition of containing glucose to produce Triandrin B;
when Triandrin B, Triandrin C and Triandrin D are required to be produced, genetic engineering bacteria of exogenous genes, namely a gene capable of coding tyrosine ammonia lyase TAL, a gene capable of coding 4-coumarate-CoA ligase 4CL, a gene capable of coding cinnamoyl-CoA reductase CCR, a gene capable of coding UDP glucosyltransferase UGT and a gene capable of coding glycosyltransferase for extending sugar chains can be selected, and the Triandrin B, the Triandrin C and the Triandrin D are cultured and produced under the condition of containing glucose;
when production of Rosavin B is desired, genetically engineered bacteria can be selected for exogenous genes as a gene capable of encoding phenylalanine ammonia lyase PAL, a gene capable of encoding 4-coumarate-coa ligase 4CL, a gene capable of encoding cinnamoyl-coa reductase CCR, and a gene capable of encoding UDP glucosyltransferase UGT and a gene capable of encoding glycosyltransferase for sugar chain extension, and cultured under conditions containing glucose to produce Rosavin B.
The present invention will be described in detail below by way of examples.
In the following examples, E.coli strain BL21(DE3) and E.coli DH 5. alpha. were purchased from Beijing Quanji Biotech, Inc., E.coli strain BL21(DE3) was used for expression of all the genes in the present invention, and E.coli DH 5. alpha. was used for cloning of all the genes in the present invention.
Coli expression vector pCDFduet-1, pRSFDuet-1, pET28a was purchased from Novagen.
Phusion high fidelity DNA polymerase was purchased from Thermo.
Clonexpress Multi One Step Cloning Kit was purchased from Vazyme.
The primers and the genes are synthesized by Shenzhen Hua Dagen science and technology Limited.
The test methods in the following examples, which are not specified under specific conditions, were carried out under conventional conditions, for example "molecular cloning: the conditions described in the laboratory manual, or the conditions recommended by the manufacturer of the corresponding biological reagents.
The phenylalanine ammonia lyase gene PAL is from Arabidopsis (AtPAL, GenBank: AY 133595.1);
the tyrosine ammonia lyase gene TAL is shown as SEQ ID No. 1;
the 4-coumaric acid coenzyme A ligase gene 4CL is shown as SEQ ID No. 2;
the cinnamoyl-CoA reductase gene CCR is derived from Arabidopsis thaliana (AtCCR, GenBank: AF 332459.1);
the UDP glucosyltransferase UGT gene UGT73C5 is shown as SEQ ID No. 3;
the glycosyl-glycosyl transferase GGT gene CaUGT3 is shown as SEQ ID No. 4.
Plasmid pKD46 (temperature sensitive, containing exo, beta and gam genes regulated by arabinose promoter, Amp)r) pKD4 (Kana resistance gene with FRT sites at both ends, Kan)r) pCP20 (temperature-sensitive, encoding an FLP recombinase capable of recognizing FRT sites, Amp)r) Are all commercially available.
Example 1
This example serves to illustrate the reconstitution of the Lussevier B synthetic pathway in E.coli
The Escherichia coli expression vector V1 is pCDFDuet-4CL-PAL-CCR, and its preparation method refers to patent application No. 201611179577.9 (Liutao, Zhouyang, Bihui Pinna, Zhenghua, Mayan, recombinant Escherichia coli for producing cinnamyl alcohol and cloisonn, construction method and application).
The Escherichia coli expression vector V2 is pRSFDuet-AtUGT73C5-CaUGT3, and the preparation method of the vector is as follows:
(1) the primer 73C5-5FPBam (SEQ ID No.5)/73C5-3RPSal (SEQ ID No.6) is used as a guide, the synthesized AtUGT73C5 gene (SEQ ID No.3) is used as a template to carry out PCR, the AtUGT73C5 gene is obtained by amplification, and the plasmid pRSDet1 cut by BamHI and SalI is connected after being cut by BamHI and SalI, so that pRSDDuet-AtUGT73C5 is constructed.
(2) 5 '-KpnI and 3' -XhoI restriction sites are added when both ends of the CaUGT3 gene (SEQ ID No.4) are synthesized, and the plasmid pRSFDuet-AtUGT73C5 after the digestion of KpnI and XhoI is connected to construct pRSFDuet-AtUGT73C5-CaUGT 3.
(3) The vectors V1(pCDFDuet-4CL-PAL-CCR) and V2(pRSFDuet-AtUGT73C5-CaUGT3) were co-transformed into E.coli strain BL21(DE3) to obtain E.coli strain BWT-PDG producing cinnamyl alcohol, loxosterin and loxosterin B. The synthetic pathway from glucose to vosevelamer B is shown in figure 1.
In the steps (1) and (2), the reaction system of the PCR amplification reaction is: 5 XPisuion HF buffer 10 u L, 2.5mM dNTP 2.5 u L, 50 u M forward primer 0.5 u L, 50 u M reverse primer 0.5 u L, template 0.5 u L, Phusion DNA polymerase 0.5 u L, water 35.5 u L.
The reaction procedure of the PCR amplification reaction is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 20 seconds, annealing at 56 ℃ for 45 seconds, extension at 72 ℃ for 2 minutes, 30 cycles; extension at 72 ℃ for 10 min.
