CN114457129A - Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone - Google Patents
Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone Download PDFInfo
- Publication number
- CN114457129A CN114457129A CN202111135083.1A CN202111135083A CN114457129A CN 114457129 A CN114457129 A CN 114457129A CN 202111135083 A CN202111135083 A CN 202111135083A CN 114457129 A CN114457129 A CN 114457129A
- Authority
- CN
- China
- Prior art keywords
- recombinant
- recombinant vector
- lactone
- dehydrogenase
- gene sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Images
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
-
- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01168—2-Dehydropantolactone reductase (A-specific) (1.1.1.168)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01169—2-Dehydropantoate 2-reductase (1.1.1.169), i.e. ketopantoate-reductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01002—Formate dehydrogenase (1.2.1.2)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone, wherein the recombinant engineering bacterium is induced to efficiently express L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase, and efficiently convert L-pantolactone to generate DL-pantolactone. The recombinant engineering bacteria of the invention obviously improve the purity and quality of DL-pantoic acid lactone, the reaction efficiency and the resource utilization rate, reduce the production cost, and have the advantages of simple operation, environmental protection and the like.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium and application thereof in efficient transformation of L-pantoic acid lactone.
Background
D-pantoic acid lactone is a medical intermediate for synthesizing vitamin medicine D-panthenol and neurotrophic medicine D-calcium homopantothenate, is used as a synthetic precursor of feed additives and daily chemical products, and has annual output of more than ten thousand tons. The industrial preparation of D-pantoic acid lactone usually adopts a method combining a chemical method and a resolution method, and comprises the following steps: isobutyraldehyde and formaldehyde are subjected to aldol condensation to generate hydroxyl pivalic aldehyde, then react with cyano to generate cyanoaldehyde, and are hydrolyzed to generate DL-pantoic acid lactone raceme, and D-pantoic acid lactone is prepared by adopting chemical resolution and enzyme resolution. However, the separation generates a large amount of L-pantoic acid lactone, and if the L-pantoic acid lactone is unreasonably utilized, resources are wasted, and the production cost is increased.
Progress in the chemical enzymatic synthesis of D-pantolactone (Chilobrachys et al, fermentation science and technology communications, vol.45, No. 4) discloses that DL-pantolactone is subjected to L-pantolactone dehydrogenase to produce ketopantolactone, which is then reduced by ketopantolactone reductase to obtain D-pantolactone, and the dehydrogenation efficiency of L-pantolactone dehydrogenase is low in this method. Also discloses that L-pantolactone is reacted by L-pantolactone dehydrogenase to obtain ketopantolactone, which is spontaneously hydrolyzed to generate ketopantoate, and then reduced by ketopantoate reductase to obtain D-pantoate, and the D-pantolactone is prepared by acidic ring closure. The method has low reaction efficiency of L-pantolactone dehydrogenase, and spontaneous hydrolysis is difficult to control in practical experiments, so that D-pantolactone is not obtained basically, and the experiments are difficult to reproduce.
CN110423717A discloses a multienzyme recombinant cell and a method for synthesizing D-pantolactone by multienzyme cascade catalysis, wherein the method uses ternary complex enzyme consisting of L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase to convert and produce D-pantolactone. However, glucose dehydrogenase is easily induced to generate a reaction byproduct gluconic acid, which causes difficulty in separation of D-pantolactone, generates a large amount of waste materials, and increases difficulty in treatment. For this reason, it is required to develop a process for producing D-pantolactone with a high conversion rate.
Disclosure of Invention
The invention aims to provide a recombinant vector, which is selected from recombinant vectors containing an L-pantoate lactone dehydrogenase modified gene sequence with a nucleotide sequence shown as SEQ ID No.1, a ketopantoate lactone reductase modified gene sequence with a nucleotide sequence shown as SEQ ID No.2 and a formate dehydrogenase modified gene sequence with a nucleotide sequence shown as SEQ ID No. 3.
In the preferred technical scheme of the invention, the nucleotide sequence of the L-pantoate lactone dehydrogenase modified gene sequence is shown as SEQ ID NO.1, the nucleotide sequence of the ketopantoate lactone reductase modified gene sequence is shown as SEQ ID NO.2, and the nucleotide sequence of the formate dehydrogenase modified gene sequence is shown as SEQ ID NO.3, and the nucleotide sequences are respectively arranged on a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector.
In a preferred technical scheme of the present invention, the recombinant vector optionally includes any one of a fourth recombinant vector or a fifth recombinant vector, wherein the fourth recombinant vector comprises a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2 and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown in SEQ ID No.3, the fifth recombinant vector comprises a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown in SEQ ID No.1, a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2, and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown in SEQ ID No. 3.
In a preferred technical scheme of the invention, the recombinant vector is selected from a first recombinant vector containing a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown in SEQ ID No.1, a fourth recombinant vector containing a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2 and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown in SEQ ID No. 3.
In the preferable technical scheme of the invention, the recombinant vector is used for preparing DL-pantoic acid lactone from L-pantoic acid lactone.
In a preferred technical scheme of the invention, the first recombinant vector comprises an L-pantoate lactone dehydrogenase modified gene sequence.
In the preferred technical scheme of the invention, the L-pantoate lactone dehydrogenase encoding gene is subjected to codon optimization to obtain an L-pantoate lactone dehydrogenase modified gene sequence.
In a preferred technical scheme of the invention, the coding gene of the L-pantolactone dehydrogenase is derived from any one of rhodococcus erythropolis, mycobacterium, nocardia, streptomyces and actinomycetes.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 1, and the nucleotide sequence of the target gene 1 is shown as SEQ ID NO. 1.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase encoding gene is subjected to codon optimization to obtain a ketopantoate lactone reductase modified gene sequence.
In a preferred technical scheme of the invention, the ketopantoate lactone reductase coding gene is derived from any one of candida magnoliae, micromonospora and streptomyces.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 2, and the nucleotide sequence of the target gene 2 is shown as SEQ ID NO. 2.
In a preferred embodiment of the present invention, the cleavage site is selected from SacI and NotI, or a combination thereof.
In the preferred technical scheme of the invention, the formate dehydrogenase encoding gene is optimized by a codon to obtain a formate dehydrogenase modified gene sequence.
