WO2024066819A1 - 发酵生产3-羟基丙酸和丙烯酸的方法 - Google Patents

发酵生产3-羟基丙酸和丙烯酸的方法 Download PDF

Info

Publication number
WO2024066819A1
WO2024066819A1 PCT/CN2023/114590 CN2023114590W WO2024066819A1 WO 2024066819 A1 WO2024066819 A1 WO 2024066819A1 CN 2023114590 W CN2023114590 W CN 2023114590W WO 2024066819 A1 WO2024066819 A1 WO 2024066819A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide sequence
gene
seq
protein
expresses
Prior art date
Application number
PCT/CN2023/114590
Other languages
English (en)
French (fr)
Inventor
孙宇
杨令霞
Original Assignee
北京绿色康成生物技术有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 北京绿色康成生物技术有限公司 filed Critical 北京绿色康成生物技术有限公司
Publication of WO2024066819A1 publication Critical patent/WO2024066819A1/zh

Links

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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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)
    • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
    • C12Y101/05003—Glycerol-3-phosphate dehydrogenase (1.1.5.3)
    • 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)
    • C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
    • 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)
    • C12Y102/01004—Aldehyde dehydrogenase (NADP+) (1.2.1.4)
    • 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)
    • C12Y102/01005—Aldehyde dehydrogenase [NAD(P)+] (1.2.1.5)
    • 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)
    • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03021—Glycerol-1-phosphatase (3.1.3.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01028—Propanediol dehydratase (4.2.1.28)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the technical field of genetic engineering and biological fermentation, and in particular relates to a method for producing 3-hydroxypropionic acid and acrylic acid by fermentation.
  • 3-Hydroxypropionic acid is an important platform compound and one of the 12 high-value-added bio-based platform chemicals announced by the U.S. Department of Energy.
  • 3-hydroxypropionic acid (3-HP) as a monomer
  • high-performance biodegradable material poly (3-hydroxypropionic acid) (PHP) can be synthesized.
  • 3-hydroxypropionic acid can be further used to synthesize acrylic acid, 1,3-propylene glycol, malonic acid, propylenediamine, etc.
  • 3-Hydroxypropionic acid can also be used as an additive and preservative for food or feed.
  • Acrylic acid is a widely used chemical and a key raw material for the synthesis of polyacrylic acid (salt).
  • Polyacrylic acid has a strong water absorption capacity and is widely used in baby diapers, sanitary napkins, incontinence products, etc.
  • the production of acrylic acid mainly relies on chemical methods, usually obtained by the segmented oxidation of propylene. This process easily produces a large amount of impurities, which will affect the safety of the product in the preparation of sanitary products and trauma materials.
  • the process relies on fossil resources, and the production process requires high temperature and high pressure, which produces a large amount of pollutants and carbon dioxide emissions. Therefore, the development of green and safe acrylic acid production technology and the direct production of acrylic acid from cheap biomass raw materials have important industrial application potential.
  • the object of the present invention is to provide a method for producing 3-hydroxypropionic acid and acrylic acid by fermentation.
  • the present invention is conceived as follows: by introducing the synthetic pathway of glycerol and 3-hydroxypropionic acid into Corynebacterium glutamicum, efficient conversion of 3-hydroxypropionic acid from various cheap carbon sources such as glucose, sucrose, molasses, etc. is achieved.
  • the present invention also provides a method for directly converting a fermentation broth containing 3-hydroxypropionic acid to produce acrylic acid.
  • the present invention provides a recombinant Corynebacterium glutamicum, wherein the aldehyde dehydrogenase gene ald in the recombinant Corynebacterium glutamicum is strengthened, and the glyceraldehyde-3-phosphate dehydrogenase gene gapA is weakened.
  • the gene enhancement method can be selected from the following 1) to 4), or any combination thereof:
  • the method of gene weakening can be selected from the following a) to c), or any combination thereof:
  • Corynebacterium glutamicum also overexpresses the diol dehydratase gene pduCDEGH.
  • Corynebacterium glutamicum also overexpresses the 3-phosphate dehydrogenase gene gpd and the glycerol 3-phosphatase gene gpp.
  • the gene ald is:
  • A2 a nucleotide sequence in which one or more nucleotides are substituted, deleted and/or added to the nucleotide sequence shown in SEQ ID NO:1 and the nucleotide sequence expresses a protein with the same function;
  • A3 a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1 ⁇ SSPE solution containing 0.1% SDS or a 0.1 ⁇ SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; or
  • A4) is a nucleotide sequence that has more than 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence of A1), A2) or A3) and expresses a protein with the same function.
  • the gene gapA is:
  • B4) is a nucleotide sequence that has more than 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence of B1), B2) or B3) and expresses a protein with the same function.
  • the gene pduCDEGH is:
  • C4) is a nucleotide sequence that has more than 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence of C1), C2) or C3) and expresses a protein with the same function.