In the above step (3), plasmids V1(pCDFDuet-4CL-PAL-CCR) and V2(pRSFDuet-AtUGT73C5-CaUGT3) were transformed into E.coli strain BL21(DE3) by chemical transformation, specifically: 100 μ L of E.coli strain BL21(DE3) competent cells were placed on ice, after 10 minutes 2 μ L of plasmid pCDFDuet-4CL-PAL-CCR and 2 μ L of pRSFDuet-AtUGT73C5-CaUGT3 were added, gently mixed, and after 30 minutes on ice, heat shock was applied at 42 ℃ for 90 seconds, and immediately after 2 minutes on ice, 600 μ L of LB liquid medium was added, and after resuscitating and culturing at 37 ℃ and 150rpm for 30 minutes in a shaker, the bacterial solution was spread on LB plates containing streptomycin and kanamycin. Screening a transformation strain BWT-PDG carrying two expression vectors simultaneously by using streptomycin and kanamycin resistance, and performing enzyme digestion verification by extracting plasmids to obtain a recombinant escherichia coli strain BWT-PDG capable of synthesizing cinnamyl alcohol, loxosterin and loxosterin B.
Example 2
This example illustrates the reconstitution of the synthetic pathway of torsavide B in E.coli with high phenylalanine production
The high-yield phenylalanine Escherichia coli does not express tyrR (DNA-binding transcriptional dual regulator TyrR), tyrA (fused chloride mutase/precursor dehydrogenase), trpE (anti kinase pathway subunit TrpE) genes, and the specific construction method is as follows:
(1) primer tyrR-5FPL (SEQ ID No: 7)/tyrR-3RPL (SEQ ID No: 8) was used to amplify the Kan gene in pKD4 to replace the tyrR gene coding region; the primer tyrRGD-5FP (SEQ ID No: 9)/tyrRGD-3RP (SEQ ID No: 10) is a recombinant bacteria identification primer after tyrR gene knockout, and the two primers are respectively positioned at the left side and the right side of a targeting sequence region. The PCR reaction system is as follows: 5 × PCR buffer, 10 μ L; dNTP (2mM each), 5. mu.L; primers tyrR-5FPL/tyrR-3RPL (10. mu.M), 2. mu.L each; phusion high-fidelity DNA polymerase (5U/. mu.L), 0.5. mu.L; pKD4, 50 ng; ddH2O make up to 50. mu.L. The reaction procedure is as follows: initial denaturation 98 ℃ for 30 sec; 8sec at 98 ℃, 30sec at 55 ℃, 30sec at 72 ℃ for 30 cycles; final extension at 72 ℃ for 10 min. Obtaining the target molecule DNA fragment.
(2) Escherichia coli BL21(DE3) containing pKD46 cultured overnight for 12h was inoculated at a ratio of 1:100, cultured with shaking to OD600 of about 0.2, and further cultured with arabinose at a final concentration of 10mmol/L to OD600 of 0.5-0.6 to prepare electrotransformation competence. 50. mu.L of the competent cells were electroporated with about 200ng of the targeting molecule DNA fragment. Immediately after electric shock, 1mL of LB liquid medium was added, shaking cultured at 37 ℃ for 2 hours, and spread on LB plates (Kan)r) Positive clones were screened. Subsequently, in a second round of in vivo recombination, plasmid pCP20 encoding FLP site-specific recombinase was transferred into TyrR knockout strains to knock out the Kan resistance gene. The cells were then incubated at 37 ℃ and pCP20 was discarded. PCR verification is carried out by using a primer tyrRGD-5FP/tyrRGD-3RP to obtain the tyrR gene knocked-out escherichia coli.
(3) The tyrA and trpE genes were knocked out in the same manner to obtain E.coli BPHE strain with the tyrR, tyrA and trpE genes knocked out. Primers for tyrA knock-out are shown in SEQ ID No: 11 and SEQ ID No: 12, the verification primer is shown as SEQ ID No: 13 and SEQ ID No: as shown at 14. The primers for knocking out trpE are shown as SEQ ID No: 15 and SEQ ID No: 16, and the verification primer is shown as SEQ ID No: 17 and SEQ ID No: 18, respectively.
The vector V1(pCDFDuet-4CL-PAL-CCR) and V2(pRSFDuet-AtUGT73C5-CaUGT3) are jointly transformed into an escherichia coli strain BPHE to obtain the escherichia coli strain BPHE-PDG with high cinnamyl alcohol, high loxel and high loxieb.
Example 3
This example illustrates the reconstitution of Triandrin B, Triandrin C and Triandrin D biosynthetic pathways in E.coli
The expression vectors of this example are escherichia coli expression vector V2 and escherichia coli expression vector V3, escherichia coli expression vector V2 is constructed as in example 1, and escherichia coli expression vector V3 contains genes TAL, CCR, and 4 CL.
The specific preparation method of the Escherichia coli expression vector V3 is as follows:
PCR is carried out by taking primers CCR-4CL-5F (SEQ ID No: 19)/CCR-4CL-3R (SEQ ID No: 20) and TAL-5F (SEQ ID No: 21)/TAL-3R (SEQ ID No: 22) as guides and taking a linearized vector V1(pCDFDuet-4CL-PAL-CCR) and a TAL gene (SEQ ID No.1) which are cut by EcoRI as templates, fragments pCDFDuet-4CL-CCR and TAL are obtained by amplification, and the two fragments are spliced into a complete vector by seamless cloning to obtain a vector V3(pCDFDuet-4 CL-TAL-CCR).
The reaction system of seamless cloning is as follows: 5 × CE MultiS Buffer 4 μ L, extension MultiS 2 μ L, linearized vector fragment pCDFDuet-4CL-CCR 4 μ L, gene fragment TAL 10 μ L. Reacting at 37 ℃ for 30 min; cooled to 4 ℃ or immediately placed on ice to cool. 20 mu L of reaction liquid is transformed into an escherichia coli competent cell DH5 alpha to obtain a positive clone, and the sequencing confirms that the positive clone is correct to obtain a constructed vector V3(pCDFDuet-4 CL-TAL-CCR).
Coli strain BL21(DE3) was co-transformed with the vectors V3(pCDFDuet-4CL-TAL-CCR) and V2(pRSFDuet-AtUGT73C5-CaUGT3) according to the method of example 1 to obtain E.coli strain BWT-TDG producing coumarol, Triandrin B, Triandrin C and Triandrin D.