In a preferred embodiment of the present invention, the formate dehydrogenase-encoding gene is derived from any one of Burkholderia, Escherichia coli and Aeromonas.
In the preferred technical scheme of the invention, the formate dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 3, and the nucleotide sequence of the target gene 3 is shown as SEQ ID NO. 3.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of EcoRI and NotI or a combination thereof.
In a preferred embodiment of the present invention, the codon optimization is performed according to the codon preference of E.coli.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In a preferred embodiment of the present invention, the method for obtaining the first recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a first vector to obtain a first recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the second recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 into a second vector to obtain a second recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the third recombinant vector comprises the following steps: modifying a gene sequence of formate dehydrogenase or a target gene 3 or a gene sequence shown as SEQ ID NO: 3 into a third vector to obtain a third recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 and a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: firstly, ketopantoate lactone reductase is modified to a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 into a fourth vector, and then modifying a gene sequence or a target gene 3 with formate dehydrogenase or a nucleotide sequence shown as SEQ ID NO: 3 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: firstly, modifying a gene sequence or a target gene 3 by formate dehydrogenase or a gene sequence shown as SEQ ID NO: 3 into a fourth vector, and then transforming the ketopantoate lactone reductase modified gene sequence or the target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1, ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2, a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3 into a fifth vector to obtain a fifth recombinant vector, wherein the cloning sequence of the 3 genes or the nucleotide sequences is not shown in sequence.
Another object of the present invention is to provide a recombinant engineered bacterium comprising any one or a combination of a first recombinant vector capable of expressing L-pantolactone dehydrogenase, a second recombinant vector expressing ketopantolactone reductase and a third recombinant vector expressing formate dehydrogenase;
and/or, the recombinant engineering bacteria comprise a fourth recombinant vector capable of co-expressing ketopantoate reductase and formate dehydrogenase;
and/or the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase.
In a preferred technical scheme of the invention, the recombinant engineering bacteria comprise a first recombinant vector capable of expressing L-pantoate lactone dehydrogenase and a fourth recombinant vector for co-expressing ketopantoate reductase and formate dehydrogenase.
In the preferred technical scheme of the invention, the recombinant engineering bacteria are used for preparing DL-pantoic acid lactone from L-pantoic acid lactone.
In a preferred technical scheme of the invention, the first recombinant vector comprises an L-pantoate lactone dehydrogenase modified gene sequence.
In the preferable technical scheme of the invention, the L-pantolactone dehydrogenase encoding gene is subjected to codon optimization to obtain an L-pantolactone dehydrogenase modified gene sequence.
In a preferred technical scheme of the invention, the L-pantoate lactone dehydrogenase encoding gene is derived from any one of rhodococcus erythropolis, mycobacterium, nocardia and streptomyces.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 1, and the nucleotide sequence of the target gene 1 is shown as SEQ ID NO. 1.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the second recombinant vector comprises a ketopantoate lactone reductase-modified gene sequence.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase encoding gene is subjected to codon optimization to obtain a ketopantoate lactone reductase modified gene sequence.
In a preferred technical scheme of the invention, the ketopantoate lactone reductase coding gene is derived from any one of candida magnoliae, micromonospora and streptomyces.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 2, and the nucleotide sequence of the target gene 2 is shown as SEQ ID NO. 2.
In a preferred embodiment of the present invention, the cleavage site is selected from SacI and NotI, or a combination thereof.
In a preferred embodiment of the present invention, the third recombinant vector comprises a formate dehydrogenase-modified gene sequence.
In the preferred technical scheme of the invention, the formate dehydrogenase encoding gene is subjected to codon optimization to obtain a formate dehydrogenase modified gene sequence.
In a preferred embodiment of the present invention, the formate dehydrogenase-encoding gene is derived from any one of Burkholderia, Escherichia coli and Aeromonas.
In the preferred technical scheme of the invention, the formate dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 3, and the nucleotide sequence of the target gene 3 is shown as SEQ ID NO. 3.
In a preferred technical scheme of the invention, the enzyme cutting site is selected from any one of EcoRI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the codon optimization is performed according to the codon preference of E.coli.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In a preferred embodiment of the present invention, the method for obtaining the first recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a first vector to obtain a first recombinant vector.
In a preferred embodiment of the present invention, the fourth recombinant vector comprises a ketopantoate lactone reductase modified gene sequence and a formate dehydrogenase modified gene sequence.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 and a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: firstly, ketopantoate lactone reductase is modified to a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 into a fourth vector, and then modifying a gene sequence or a target gene 3 with formate dehydrogenase or a nucleotide sequence shown as SEQ ID NO: 3 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: firstly, modifying a gene sequence or a target gene 3 by formate dehydrogenase or a gene sequence shown as SEQ ID NO: 3 into a fourth vector, and then transforming the ketopantoate lactone reductase gene sequence or the target gene 2 or the nucleotide sequence shown as SEQ ID NO: 2 into a fourth vector to obtain a fourth recombinant vector.
In the preferable technical scheme of the invention, the first recombinant vector and the fourth recombinant vector are sequentially or synchronously introduced into a host cell to obtain the recombinant engineering bacteria.
In a preferred embodiment of the present invention, the host cell is selected from any one of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, pantoea ananatis, or a combination thereof.
In a preferred technical scheme of the invention, the induction method of the recombinant engineering bacteria comprises the following steps:
s-1, inoculating the recombinant engineering bacteria into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-12h at the temperature of 30-40 ℃ and the rpm of 50-500 to obtain a first-stage seed solution;
s-2, inoculating the primary seed solution into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-10h at the temperature of 30-40 ℃ and at the speed of 50-500rpm to obtain a secondary seed solution;
s-3, inoculating the secondary seed liquid into a fermentation culture medium according to the inoculation amount of 0.1-0.5%, feeding glucose solution, maintaining the glucose concentration in the fermentation liquid to be not higher than 5g/L, performing fermentation culture for 12-20h under the aerobic condition at the pH of 7 and 37 ℃, collecting wet thalli, and performing freezing storage at the temperature of-20 ℃.
In the preferred technical scheme of the invention, the fermentation temperature of the S-1 and S-2 steps is 35-38 ℃.
In the preferred technical scheme of the invention, the rotating speed of the S-1 and S-2 steps is 100-400rpm, preferably 200-300 rpm.