Example 4
This example serves to illustrate the reconstitution of the biosynthetic pathway of Triandrin B, Triandrin C and Triandrin D in tyrosine-producing E.coli
The high-yield tyrosine Escherichia coli does not express tyrR (DNA-binding transcriptional dual regulator TyrR), pheA (fused chloride mutase/precursor dehydrogenase), trpE (anti kinase synthase subunit TrpE) genes, and the construction method is as follows:
the tyrR, pheA and trpE genes were knocked out in the same manner as in example 3 to obtain E.coli strain BTAL in which the genes tyrR, pheA and trpE were knocked out. Primers for knocking pheA out are shown as SEQ ID No: 23 and SEQ ID No: 24, the verification primer is shown as SEQ ID No: 25 and SEQ ID No: shown at 26.
The vectors V3(pCDFDuet-4CL-TAL-CCR) and V2(pRSFDuet-AtUGT73C5-CaUGT3) were co-transformed into E.coli strain BTAL according to the method of example 1 to obtain E.coli strain BTAL-TDG which produces coumarol, Triandrin B, Triandrin C and Triandrin D at high yield.
Test example
This example illustrates the fermentation culture of recombinant Escherichia coli strains BWT-PDG, BPHE-PDG, BWT-TDG and BTAL-TDG and the identification of the products
(1) Fermentation culture
1) Escherichia coli expression strains BWT-PDG, BPHE-PDG, BWT-TDG or BTAL-TDG constructed as described above in examples 1 to 4 were inoculated into 2ml of LB liquid medium (tryptone 10g/L, sodium chloride 10g/L, yeast extract 5g/L) containing antibiotics (streptomycin and kanamycin, final concentrations of 100mg/L and 50mg/L, respectively) as selection markers, respectively, and cultured at 37 ℃ for 12 hours to give an Escherichia coli seed solution.
2) The E.coli seed solutions obtained in step (1) were inoculated in 1 vol% of each of LB liquid media containing the antibiotics (streptomycin and kanamycin, final concentrations of 100mg/L and 50mg/L, respectively) as selection markers in an amount of 1mL, and cultured at 37 ℃ and isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1mM when OD600 reached 0.6, and induced at 16 ℃ for 16 hours. After the thalli is centrifugally collected at 4000rpm, 50mL of the thalli containing the sieve is inoculatedM9Y (Na) for selection of antibiotic for marker2HPO4·12H2O 15.12g/L，KH2PO4 3.0g/L，NaCl 0.5g/L，MgSO4·7H2O 0.5g/L，CaCl2 0.011g/L，NH4Cl 1.0g/L, yeast extract 0.25g/L, glucose 20g/L) in a liquid medium, and culturing at 30 ℃ for 24-72 hours to obtain fermentation liquids a (example 1) and B (example 2) containing cinnamyl alcohol, rosavin and rosavin B, and fermentation liquids C (example 3) and D (example 4) producing coumaryl alcohol, Triandrin B, Triandrin C and Triandrin D.
(2) Detection of product
1) HPLC detection of the product: after fermentation liquids a, b, c and d are respectively 1mL and centrifuged at 12000rpm for 5min, supernatants are taken for HPLC analysis and detection. The analysis conditions were as follows: the instrument is as follows: an Agilent liquid chromatograph, and the determination conditions comprise: a C18 column (4.6X 250 mm); the detection wavelength is 254 nm; mobile phase a ═ water (containing 0.1% by volume of formic acid), B ═ methanol; the flow rate is 1 mL/min; gradient elution conditions: 0-5min 20% volume B; 6-25min 20% volume B to 100% volume B (the concentration of B increases uniformly within 6-25 min); 25-30min, 100% B; 31-40min, 20% B. The amount of sample was 50. mu.L.
The results for fermentation broths a, b, c and d are shown in FIG. 2. Wherein, 3 product peaks ( peaks 1, 2 and 3) appear in the fermentation liquor a (strain BWT-PDG) and the fermentation liquor b (strain BPHE-PDG), and the peaks 1, 2 and 3 in the fermentation liquor b are all higher than the fermentation liquor a. In fermentation broth c (strain BWT-TDG), there were 7 product peaks (peaks 1-7, wherein peak 8 was smaller and ignored), and in fermentation broth d (strain BTAL-TDG), because phenylalanine synthesis was blocked, peaks 1-3 disappeared, and there were 5 product peaks (peaks 4-8).
2) LC-MS and NMR analysis of the product: performing LC-MS analysis on the peaks monitored in each fermentation broth in the step (1), wherein the conditions for performing LC-MS analysis comprise: a C18 column (4.6X 250 mm); the detection wavelength is 254 nm; gradient elution conditions: 0-5min 20% volume B; 6-25min 20% volume B to 100% volume B (the concentration of B increases uniformly within 6-25 min); 25-30min, 100% B; 31-40min, 20% B. The amount of sample was 50. mu.L. ESI positive ion source, molecular weight scan range 50-1000.
The results are shown in FIG. 3, and the results of the product peaks 1-3 in fermentation broth a are shown in FIGS. 3 a-c. The MS spectrum of peak 1 (fig. 3a) has MS characteristic peak 117.0700 of cinnamyl alcohol; MS characteristic peak 319.1151 with cloison on MS spectrum of peak 2 (fig. 3 b); the MS pattern of peak 3 (fig. 3c) has MS characteristic peaks 476.2126 and 481.1686 of torsavid B. Through further one-dimensional and two-dimensional NMR, the peak 3 can be determined to be Trans-Cinnamyl- (6' -O-beta-D-glucopyranosyl) -O-beta-D-glucopyranoside, which is an analogue of the important active ingredient of rhodiola rosea, and is a novel compound named as Trachelospmaintenance B. The NMR data are as follows, and are shown in FIG. 4.