In the preferable technical scheme of the invention, the LB culture medium comprises antibiotics, yeast powder, tryptone and sodium chloride.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 50-200mg/L of antibiotic, 4-6g/L of yeast powder, 8-12g/L of tryptone and 8-12g/L of sodium chloride.
In a preferred technical scheme of the invention, the antibiotic is selected from any one of penicillin, amphotericin B, nystatin, polymyxin B, streptomycin, gentamicin, tetracycline, neomycin, ampicillin and kanamycin or a combination thereof.
In the preferable technical scheme of the invention, the components of the LB culture medium comprise 50-200mg/L of antibiotics, 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
In the preferable technical scheme of the invention, the fermentation medium comprises magnesium sulfate heptahydrate, potassium dihydrogen phosphate, citric acid monohydrate, ammonium sulfate, yeast powder and glucose.
In the preferable technical scheme of the invention, the fermentation medium comprises 1-3g/L magnesium sulfate heptahydrate, 6-8g/L potassium dihydrogen phosphate, 1-3g/L citric acid monohydrate, 2-4g/L ammonium sulfate, 0.5-2g/L yeast powder and 5-7g/L glucose.
In the preferable technical scheme of the invention, the fermentation medium comprises 2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 2g/L citric acid monohydrate, 3g/L ammonium sulfate, 1g/L yeast powder and 6g/L glucose.
The invention also aims to provide a construction method of the recombinant engineering bacteria, which comprises the following steps:
(1) introducing any one of the L-pantoate lactone dehydrogenase modified gene sequence, the ketopantoate lactone reductase modified gene sequence and the formate dehydrogenase modified gene sequence or the combination sequence or the synchronization thereof into a vector to obtain a recombinant vector;
(2) and (3) introducing the obtained recombinant vector into a host cell to obtain the recombinant engineering bacterium.
In a preferable technical scheme of the invention, the recombinant vector is selected from a first recombinant vector containing a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown in SEQ ID No.1, a fourth recombinant vector ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2 and a formate dehydrogenase modified gene sequence shown in SEQ ID No. 3.
In a preferred embodiment of the present invention, the method for obtaining the first recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a first vector to obtain a first recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 and a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3 into a fourth vector to obtain a fourth recombinant vector, wherein the cloning sequence of the 2 genes or the nucleic acid sequences is not shown in sequence.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In the preferable technical scheme of the invention, the first recombinant vector and the fourth recombinant vector are sequentially or synchronously introduced into host cells to obtain the recombinant engineering bacteria.
In the preferable technical scheme of the invention, the recombinant engineering bacteria can co-express L-pantoate lactone dehydrogenase, ketopantoate lactone reductase and formate dehydrogenase.
In a preferred embodiment of the present invention, the host cell is any one selected from the group consisting of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, and pantoea ananatis.
The invention also aims to provide a preparation method of DL-pantoic acid lactone, which comprises the following steps: putting the L-pantoic acid lactone with the concentration of 10-300g/L into a reaction system of ammonium formate with the concentration of 1-100g/L and recombinant engineering bacteria with the thallus OD value of 1-5, and reacting for 20-40h under the conditions of 30-40 ℃ and pH4-7 to prepare the DL-pantoic acid lactone.
In the preferable technical scheme of the invention, the OD value of the recombinant engineering bacteria is 2-3.
In a preferred embodiment of the invention, the concentration of L-pantoic acid lactone is 100-250g/L, preferably 150-200 g/L.
In a preferred embodiment of the invention, the ammonium formate concentration is from 20 to 80g/L, preferably from 30 to 50 g/L.
In a preferred embodiment of the present invention, a metal salt or a phosphate solution may be further added to the reaction system.
In a preferred embodiment of the present invention, the metal salt or phosphate is selected from any one of zinc salt, calcium salt, copper salt, magnesium salt, sodium salt, and potassium salt, or a combination thereof, and is preferably selected from any one of magnesium chloride, zinc chloride, calcium chloride, copper chloride, sodium chloride, potassium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, magnesium sulfate, zinc sulfate, calcium sulfate, copper sulfate, magnesium phosphate, zinc phosphate, calcium phosphate, copper phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium hydrogen phosphate, calcium pyrophosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acid pyrophosphate, sodium phosphate, and sodium pyrophosphate, or a combination thereof.
In the preferred technical scheme of the invention, the concentration of the metal salt or phosphate solution is 0-50mmol/L, preferably 1-40mmol/L, and more preferably 5-30 mmol/L.
In the preferred technical scheme of the invention, Zn in the metal salt solution2+The concentration is 0-15mM, preferably 5-10 mM.
In the preferred technical scheme of the invention, Cu in the metal salt solution2+The concentration is 0-15mM, preferably 5-10 mM.
In the preferred technical scheme of the invention, Ca in the metal salt solution2+The concentration is 0-9mM, preferably 2-7 mM.
In the preferred technical scheme of the invention, HPO in inorganic salt solution4 2-Or H2PO4-The concentration is 1-50mM, preferably 5-40 mM.
In the preferred technical scheme of the invention, the temperature of the reaction system is 35-37 ℃.
In the preferred technical scheme of the invention, the pH of the reaction system is 4.5-6.5, and the pH is 5-6 preferably.
In the preferred technical scheme of the invention, the reaction time is 25-35 h.
In a preferred embodiment of the present invention, the pH adjuster is any one selected from the group consisting of ammonia water, sodium hydroxide, sodium bicarbonate, triethylamine, potassium hydroxide, sodium phosphate, sodium citrate, sodium malate, phosphate buffer, Tris buffer, sulfuric acid, and hydrochloric acid.
The invention also aims to provide application of the recombinant engineering bacteria in preparing DL-pantoic acid lactone from L-pantoic acid lactone.
In a preferred embodiment of the present invention, the use of the obtained DL-pantolactone for the preparation of a panto-compound, preferably selected from any one of D-pantolactone, D-pantothenic acid, calcium D-pantothenate, D-panthenol, pantethine.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Unless otherwise stated, the present invention was examined as follows.
1. Conversion rate
Instruments and working conditions: shimadzu LC-16 liquid chromatograph, colorSpectrum column
IE5 μm, 4.6 × 250mm, column temperature 30 deg.C, collection time 30min, wavelength 210nm, flow rate 1.0ml/min, mobile phase 0.05mol/L sodium dihydrogen phosphate water solution: methanol 60: 40.