[1H-NMR(600MHz,D2O) data:1H NMR(600MHz,)δ7.42(d,J＝7.5Hz,H-5,9),7.32(d,J＝7.5Hz,H-6,8),7.26(d,J＝7.5Hz,H-7),6.65(d,J＝15.9Hz,H-3),6.31(dt,J＝15.9Hz,6.4Hz,H-2),4.45(d,J＝7.9Hz,H-1′),4.42(dd,J＝12.7,6.8Hz,Hα-1),4.39(d,J＝7.9Hz,H-1″),4.31(dd,J＝12.7,6.8Hz,Hβ-1),4.08(dd,J＝11.8,2.0Hz,Hα-6′),3.80(dd,J＝12.4,2.1Hz,Hα-6″),3.76(dd,J＝11.8,5.7Hz,Hβ-6′),3.61(dd,J＝12.4,5.6Hz,Hβ-6′),3.51～3.22(m,H-2′,2″,3′,3″,4′,4″,5′,5″).13C NMR(150MHz,D2o) data: δ 135.8(s, C-4),133.0(d, C-4),128.3(d, C-5,9),127.6(d, C-7),126.0(d, C-6,8),102.2(d, C-1 "), 100.7(d, C-1 '), 75.3/75.2/75.1/74.3/72.5/72.4/69.0/68.8(d, C-3'/C-3 '-5'), 69.8(t, C-1),68.0(t, C-6 '), 60.1(t, C-6').
The MS spectrum of the peak 4 (figure 3d) has a characteristic peak 133.0690 of coumaryl alcohol; the MS spectrum of the peak 5 (figure 3e) has characteristic peaks 313.1408, 330.1536 and 335.1095 of the coumaryl alcohol hydroxyl monoglucoside Triandrin; the MS spectrum of the peak 6 (figure 3f) has characteristic peaks 330.1528 and 335.1089 of coumarol phenolic hydroxyl monoglucoside Triandrin B; the MS spectrum of the peak 7 (figure 3g) has characteristic peaks 475.1794 and 492.2069,457.1615 of coumaryl alcohol hydroxyl diglucoside Triandrin C; the MS spectrum of the peak 8 (figure 3h) has characteristic peaks 492.2056 and 497.1608 of coumarol-phenolic hydroxyl-diglucoside Triandrin D.
By means of further1H and13c NMR can determine Peak 5 as 4-hydroxyl-cinnamyl-O-beta-D-glucopyranoside, coumaryl alcohol hydroxyl mono glucoside, one of the important active ingredients of rhodiola rosea, English name is Triandrin (Sachaliside), NMR data are shown as follows, and see figure 5 specifically;
1H NMR(600MHz,CD3OD) data δ 7.29(d, J ═ 8.5Hz, H-5,9),6.78(d, J ═ 8.6Hz, H-6,8),6.61(d, J ═ 15.9Hz, H-3),6.21(dt, J ═ 15.9,6.4Hz, H-2),4.53(dd, J ═ 12.8,5.4Hz, H-3), d, J, H, c, d, J, c, d, c, d, c, d, cα-1),4.41(d,J＝7.8Hz,H-1′),4.32(dd,J＝12.4,6.8Hz,Hβ-1),3.93(dd,J＝11.9,1.7Hz,Hα-6′),3.73(dd,J＝11.9,5.1Hz,Hβ-6′),3.44～3.23(m,H-2′,3′,4′,5′).
Peak 6 is 4-hydroxy-cinnamyl alcohol-4-O- β -D-glucopyranoside, coumarol phenolic hydroxyl monoglucoside, a novel compound designated Triandrin B, NMR data are shown below, see fig. 6 in particular;
1H NMR(600MHz,CD3OD) data: δ 7.33(d, J ═ 8.6Hz, H-5,9),7.04(d, J ═ 8.6Hz, H-6,8),6.54(d, J ═ 15.9Hz, H-3),6.24(dt, J ═ 5.8Hz,15.9Hz, H-2),4.89(d, J ═ 7.4Hz, H-1'), 4.19(d, J ═ 5.8Hz, H-1),3.88(dd, J ═ 12.0,1.7Hz, H-1)α-6′),3.69(dd,J＝12.0,5.2Hz,Hβ-6′),3.46～3.38(m,H-2′,H-3′,H-4′,H-5′).13C NMR(150MHz,CD3OD) data: δ 159.1(s, C-7),133.3(s, C-4),131.7(d, C-3),129.0(d, C-5,9),128.9(d, C-2),118.3(d, C-6,8),102.7(d, C-1 '), 78.6(d, C-3'), 78.5(d, C-5 '), 75.4(d, C-2'), 71.8(d, C-4 '), 64.3(t, C-1),63.0(t, C-6').
Peak 7 is 4-hydroxy-cinnamyl- (6' -O- β -D-glucopyranosyl) -O- β -D-glucopyranoside, coumaryl alcohol hydroxy diglucoside, which is a novel compound designated Triandrin C, with NMR data shown below, see fig. 7 in particular;
1H NMR(600MHz,CD3OD) data: δ 7.31(d, J ═ 8.5Hz, H-5,9),6.78(d, J ═ 8.5Hz, H-6,8),6.64(d, J ═ 15.9Hz, H-3),6.21(dt, J ═ 15.9,6.4Hz, H-2),4.53(dd, J ═ 12.3,5.9Hz, H-5,9)α-1),4.46(d,J＝7.7Hz,H-1′),4.42(d,J＝7.8Hz,H-1″),4.32(dd,J＝12.3,6.7Hz,Hβ-1),4.21(dd,J＝11.4,1.5Hz,Hα-6′),3.92(brd,J＝11.6Hz,Hα-6″),3.85(dd,J＝11.5,5.7Hz,Hβ-6′),3.72(dd,J＝11.8,4.9Hz,Hβ-6″),3.51～3.27(m,H-2′,2″,3′,3″,4′,4″,5′,5″).
The compound amount of the peak 8 is low, NMR detection is not carried out, and the MS characteristic peak and the relations with the compounds 5, 6 and 7 are combined to determine that the peak 8 is coumarol-phenolic hydroxyl-diglucoside, which is a new compound and is named as Triandrin D.
The test shows that the highest output of the torsavide B in the strain BWT-PDG fermentation liquor a can reach 1107.9 +/-109.2 mg/L, and the amounts of the residual torsavide and cinnamyl alcohol are respectively 256.8 +/-26.9 mg/L and 161.1 +/-20.1 mg/L;
the yield of the lasiobuxine B in the high-yield phenylalanine strain BPHE-PDG fermentation liquor B can reach 1609.3 +/-7.0 mg/L, is improved by 45.3 percent compared with the wild strain BWT-PDG, and the yields of the residual lasiobuxine and cinnamyl alcohol are respectively 180.7 +/-9.0 mg/L and 346.7 +/-20.1 mg/L;
the highest yield of Triandrin in the strain BWT-TDG fermentation broth c reaches 432.7 +/-39.4 mg/L, and the highest yield of Triandrin B reaches 302.5 +/-49.1 mg/L; the yields of coumaryl alcohol, Triandrin C and Triandrin D were not determined.
The highest yield of Triandrin in the fermentation liquid d of the high-yield tyrosine strain BTAL-TDG reaches 1686.4 +/-135.6 mg/L, which is improved by 2.9 times compared with the strain BWT-TDG; the highest yield of Triandrin B reaches 1565.6 +/-53.2 mg/L, which is 4.2 times higher than that of BWT-TDG strain. The yields of coumaryl alcohol, Triandrin C and Triandrin D were not determined.
The escherichia coli with high cinnamyl alcohol glucoside yield can be used for producing 3 compounds such as cinnamyl alcohol, Rosavin B and the like through fermentation, wherein the highest yield of the novel compound Rosavin B can reach 1.6 g/L. The escherichia coli with high coumarol glucoside yield can be used for producing 5 compounds such as coumarol, Triandrin B, Triandrin C, Triandrin D and the like through fermentation, wherein the 5 compounds comprise 3 new compounds, the highest yield of Triandrin reaches 1.7g/L, and the highest yield of Triandrin B reaches 1.6 g/L. Therefore, the invention provides a new biosynthesis way for high yield of cinnamyl alcohol glucoside and coumaryl alcohol glucoside, and the compounds have good application prospect as the important active ingredients of rhodiola rosea or the analogues thereof, lay the foundation for large-scale industrial production and have important economic value and social benefit.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> trametes derivative, genetic engineering bacterium for producing the trametes derivative and application thereof
<130> I56961TIB
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 2082
<212> DNA
<213> Gene encoding tyrosine ammonia lyase TAL
<400> 1
atggctccgc gtccgacctc tcagtctcag gctcgtacct gcccgaccac ccaggttacc 60
caggttgaca tcgttgaaaa aatgctggct gctccgaccg actctaccct ggaactggac 120
ggttactctc tgaacctggg tgacgttgtt tctgctgctc gtaaaggtcg tccggttcgt 180
gttaaagact ctgacgaaat ccgttctaaa atcgacaaat ctgttgaatt tctgcgttct 240
cagctgtcta tgtctgttta cggtgttacc accggtttcg gtggttctgc tgacacccgt 300
accgaagacg ctatctctct gcaaaaagct ctgctggaac accagctgtg cggtgttctg 360
ccgtcttctt tcgactcttt ccgtctgggt cgtggtctgg aaaactctct gccgctggaa 420
gttgttcgtg gtgctatgac catccgtgtt aactctctga cccgtggtca ctctgctgtt 480
cgtctggttg ttctggaagc tctgaccaac ttcctgaacc acggtatcac cccgatcgtt 540
ccgctgcgtg gtactatctc tgcttctggt gacctgtctc cgctgtctta catcgctgct 600
gctatctctg gtcacccgga ctctaaagtt cacgttgttc acgaaggtaa agaaaaaatc 660
ctgtacgctc gtgaagctat ggctctgttc aacctggaac cggttgttct gggtccgaaa 720
gaaggtctgg gtctggttaa cggtactgct gtttctgctt ctatggctac cctggctctg 780
cacgacgctc acatgctgtc tctgctgtct cagtctctga ccgctatgac cgttgaagct 840
atggttggtc acgctggttc tttccacccg ttcctgcacg acgttacccg tccgcacccg 900
acccagatcg aagttgctgg taacatccgt aaactgctgg aaggttctcg tttcgctgtt 960
caccacgaag aagaagttaa agttaaagac gacgaaggta tcctgcgtca ggaccgttac 1020
ccgctgcgta cctctccgca gtggctgggt ccgctggttt ctgacctgat ccacgctcac 1080
gctgttctga ccatcgaagc tggtcagtct accaccgaca acccgctgat cgacgttgaa 1140