The experimental steps are as follows: respectively diluting the reaction solution by 100 times when the conversion time T is 0 and T is M (M is any value more than 0), filtering, injecting sample with the sample amount of 10ul, and respectively recording the peak area S of L-pantolactone0And SM。
Conversion rate(M time)＝(S0-SM)/S0。
Compared with the prior art, the invention has the following beneficial technical effects:
1. the recombinant engineering bacteria of the invention are induced to express L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase, and efficiently convert L-pantolactone to generate DL-pantolactone, thereby improving the reaction efficiency and reducing the production cost.
2. The preparation method of DL-pantolactone has the advantages of high reaction efficiency, simple operation, suitability for large-scale industrial production and the like.
3. The method uses formate dehydrogenase to convert formic acid into volatile carbon dioxide, improves reaction efficiency, avoids complicated steps caused by separating and removing sodium gluconate, shortens production period, avoids producing a large amount of sodium gluconate, reduces the generation of three wastes and recovery and treatment cost thereof, and is suitable for industrial production.
Drawings
FIG. 1 is a scheme for the preparation of DL-pantolactone from L-pantolactone.
Detailed Description
The present invention will be further described with reference to the following examples.
Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and the materials, reagents and the like used therein are commercially available.
Example 1Construction of recombinant engineering bacteria
1. Design and Synthesis of Gene of interest 1-3
Step one, carrying out codon optimization on a nucleotide sequence of an L-pantoate lactone dehydrogenase encoding gene derived from Humibacter sp.BT305 (actinomycetes) according to codon preference of escherichia coli (E.coli) to obtain an L-pantoate lactone dehydrogenase modified gene sequence, wherein the nucleotide sequence is shown as SEQ ID No. 1;
performing codon optimization on a nucleotide sequence of a ketopantoate lactone reductase encoding gene derived from candida magnolifolia according to codon preference of escherichia coli (E.coli) to obtain a D-ketopantoate lactone modified gene sequence, wherein the nucleotide sequence is shown as SEQ ID No. 2;
performing codon optimization on the nucleotide sequence of the formate dehydrogenase encoding gene derived from Burkholderia according to the codon preference of escherichia coli (E.coli) to obtain a formate dehydrogenase modified gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 3;
step four, synthesizing nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and respectively adding XhoI and NdeI enzyme cutting sites, XhoI and NdeI enzyme cutting sites and EcoRI and NotI enzyme cutting sites to obtain a target gene 1, a target gene 2 and a target gene 3.
2. Construction of recombinant engineering bacteria
Step one, taking a DNA molecule of a target gene 1 as a template, carrying out PCR amplification on lpldh-for and lpldh-rev by using primers, carrying out electrophoresis separation on a 1% agarose gel, and recovering a gene fragment of the target gene 1 by using a gel recovery kit.
The primer sequence is as follows: (restriction sites underlined)
lpldh-for：GGAATTCCATATGATGAACCCGTGGTTTGAAAC
lpldh-rev：CCGCTCGAGTGCGCTTTCTGCTTCTGC
And (3) PCR system:
and (3) PCR process: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 57 deg.C for 30s, extension at 72 deg.C for 1.5min, circulating for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And step two, double-enzyme cutting pET-21a plasmid and gene fragment of genome 1 by restriction endonuclease XhoI and NdeI, recovering vector skeleton and enzyme cutting products, connecting the vector skeleton and the enzyme cutting products by using T4 DNA ligase, chemically transforming the connecting products (named as pET-21a-LPLDH) into E.coli BL21(DE3) competent cells, screening positive clones, extracting plasmids for sequencing identification, and naming the correct clones as E-21 a-LPLDH.
And step three, taking the DNA molecule of the target gene 2 as a template, carrying out PCR amplification on KPR-for and KPR-rev by adopting a primer pair, carrying out electrophoresis separation on a PCR product by using 1% agarose gel, and recovering the gene fragment of the target gene 2 by using a gel recovery kit.
The primer sequence is as follows: (restriction sites underlined)
KPR-for：
GGAATTCCATATGATGGCTAAAAACTTCTCTAACGTTGAATACC
KPR-rev：CCGCTCGAGCGGCAGGGTGTAACCACCGTCAAC
And (3) PCR system:
and (3) PCR process: pre-denaturing at 95 deg.C for 5min, denaturing at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extending at 72 deg.C for 1min, circulating for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in 4 deg.C refrigerator.
And step four, double-digesting pRSFDuet-I plasmid and gene fragment of the gene 2 by using restriction endonuclease XhoI and NdeI, recovering a vector framework and a digestion product, connecting the vector framework and the digestion product by using T4 DNA ligase, chemically transforming the ligation product (named as pRSFDuet-kpr) into E-21a-lpldh competent cells, screening positive clones, extracting plasmids, sequencing and identifying, and naming the correct clone as E-lpldh-kpr.
And step five, taking the DNA molecule of the target gene 3 as a template, performing PCR amplification on FDH-for and FDH-rev by adopting a primer pair, separating a PCR product by 1% agarose gel electrophoresis, and recovering the gene fragment of the target gene 3 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
FDH-for：CGGAATTCATGGCTACCGTTCTGTGCG
FDH-rev：ATAAGAATGCGGCCGCGGTCAGACGGTAAGACTGAGC
The PCR system was as follows:
and (3) PCR process:
pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min, circulation for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And sixthly, double-digesting pRSFDuet-kpr plasmid and the gene fragment of the genome 3 by using restriction enzymes EcoRI and NotI, recovering a vector skeleton and a digestion product, connecting the vector skeleton and the digestion product by using T4 DNA ligase, chemically converting the connection product (named as pRSFDuet-kpr-FDH) into E-lpldhh-kpr competent cells, screening positive clones, extracting the plasmid for sequencing identification, and naming the correct clone as E-lpldhh-kpr-FDH to prepare the recombinant engineering bacteria for co-expressing L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase.