aacaaaacct ctcaccacgg tggtaacttc caggctgctg ctgttgctaa cactatggaa 1200
aaaacccgtc tgggtctggc tcagatcggt aaactgaact tcacccagct gaccgaaatg 1260
ctgaacgctg gtatgaaccg tggtctgccg tcttgcctgg ctgctgaaga cccgtctctg 1320
tcttaccact gcaaaggtct ggacatcgct gctgctgctt acacctctga actgggtcac 1380
ctggctaacc cggttaccac ccacgttcag ccggctgaaa tggctaacca ggctgttaac 1440
tctctggctc tgatctctgc tcgtcgtacc accgaatcta acgacgttct gtctctgctg 1500
ctggctaccc acctgtactg cgttctgcaa gctatcgacc tgcgtgctat cgaatttgaa 1560
tttaaaaaac agttcggtcc ggctatcgtt tctctgatcg accagcactt cggttctgct 1620
atgaccggtt ctaacctgcg tgacgaactg gttgaaaaag ttaacaaaac cctggctaaa 1680
cgtctggaac agaccaactc ttacgacctg gttccgcgtt ggcacgacgc tttctctttc 1740
gctgctggta ctgttgttga agttctgtct tctacctctc tgtctctggc tgctgttaac 1800
gcttggaaag ttgctgctgc tgaatctgct atctctctga cccgtcaggt tcgtgaaacc 1860
ttctggtctg ctgcttctac ctcttctccg gctctgtctt acctgtctcc gcgtacccag 1920
atcctgtacg ctttcgttcg tgaagaactg ggtgttaaag ctcgtcgtgg tgacgttttc 1980
ctgggtaaac aggaagttac catcggttct aacgtttcta aaatctacga agctatcaaa 2040
tctggtcgta tcaacaacgt tctgctgaaa atgctggctt aa 2082
<210> 2
<211> 1686
<212> DNA
<213> Gene encoding p-hydroxycinnamic acid coenzyme A ligase 4CL
<400> 2
atggcgccac aagaacaagc agtttctcag gtgatggaga aacagagcaa caacaacaac 60
agtgacgtca ttttccgatc aaagttaccg gatatttaca tcccgaacca cctatctctc 120
cacgactaca tcttccaaaa catctccgaa ttcgccacta agccttgcct aatcaacgga 180
ccaaccggcc acgtgtacac ttactccgac gtccacgtca tctcccgcca aatcgccgcc 240
aattttcaca aactcggcgt taaccaaaac gacgtcgtca tgctcctcct cccaaactgt 300
cccgaattcg tcctctcttt cctcgccgcc tccttccgcg gcgcaaccgc caccgccgca 360
aaccctttct tcactccggc ggagatagct aaacaagcca aagcctccaa caccaaactc 420
ataatcaccg aagctcgtta cgtcgacaaa atcaaaccac ttcaaaacga cgacggagta 480
gtcatcgtct gcatcgacga caacgaatcc gtgccaatcc ctgaaggctg cctccgcttc 540
accgagttga ctcagtcgac aaccgaggca tcagaagtca tcgactcggt ggagatttca 600
ccggacgacg tggtggcact accttactcc tctggcacga cgggattacc aaaaggagtg 660
atgctgactc acaagggact agtcacgagc gttgctcagc aagtcgacgg cgagaacccg 720
aatctttatt tccacagcga tgacgtcata ctctgtgttt tgcccatgtt tcatatctac 780
gctttgaact cgatcatgtt gtgtggtctt agagttggtg cggcgattct gataatgccg 840
aagtttgaga tcaatctgct attggagctg atccagaggt gtaaagtgac ggtggctccg 900
atggttccgc cgattgtgtt ggccattgcg aagtcttcgg agacggagaa gtatgatttg 960
agctcgataa gagtggtgaa atctggtgct gctcctcttg gtaaagaact tgaagatgcc 1020
gttaatgcca agtttcctaa tgccaaactc ggtcagggat acggaatgac ggaagcaggt 1080
ccagtgctag caatgtcgtt aggttttgca aaggaacctt ttccggttaa gtcaggagct 1140
tgtggtactg ttgtaagaaa tgctgagatg aaaatagttg atccagacac cggagattct 1200
ctttcgagga atcaacccgg tgagatttgt attcgtggtc accagatcat gaaaggttac 1260
ctcaacaatc cggcagctac agcagagacc attgataaag acggttggct tcatactgga 1320
gatattggat tgatcgatga cgatgacgag cttttcatcg ttgatcgatt gaaagaactt 1380
atcaagtata aaggttttca ggtagctccg gctgagctag aggctttgct catcggtcat 1440
cctgacatta ctgatgttgc tgttgtcgca atgaaagaag aagcagctgg tgaagttcct 1500
gttgcatttg tggtgaaatc gaaggattcg gagttatcag aagatgatgt gaagcaattc 1560
gtgtcgaaac aggttgtgtt ttacaagaga atcaacaaag tgttcttcac tgaatccatt 1620
cctaaagctc catcagggaa gatattgagg aaagatctga gggcaaaact agcaaatgga 1680
ttgtga 1686
<210> 3
<211> 1488
<212> DNA
<213> Gene encoding UDP glucosyltransferase UGT73C5
<400> 3
atggtgagcg aaaccaccaa atctagtccg ttacattttg tgctgtttcc gtttatggca 60
cagggtcaca tgattccgat ggtggatatt gcacgcttac tggcccagcg cggcgttatt 120
attaccattg tgaccacccc gcataatgca gcacgcttta aaaatgtgct gaatcgcgcc 180
attgaaagtg gcctgccgat taacctagtt caggttaaat ttccgtatct ggaagccggc 240
ttacaggaag gccaggaaaa tattgatagc ttagatacca tggaacgcat gattccgttt 300
tttaaagcag ttaattttct ggaagaaccg gttcagaaac tgattgaaga gatgaatccg 360
cgcccgtctt gtctgattag cgatttttgt ctgccgtata cctctaaaat tgccaaaaaa 420
ttcaatattc cgaaaattct gtttcatggc atgggctgtt tttgtctgtt atgtatgcat 480
gtgctgcgca aaaatcgcga aattctggat aatctgaaat cagataaaga actgtttacc 540
gtgccggatt ttccggatcg tgttgagttc acccgcaccc aggttccggt ggaaacctat 600
gttccggcag gcgattggaa agatattttt gatggtatgg ttgaagccaa cgagacctct 660
tatggcgtga ttgttaatag ctttcaggaa ctggaaccgg cctatgccaa agattataag 720
gaagttcgct caggcaaagc ctggaccatt ggcccggtga gcctgtgtaa taaggtgggt 780
gcagataaag ccgaacgcgg caacaaatca gatattgatc aggatgaatg tctgaaatgg 840
ttagatagca agaaacatgg tagtgtgctg tatgtgtgtc tgggtagcat ttgtaatctg 900
ccgctgtcac agctgaaaga actgggctta ggcttagaag aatcacagcg cccgtttatt 960
tgggtaattc gcggttggga aaaatacaag gaattagttg aatggtttag cgaatcaggc 1020
tttgaagatc gtattcagga tcgcggctta ctgattaaag gttggtcacc gcagatgctg 1080
attctgagtc atccgagcgt gggcggcttt ctgacccatt gtggttggaa tagtacctta 1140
gaaggcatta ccgccggcct gccgttactg acctggccgc tgtttgcaga tcagttttgt 1200
aacgagaaac tggttgttga agtgctgaaa gcaggcgttc gtagcggcgt ggaacagccg 1260
atgaaatggg gcgaagaaga aaaaattggc gtgttagtgg ataaagaagg tgttaaaaaa 1320
gcagtggaag aactgatggg cgaatcagat gatgccaaag aacgtcgtcg tcgcgccaaa 1380
gaattaggcg atagcgcaca taaagcagtt gaagaaggtg gctcaagtca tagcaatatt 1440
agctttctgc tgcaggatat tatggaactg gccgaaccga ataattaa 1488
<210> 4
<211> 1365
<212> DNA
<213> Gene encoding sugar-glycosyltransferase CaUGT3
<400> 4
atggccaccg aacagcagca ggcaagcatt agctgtaaaa ttctgatgtt tccgtggctg 60
gcatttggtc atattagtag ttttctgcaa ctggcaaaaa agctgagcga tcgcggcttt 120
tatttttata tttgcagcac cccgattaat ctggatagca ttaagaataa gatcaaccag 180
aattacagca gcagcattca gctggttgat ctgcatctgc cgaatagtcc gcagctgccg 240
ccgagcctgc ataccaccaa tggcctgccg ccgcatctga tgagtaccct gaaaaatgcc 300
ctgattgatg caaatccgga tctgtgtaaa attattgcca gcattaagcc ggatctgatt 360
atctatgatc tgcatcagcc gtggaccgaa gcactggcaa gtcgtcataa tattccggca 420
gttagtttta gtaccatgaa tgcagttagc tttgcctatg tgatgcacat gtttatgaat 480
ccgggcattg aatttccgtt taaagccatt catctgagtg attttgaaca ggcacgcttt 540
ctggaacagc tggaaagcgc caaaaatgat gccagtgcca aagatccgga actgcaaggc 600
agcaaaggtt tctttaatag tacctttatc gtgcgcagca gccgcgaaat tgaaggtaaa 660
tatgttgatt atctgagcga aattctgaaa agtaaagtga ttccggtgtg tccggttatt 720
agcctgaata ataatgatca gggccagggt aataaggatg aagatgaaat tattcagtgg 780
ctggataaaa aatctcatcg cagtagcgtg tttgttagtt ttggtagtga atatttcctg 840
aatatgcagg aaattgaaga aattgcaatc ggcctggaac tgagcaatgt gaattttatt 900
tgggtgctgc gctttccgaa aggtgaagat accaaaattg aagaagtgct gccggaaggc 960
tttctggatc gtgttaaaac caaaggccgt attgttcatg gctgggcacc gcaggcacgc 1020
attctgggcc atccgagtat tggtggtttt gtgagccatt gtggttggaa tagcgtgatg 1080
gaaagcattc agattggcgt gccgattatt gcaatgccga tgaatctgga tcagccgttt 1140
aatgcccgcc tggtggtgga aattggtgtg ggtattgaag ttggccgcga tgaaaatggc 1200
aaactgaaac gcgaacgtat tggtgaagtg attaaggaag tggcaattgg caaaaaaggc 1260
gaaaaactgc gcaaaaccgc caaagatctg ggtcagaaac tgcgtgatcg cgaaaaacag 1320
gattttgatg aactggcagc aaccctgaaa cagctgtgtg tgtaa 1365
<210> 5
<211> 40
<212> DNA
<213> primer 73C5-5FPBam
<400> 5
actggatccg atggtgagcg aaaccaccaa atctagtccg 40
<210> 6
<211> 39
<212> DNA
<213> primer 73C5-3RPSal
<400> 6
tgagtcgact tagtggtggt ggtggtggtg attattcgg 39
<210> 7
<211> 70
<212> DNA
<213> primer tyrR-5FPL
<400> 7
gtgtcatatc atcatattaa ttgttctttt ttcaggtgaa ggttcccatg attccgggga 60
tccgtcgacc 70
<210> 8
<211> 71
<212> DNA
<213> primer tyrR-3RPL
<400> 8
gcataattta atatgcctga tggtgttgca ccatcaggca tattcgcgct tatgtaggct 60
ggagctgctt c 71
<210> 9
<211> 20
<212> DNA
<213> primer tyrRGD-5FP
<400> 9
tggattgacg atgacaaacc 20
<210> 10
<211> 20
<212> DNA
<213> primer tyrRGD-3RP
<400> 10
ttctgcaagc ctacggtgtg 20
<210> 11
<211> 69
<212> DNA
<213> primer F for tyrA knock-out
<400> 11
tattatggtt gctgaattga ccgcattacg cgatcaaatt gatgaagtca ttccggggat 60
ccgtcgacc 69
<210> 12
<211> 67
<212> DNA
<213> primer R for tyrA knock-out
<400> 12
ttattactgg cggttgtcat tcgcctgacg caataacacg cggctttctg taggctggag 60
ctgcttc 67
<210> 13
<211> 22
<212> DNA
<213> tyrA knock-out verification primer F
<400> 13
tgtatccgta accgatgcct gc 22
<210> 14
<211> 21
<212> DNA
<213> tyrA knock-out verification primer R
<400> 14
aaccacaatc tgattatgac c 21
<210> 15
<211> 70
<212> DNA
<213> primer F for knockout of trpE
<400> 15
agagaataac aatgcaaaca caaaaaccga ctctcgaaca gctaacctgc attccgggga 60
tccgtcgacc 70
<210> 16
<211> 67
<212> DNA
<213> primer R for knockout of trpE
<400> 16
acgaccttcc gcctcggcaa ttaactgcat agcgcgtact ttcggcgctg taggctggag 60
ctgcttc 67
<210> 17
<211> 21
<212> DNA
<213> verification primer F for trpE knockout
<400> 17
aagggtatcg acaatgaaag c 21