The preparation of chemically transformed to competent cells is carried out by conventional methods comprising the following steps:
selecting a single colony, inoculating the single colony into an LB culture medium containing 50ug/mL ampicillin, and performing shake culture at 37 ℃ and 200rpm for 8h to obtain a seed solution;
inoculating the obtained seed liquid into 50mL LB culture medium containing 50ug/mL ampicillin, and shake culturing at 37 deg.C and 200rpm until the OD600 of the bacterial liquid is 0.2-0.4;
subpackaging 50ml of shake culture solution into 4 10ml centrifuge tubes, carrying out ice bath for 10min, centrifuging at 4000r/min for 10min at 4 ℃, and removing supernatant;
2ml of precooled 0.1mol/l CaCl per tube2Suspending thallus in the solution, carrying out ice bath for 10min, centrifuging at 4000r/min for 10min at 4 ℃, and removing supernatant;
with 1.6ml of pre-cooled 0.1mol/l CaCl containing 15% glycerol2Suspending thallus in the solution to obtain competent cell, and storing at-70 deg.C;
placing competent cells on ice, adding 1uL recombinant plasmid, mixing, placing on ice for 30min, heat-shocking at 42 deg.C for 90s, taking out, placing on ice for 2min, adding 800uL LB culture medium, culturing at 37 deg.C and 200rpm for 2h, taking out, sucking 300uL, coating on antibiotic-containing plate, and culturing overnight.
Example 2Induced expression of recombinant engineering bacteria
Inoculating the recombinant engineering bacteria prepared in the example 1 into 5mL LB culture medium according to the inoculation amount of 2%, and culturing the recombinant engineering bacteria for 10-15h at 37 ℃ and 200rpm to obtain a first-stage seed solution;
inoculating the primary seed solution into 100mL LB culture medium according to the inoculation amount of 2%, and culturing at 37 ℃ under 200rpm for 8h to obtain a secondary seed solution;
inoculating the secondary seed liquid into a fermentation tank containing 6L of fermentation culture medium according to the inoculation amount of 0.2%, wherein the fermentation culture medium comprises 2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 2g/L citric acid monohydrate, 3g/L ammonium sulfate, 1g/L yeast powder and 6g/L glucose. Adding glucose solution, maintaining the glucose concentration in the fermentation liquid to be less than 5g/L, fermenting and culturing at pH7 and 37 deg.C under aerobic condition for 18h, and freezing and storing the prepared recombinant engineering bacteria at-20 deg.C for use.
Example 3 conversion of L-pantoic acid lactone
Adding 130g of L-pantolactone and 31.5g of ammonium formate into a reaction vessel, adding water until the total volume is 1L, stirring to dissolve, adjusting the pH value of the solution to 6.2 by using 20-25% ammonia water, adding the recombinant engineering bacteria (OD is 2) collected in the example 2, placing the mixed solution at 37 ℃ and stirring for reaction for 32 hours to obtain a reaction solution, wherein the final concentration of the D-pantolactone is 91g/L, the final concentration of the L-pantolactone is 39g/L, and the conversion rate of the L-pantolactone is 70%.
Example 4 conversion of L-pantolactone
Adding 130g of L-pantolactone and 63.06g of ammonium formate into a reaction vessel, adding water until the total volume is 1L, stirring to dissolve, adjusting the pH value of the solution to 6.2 by using 20-25% ammonia water, adding the recombinant engineering bacteria (OD is 2) collected in example 2, and stirring the mixed solution at the constant temperature of 37 ℃ for 32 hours to obtain a reaction solution, wherein the final concentration of the D-pantolactone is 84.63g/L, the final concentration of the L-pantolactone is 45.37g/L, and the conversion rate of the L-pantolactone is 65.1%.
Example 5 conversion of L-pantoic acid lactone
Adding 130g of L-pantolactone, 31.5g of ammonium formate and 2g of copper chloride into a reaction vessel, adding water until the total volume is 1L, stirring to dissolve, adjusting the pH value of the solution to 6.2 by using 20-25% ammonia water, adding the recombinant engineering bacteria (OD is 2) collected in example 2, placing the mixed solution at 37 ℃ and stirring for reaction for 32 hours to obtain a reaction solution, wherein the final concentration of the D-pantolactone is 96.85g/L, the final concentration of the L-pantolactone is 33.15g/L, and the conversion rate of the L-pantolactone is 74.5%.
Example 6 conversion of L-pantolactone
Adding 130g of L-pantolactone, 31.5g of ammonium formate and 2g of zinc chloride into a reaction vessel, adding water until the total volume is 1L, stirring to dissolve, adjusting the pH value of the solution to 6.2 by using 20-25% ammonia water, adding the recombinant engineering bacteria (OD is 2) collected in the example 2, placing the mixed solution at 37 ℃ and stirring for reaction for 32 hours to obtain a reaction solution, wherein the final concentration of the D-pantolactone is 93.73g/L, the final concentration of the L-pantolactone is 36.27g/L, and the conversion rate of the L-pantolactone is 72.1%.
Example 7 conversion of L-pantolactone
Adding 130g of L-pantolactone, 31.5g of ammonium formate and 5g of disodium hydrogen phosphate into a reaction vessel, adding water until the total volume is 1L, stirring to dissolve, adjusting the pH value of the solution to 6.2 by using 20-25% ammonia water, adding the recombinant engineering bacteria (OD is 2) collected in the example 2, placing the mixed solution at 37 ℃ and stirring for reaction for 32 hours to obtain a reaction solution, wherein the final concentration of the D-pantolactone is 95.42g/L, the final concentration of the L-pantolactone is 34.58g/L, and the conversion rate of the L-pantolactone is 73.4%.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.
Sequence listing
<110> Anhui Hua constant Biotech, Inc
Bayannur Huaheng Biotechnology Co.,Ltd.
QINHUANGDAO HUAHENG BIOENGINEERING Co.,Ltd.
HEFEI HUAHENG BIOLOGICAL ENGINEERING Co.,Ltd.
<120> recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone
<150> 2020110463081
<151> 2020-09-29
<150> 2021109421716
<151> 2021-08-17
<150> 2021110851274
<151> 2021-09-16
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1176
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggctaaaa acgctttctt cgaaaccgtt gctgaagctc agcgtcgtgc taaaaaacgt 60
ctgccgaaat ctgtttacgc tgctctggtt gctggttctg aaaaaggtct gaccgttgac 120
gacaacgttg ctgctttctc tgaactgggt ttcgctccgc acgctgctgg tctgtctgac 180
aaacgtgaaa tgtctaccac catcatgggt caggacatct ctctgccggt tatgatctct 240
ccgaccggtg ttcaggctgt tcacccggac ggtgaagttg ctgttgctcg tgctgctgct 300
gctcgtggta ccgctatcgg tctgtcttct ttcgcttcta aatctatcga agaagttgct 360
gctgctaacc cgcaggtttt cttccagatg tactgggttg gttctcgtga cgttctgctg 420
cagcgtatgg aacgtgctcg tgctgctggt gctaaaggtc tgatcatcac caccgactgg 480
tctttctctt acggtcgtga ctggggttct ccgtctatcc cggaaaaaat ggacctgaaa 540
gctatgttcc agttcgctcc ggaaggtatc atgcgtccga aatggctgct ggaattcgct 600
aaaaccggta aaatcccgga cctgaccacc ccgaacctgg ctgctccggg tcagccggct 660
ccgaccttct tcggtgctta cggtgaatgg atgcagaccc cgctgccgac ctgggaagac 720
atcgcttggc tgcgtgaaca gtggggtggt ccgttcatgc tgaaaggtat catgcgtatc 780
gacgacgcta aacgtgctgt tgacgctggt gtttctgcta tctctgtttc taaccacggt 840
ggtaacaacc tggacggtac cccggctccg atccgtgttc tgccgggtat cgctgaagct 900
gttggtgacc aggttgaagt tgttctggac ggtggtatcc gtcgtggtgg tgacgttgtt 960
aaagctctgg ctctgggtgc taaagctgtt atgctgggtc gtgcttacct gtggggtctg 1020
tctgctaacg gtcaggctgg tgttgaaaac gttctggacc tgatgcgtat gggtatcgac 1080
tctggtctga tgggtctggg tcactcttct atcaccgaac tgtctccggc tgacctggtt 1140
atcccggaag gtttcacccg taccctgggt gcttct 1176
<210> 2
<211> 849
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggctaaaa acttctctaa cgttgaatac ccggctccgc cgccggctca caccaaaaac 60
gaatctctgc aggttctgga cctgttcaaa ctgaacggta aagttgcttc tatcaccggt 120
tcttcttctg gtatcggtta cgctctggct gaagctttcg ctcaggttgg tgctgacgtt 180
gctatctggt acaactctca cgacgctacc ggtaaagctg aagctctggc taaaaaatac 240
ggtgttaaag ttaaagctta caaagctaac gtttcttctt ctgacgctgt taaacagacc 300
atcgaacagc agatcaaaga cttcggtcac ctggacatcg ttgttgctaa cgctggtatc 360
ccgtggacca aaggtgctta catcgaccag gacgacgaca aacacttcga ccaggttgtt 420
gacgttgacc tgaaaggtgt tggttacgtt gctaaacacg ctggtcgtca cttccgtgaa 480
cgtttcgaaa aagaaggtaa aaaaggtgct ctggttttca ccgcttctat gtctggtcac 540
atcgttaacg ttccgcagtt ccaggctacc tacaacgctg ctaaagctgg tgttcgtcac 600
ttcgctaaat ctctggctgt tgaattcgct ccgttcgctc gtgttaactc tgtttctccg 660
ggttacatca acaccgaaat ctctgacttc gttccgcagg aaacccagaa caaatggtgg 720
tctctggttc cgctgggtcg tggtggtgaa accgctgaac tggttggtgc ttacctgttc 780
ctggcttctg acgctggttc ttacgctacc ggtaccgaca tcatcgttga cggtggttac 840
accctgccg 849
<210> 3
<211> 1152
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggctaccg ttctgtgcgt tctgtacccg gacccggttg acggttaccc gccgcactac 60
gttcgtgaca ccatcccggt tatcacccgt tacgctgacg gtcagaccgc tccgaccccg 120
gctggtccgc cgggtttccg tccgggtgaa ctggttggtt ctgtttctgg tctgggtctg 180
cgtggttacc tggaagctca cggtcacacc ctgatcgtta cctctgacaa agacggtccg 240
gactctgaat tcgaacgtcg tctgccggac gctgacgttg ttatctctca gccgttctgg 300
ccggcttacc tgaccgctga acgtatcgct cgtgctccga aactgcgtct ggctctgacc 360
gctggtatcg gttctgacca cgttgacctg gacgctgctg ctcgtgctca catcaccgtt 420
gctgaagtta ccggttctaa ctctatctct gttgctgaac acgttgttat gaccaccctg 480
gctctggttc gtaactacct gccgtctcac gctatcgctc agcagggtgg tatgaacatc 540
gctgactgcg tttctcgttc ttacgacgtt gaaggtatgc acttcggtac cgttggtgct 600
ggtcgtatcg gtctggctgt tctgcgtcgt ctgccgttcg gtctgcacct gcactacacc 660
cagcgtcacc gtctggacgc tgctatcgaa caggaactgg gtctgaccta ccacgctgac 720
ccggcttctc tggctgctgc tgttgacatc gttaacctgc agatcccgct gtacccgtct 780
accgaacacc tgttcgacgc tgctatgatc gctcgtatga aacgtggtgc ttacctgatc 840
aacaccgctc gtgctaaact ggttgaccgt gacgctgttg ttcgtgctgt tacctctggt 900
cacctggctg gttacggtgg tgacgtttgg ttcccgcagc cggctccggc tgaccacccg 960
tggcgtgcta tgccgttcaa cggtatgacc ccgcacatct ctggtacctc tctgtctgct 1020
caggctcgtt acgctgctgg taccctggaa atcctgcagt gctggttcga cggtcgtccg 1080
atccgtaacg aatacctgat cgttgacggt ggtaccctgg ctggtaccgg tgctcagtct 1140
taccgtctga cc 1152
Claims (10)
1. A recombinant vector is selected from recombinant vectors which contain a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown as SEQ ID No.1, a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown as SEQ ID No.2 and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown as SEQ ID No. 3.
2. The recombinant vector according to claim 1, wherein the recombinant vector optionally comprises any one of a fourth recombinant vector and a fifth recombinant vector, wherein the fourth recombinant vector comprises a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2 and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown in SEQ ID No.3, the fifth recombinant vector comprises a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown in SEQ ID No.1, a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown in SEQ ID No.2 and a nucleotide sequence of a formate dehydrogenase modified gene sequence shown in SEQ ID No. 3.