<210> 18
<211> 21
<212> DNA
<213> verification primer R for TrpE knockout
<400> 18
aaagtctcct gtgcatgatg c 21
<210> 19
<211> 39
<212> DNA
<213> primer CCR-4CL-5F
<400> 19
tgcgatttaa gcggccgcat aatgcttaag tcgaacaga 39
<210> 20
<211> 39
<212> DNA
<213> primer CCR-4CL-3R
<400> 20
cgcgggatcg ggatccttat ttcggcaggt caccagacg 39
<210> 21
<211> 40
<212> DNA
<213> primer RgTAL-5F
<400> 21
ataaggatcc cgatcccgcg aaattaatac gactcactat 40
<210> 22
<211> 39
<212> DNA
<213> primer RgTAL-3R
<400> 22
tcgattatgc ttatgccagc atcttcagca gaacgttgt 39
<210> 23
<211> 70
<212> DNA
<213> primer F for knockout of pheA
<400> 23
aacactatga catcggaacc atggaacccg ttactggcgc tgcgagagag cgattgtgta 60
ggctggagct 70
<210> 24
<211> 70
<212> DNA
<213> primer R for knocking out pheA
<400> 24
tcaggttgga tcaacaggca cccatggtac gttctcactt gggtaacata acggctgaca 60
tgggaattag 70
<210> 25
<211> 22
<212> DNA
<213> verification primer F for pheA knock-out
<400> 25
tgaatgggag gcgtttcgtc gt 22
<210> 26
<211> 22
<212> DNA
<213> verification primer R for pheA knock-out
<400> 26
gttattgcgt caggcgaatg ac 22

Claims (8)

1. A genetically engineered bacterium for producing a rosavin analogue, which contains a gene capable of encoding phenylalanine ammonia lyase PAL or a gene encoding tyrosine ammonia lyase TAL, a gene capable of encoding 4-coumarate-CoA ligase 4CL, a gene capable of encoding cinnamoyl-CoA reductase CCR, and a gene capable of encoding UDP-glucosyltransferase UGT;
wherein the genetically engineered bacterium further contains a gene capable of encoding a glycosyltransferase that extends a sugar chain;
the UDP glucosyltransferase UGT is UDP glucosyltransferase UGT73C5, the coding gene is AtUGT73C5 gene from Arabidopsis thaliana, and GenBank is KJ 138865.1;
the glycosyltransferase extending a sugar chain is a sugar-glycosyltransferase CaUGT3, and a coding gene thereof is a CaUGT3 gene from catharanthus roseus, and GenBank is AB 443870;
the gene encoding phenylalanine ammonia lyase PAL is the AtPAL gene from Arabidopsis thaliana, GenBank: AY 133595.1;
the gene for coding tyrosine ammonia lyase TAL is an RgTAL gene from rhodotorula glutinis, GeneBank: AAA 33883.1;
the gene for coding 4-coumaric acid coenzyme A ligase 4CL is Pc4CL gene from parsley, and GenBank is X13325.1;
the gene for coding the cinnamoyl coenzyme A reductase CCR is an AtCCR gene from arabidopsis thaliana, and GenBank is AF 332459.1;
constructing the genetic engineering bacteria by using escherichia coli;
the loservib analogs have a structure shown in formula I, formula II, formula III or formula IV;
Figure FDA0003451802830000011
Figure FDA0003451802830000021
2. the genetically engineered bacterium of claim 1, wherein the gene encoding UDP glucosyltransferase UGT73C5 is represented by SEQ ID No. 3.
3. The genetically engineered bacterium of claim 1, wherein the gene encoding the sugar-glycosyltransferase CaUGT3 is represented by SEQ ID No. 4.
4. The genetically engineered bacterium of claim 1, wherein the gene encoding 4-coumarate-CoA ligase 4CL is represented by SEQ ID No. 2.
5. The genetically engineered bacterium of claim 1, wherein the Escherichia coli is Escherichia coli strain BL21(DE3) and Escherichia coli DH5 a.
6. The genetically engineered bacterium of claim 1, wherein the escherichia coli is a phenylalanine-producing escherichia coli or a tyrosine-producing escherichia coli;
wherein the E.coli producing phenylalanine at high yield does not express tyrR, tyrA and trpE genes;
the tyrosine-producing E.coli does not express tyrR, pheA and trpE genes.
7. Use of the genetically engineered bacterium of any one of claims 1 to 6 for the production of a rosavin analogue.
8. A method for producing a voseville analog, characterized by culturing the genetically engineered bacterium according to any one of claims 1 to 6 in a medium containing glucose, and inducing expression of a foreign gene to produce the voseville analog.
CN201910403327.6A 2019-05-15 2019-05-15 Trachelospermi analogue, genetic engineering bacterium for producing same and application thereof Active CN111943996B (en)

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