3. A recombinant engineered bacterium comprising any one or a combination of a first recombinant vector capable of expressing L-pantolactone dehydrogenase, a second recombinant vector expressing ketopantolactone reductase and a third recombinant vector expressing formate dehydrogenase;
and/or, the recombinant engineering bacteria comprise a fourth recombinant vector capable of co-expressing ketopantoate reductase and formate dehydrogenase;
and/or the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase, ketopantoate lactone reductase and formate dehydrogenase.
4. The recombinant engineered bacterium of claim 3, wherein the recombinant engineered bacterium comprises a first recombinant vector capable of expressing L-pantolactone dehydrogenase, and a fourth recombinant vector co-expressing ketopantolactone reductase and formate dehydrogenase.
5. A construction method of recombinant engineering bacteria comprises the following steps:
(1) introducing any one of the L-pantoate lactone dehydrogenase modified gene sequence, the ketopantoate lactone reductase modified gene sequence and the formate dehydrogenase modified gene sequence or the combination sequence or the synchronization thereof into a vector to obtain a recombinant vector;
(2) and (3) introducing the obtained recombinant vector into a host cell to obtain the recombinant engineering bacterium.
6. A preparation method of DL-pantoic acid lactone comprises the following steps: putting the L-pantoic acid lactone with the concentration of 10-300g/L into a reaction system of ammonium formate with the concentration of 1-100g/L and recombinant engineering bacteria with the thallus OD value of 1-5, and reacting for 20-40h under the conditions of 30-40 ℃ and pH4-7 to prepare the DL-pantoic acid lactone.
7. The preparation method according to claim 6, wherein the concentration of L-pantolactone is 100-250g/L, preferably 150-200 g/L.
8. The process according to any one of claims 6 to 7, wherein the ammonium formate concentration is between 20 and 80g/L, preferably between 30 and 50 g/L.
9. The production process according to any one of claims 6 to 8, wherein a metal salt or a phosphate solution is further added to the reaction system; preferably, the metal salt or phosphate is selected from any one of or a combination of zinc salt, calcium salt, copper salt, magnesium salt, sodium salt, potassium salt, and preferably any one of or a combination of magnesium chloride, zinc chloride, calcium chloride, copper chloride, sodium chloride, potassium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, magnesium sulfate, zinc sulfate, calcium phosphate, copper phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium hydrogen phosphate, calcium pyrophosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acid pyrophosphate, sodium phosphate, and sodium pyrophosphate.
10. The use of a recombinant engineered bacterium according to any one of claims 3-4 in the preparation of DL-pantoic acid lactone from L-pantoic acid lactone.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020110463081 | 2020-09-29 | ||
| CN202011046308.1A CN113106129A (en) | 2020-09-29 | 2020-09-29 | Preparation method of D pantolactone with high conversion rate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114457129A true CN114457129A (en) | 2022-05-10 |
Family
ID=76709094
Family Applications (13)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011046308.1A Withdrawn CN113106129A (en) | 2020-09-29 | 2020-09-29 | Preparation method of D pantolactone with high conversion rate |
| CN202110940857.1A Pending CN114426977A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110941962.7A Pending CN114426978A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942787.3A Pending CN114426979A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942171.6A Pending CN115044594A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111084984.2A Pending CN115011623A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111087303.8A Pending CN114774446A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111087987.1A Pending CN115029364A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111085127.4A Pending CN115011624A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111133610.5A Withdrawn CN114457128A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111135111.XA Withdrawn CN114457130A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111132818.5A Withdrawn CN114457127A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111135083.1A Withdrawn CN114457129A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
Family Applications Before (12)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011046308.1A Withdrawn CN113106129A (en) | 2020-09-29 | 2020-09-29 | Preparation method of D pantolactone with high conversion rate |
| CN202110940857.1A Pending CN114426977A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110941962.7A Pending CN114426978A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942787.3A Pending CN114426979A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942171.6A Pending CN115044594A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111084984.2A Pending CN115011623A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111087303.8A Pending CN114774446A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111087987.1A Pending CN115029364A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111085127.4A Pending CN115011624A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111133610.5A Withdrawn CN114457128A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111135111.XA Withdrawn CN114457130A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111132818.5A Withdrawn CN114457127A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (13) | CN113106129A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604371B (en) * | 2021-08-03 | 2023-06-16 | 合肥学院 | Yeast strain HF-21 and method for preparing D-pantolactone by double-phase catalysis thereof |
| CN115044638A (en) * | 2022-07-21 | 2022-09-13 | 上海奥萝拉医药科技有限公司 | Preparation method of D-pantolactone with high optical selectivity |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388373A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Construction of escherichia coli system with coexpression of carbonyl reductase Sys1 and glucose dehydrogenase Sygdh |
| WO2017016442A1 (en) * | 2015-07-24 | 2017-02-02 | 雅本化学股份有限公司 | Coenzyme regeneration system and preparation method thereof |
| CN110396505A (en) * | 2019-05-05 | 2019-11-01 | 杭州鑫富科技有限公司 | Ketone group pantoic acid lactone reductase and its application |
| CN110408604A (en) * | 2019-08-14 | 2019-11-05 | 华东理工大学 | The formic dehydrogenase mutant that substrate affinity and coenzyme affinity improve |
| CN110423717A (en) * | 2019-05-05 | 2019-11-08 | 杭州鑫富科技有限公司 | Multienzyme recombinant cell and multienzyme cascade the method for catalyzing and synthesizing D-pantoyl lactone |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293194C (en) * | 2004-08-03 | 2007-01-03 | 江南大学 | D-Pantothe internal ester hydrolase c DNA and its clone expression and application |
| EP1838837B1 (en) * | 2004-12-22 | 2015-04-15 | The Michigan Biotechnology Institute | Recombinant microorganisms for increased production of organic acids |
| CN1935977B (en) * | 2006-10-19 | 2010-06-09 | 华东理工大学 | Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid |
| CN106676051B (en) * | 2016-10-31 | 2019-07-05 | 中国科学院微生物研究所 | It is a kind of to prepare the method and its application for efficiently synthesizing pantothenic acid genetic engineering bacterium |
| CN109456908A (en) * | 2018-11-15 | 2019-03-12 | 江南大学 | A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase and its construction method and application |
| CN115044638A (en) * | 2022-07-21 | 2022-09-13 | 上海奥萝拉医药科技有限公司 | Preparation method of D-pantolactone with high optical selectivity |
-
2020
- 2020-09-29 CN CN202011046308.1A patent/CN113106129A/en not_active Withdrawn
-
2021
- 2021-08-17 CN CN202110940857.1A patent/CN114426977A/en active Pending
- 2021-08-17 CN CN202110941962.7A patent/CN114426978A/en active Pending
- 2021-08-17 CN CN202110942787.3A patent/CN114426979A/en active Pending
- 2021-08-17 CN CN202110942171.6A patent/CN115044594A/en active Pending
- 2021-09-16 CN CN202111084984.2A patent/CN115011623A/en active Pending
- 2021-09-16 CN CN202111087303.8A patent/CN114774446A/en active Pending
- 2021-09-16 CN CN202111087987.1A patent/CN115029364A/en active Pending
- 2021-09-16 CN CN202111085127.4A patent/CN115011624A/en active Pending
- 2021-09-27 CN CN202111133610.5A patent/CN114457128A/en not_active Withdrawn
- 2021-09-27 CN CN202111135111.XA patent/CN114457130A/en not_active Withdrawn
- 2021-09-27 CN CN202111132818.5A patent/CN114457127A/en not_active Withdrawn
- 2021-09-27 CN CN202111135083.1A patent/CN114457129A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388373A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Construction of escherichia coli system with coexpression of carbonyl reductase Sys1 and glucose dehydrogenase Sygdh |
| WO2017016442A1 (en) * | 2015-07-24 | 2017-02-02 | 雅本化学股份有限公司 | Coenzyme regeneration system and preparation method thereof |
| CN110396505A (en) * | 2019-05-05 | 2019-11-01 | 杭州鑫富科技有限公司 | Ketone group pantoic acid lactone reductase and its application |
| CN110423717A (en) * | 2019-05-05 | 2019-11-08 | 杭州鑫富科技有限公司 | Multienzyme recombinant cell and multienzyme cascade the method for catalyzing and synthesizing D-pantoyl lactone |
| CN110408604A (en) * | 2019-08-14 | 2019-11-05 | 华东理工大学 | The formic dehydrogenase mutant that substrate affinity and coenzyme affinity improve |
Non-Patent Citations (2)
| Title |
|---|
| DAYONG SI 等: "L-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of L-pantoyl lactone", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 95, pages 433 * |
| 汪钊 等: "化学酶法合成D-泛解酸内酯的研究进展", 发酵科技通讯, no. 04, pages 193 - 198 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114457127A (en) | 2022-05-10 |
| CN114457130A (en) | 2022-05-10 |
| CN114426979A (en) | 2022-05-03 |
| CN115029364A (en) | 2022-09-09 |
| CN115044594A (en) | 2022-09-13 |
| CN115011624A (en) | 2022-09-06 |
| CN113106129A (en) | 2021-07-13 |
| CN114774446A (en) | 2022-07-22 |
| CN114426977A (en) | 2022-05-03 |
| CN115011623A (en) | 2022-09-06 |
| CN114457128A (en) | 2022-05-10 |
| CN114426978A (en) | 2022-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108424900B (en) | Nitrilase mutant and construction method and application thereof | |
| CN106754806B (en) | Improved transaminase and application thereof in preparation of (R) -3-aminobutanol | |
| CN114457129A (en) | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone | |
| CN106868030B (en) | Recombinant vector, engineering bacterium containing recombinant vector and application of recombinant vector in production of alpha-ketoglutaric acid | |
| CN114621968A (en) | Tetrahydropyrimidine biosynthesis gene cluster, mutant and method for preparing tetrahydropyrimidine | |
| CN114874964A (en) | Construction method and application of recombinant escherichia coli for high yield of 2' -fucosyllactose | |
| CN115927513A (en) | Method for preparing beta-nicotinamide mononucleotide by using biological enzyme | |
| CN105420265B (en) | Genetically engineered acetic acid bacteria for over-expression of ATP enzyme and construction method and application thereof | |
| CN109852593A (en) | A kind of recombination ketoreductase and preparing the application in R-3- hydroxybutyric acid and its salt | |
| CN114525291B (en) | Carbonyl reductase and method for preparing (R) -4-chloro-3-hydroxybutyrate by using same | |
| CN114657199B (en) | Recombinant engineering bacterium and application thereof in preparation of D-pantothenic acid | |
| CN115725520A (en) | Preparation method of glutathione synthetase and method for catalytically producing glutathione | |
| CN114657198B (en) | Recombinant engineering bacterium and application thereof in preparation of panto-compound | |
| CN115975964A (en) | High-activity ketopantolactone reductase mutant and coding gene and application thereof | |
| CN114657200B (en) | Recombinant engineering bacterium and method for preparing D-pantoic acid by using same | |
| CN117866868B (en) | L-high proline production strain and construction method and application thereof | |
| CN114958703B (en) | Recombinant bacterium for synthesizing succinic acid by utilizing grease, construction method and application thereof | |
| CN111575258B (en) | Carbonyl reductase EbSDR8 mutant and construction method and application thereof | |
| CN117384933B (en) | Strain for producing 3-hydroxy propionic acid by utilizing xylose, construction method and application thereof | |
| CN114480234A (en) | Strain for producing L-alanine by high-efficiency fermentation and construction method and application thereof | |
| CN118685378A (en) | Carbonyl reductase mutant and application thereof in synthesis of 4-AA chiral intermediate | |
| CN116179380A (en) | Yarrowia lipolytica engineering bacterium WSMHP capable of producing pyruvic acid in high yield, construction method and application | |
| CN116179459A (en) | Multienzyme synergistic expression recombinant genetic engineering bacterium and application thereof | |
| CN117965484A (en) | Construction method and application of engineering escherichia coli for efficiently synthesizing lactoyl-N-fucosyl pentasaccharide-V | |
| CN118374465A (en) | Imine reductase and method for preparing (S) -nicotine by using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20220510 |
|
| WW01 | Invention patent application withdrawn after publication |