  • the gene gpd is:
  • D3 a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1 ⁇ SSPE solution containing 0.1% SDS or a 0.1 ⁇ SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or
  • D4) is a nucleotide sequence that has more than 90%, for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence of D1), D2) or D3) and expresses a protein with the same function.
  • the gene gpp is:
  • E2 A nucleotide sequence in which one or more nucleotides are replaced, deleted and/or added to the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence expresses a protein with the same function;
  • E3 a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:5 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1 ⁇ SSPE solution containing 0.1% SDS or a 0.1 ⁇ SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or
  • E4) has more than 90%, for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence of E1), E2) or E3) and expresses a nucleotide sequence of the same functional protein.
  • the present invention provides a recombinant Corynebacterium glutamicum having enhanced, for example, overexpressed, aldehyde dehydrogenase, and weakened glyceraldehyde-3-phosphate dehydrogenase.
  • the aldehyde dehydrogenase comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:1 or an amino acid sequence having more than 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology thereto and having the same function.
  • the gene encoding the glyceraldehyde-3-phosphate dehydrogenase is a gene comprising SEQ ID NO: 2.
  • the nucleotide sequence of the gene or its homologous gene is shown.
  • the recombinant Corynebacterium glutamicum further has enhanced, for example, overexpressed, diol dehydratase, 3-phosphate dehydrogenase and/or glycerol 3-phosphatase.
  • the diol dehydratase comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:3 or an amino acid sequence having more than 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology thereto and having the same function.
  • the 3-phosphate dehydrogenase comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:4 or an amino acid sequence that has more than 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology thereto and has the same function.
  • the glycerol 3-phosphatase comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:5 or an amino acid sequence that has more than 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology thereto and has the same function.
  • the recombinant Corynebacterium glutamicum is constructed by overexpressing genes ald, pduCDEGH, gpd and gpp in Corynebacterium glutamicum, and replacing the start codon of gene gapA from ATG to GTG.
  • the present invention provides a method for constructing the recombinant Corynebacterium glutamicum, and conventional genetic engineering methods can be used to transform or modify the above genes.
  • the method comprises enhancing, for example, overexpressing, the aldehyde dehydrogenase described herein and attenuating the glyceraldehyde-3-phosphate dehydrogenase described herein in Corynebacterium glutamicum.
  • the method further comprises enhancing, eg, overexpressing, a diol dehydratase, a 3-phosphate dehydrogenase, and/or a glycerol 3-phosphatase as described herein.
  • the present invention provides the use of the recombinant Corynebacterium glutamicum in the fermentation production of 3-hydroxypropionic acid.
  • Corynebacterium glutamicum is used to ferment and produce 3-hydroxypropionic acid using a cheap carbon source as a raw material.
  • the cheap carbon source can be selected from at least one of molasses, sucrose, glucose, cellobiose and the like.
  • the present invention provides a method for producing acrylic acid, wherein the recombinant Corynebacterium glutamicum is fermented and cultured, and the 3-hydroxypropionic acid produced by the fermentation is acidified and heated for dehydration to obtain acrylic acid.
  • the present invention provides a method for producing acrylic acid, comprising the step of fermenting and culturing the recombinant Corynebacterium glutamicum described herein. In one embodiment, the method further comprises an acidification and/or heating dehydration step.
  • the method comprises: adding concentrated phosphoric acid solution to the fermentation broth containing 3-hydroxypropionic acid, adjusting the pH value to 1-2, and heating the fermentation broth to 100-140° C. for reaction for 1-5 hours (preferably 140° C. for reaction for 2 hours).
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention provides a recombinant Corynebacterium glutamicum, in which the aldehyde dehydrogenase gene ald of the microorganism itself is upregulated, the expression of glyceraldehyde-3-phosphate dehydrogenase gapA is downregulated, and the heterologous diol dehydratase gene pduCDEGH, heterologous 3-phosphate dehydrogenase gpd and glycerol 3-phosphatase gpp are expressed at the same time.
  • the recombinant microorganism is fermented and cultured in a shake flask or a fermenter with an organic carbon source such as glucose as a substrate to obtain 3-hydroxypropionic acid.
  • the 3-hydroxypropionic acid in the fermentation culture medium is acidified and heated and dehydrated to further obtain acrylic acid.
  • the recombinant microorganism of the present invention can use cheap glucose, sucrose, molasses, etc. as raw materials to efficiently produce 3-hydroxypropionic acid and acrylic acid, and the production process is green, safe and simple, and has good market application prospects.
  • transformation and “recombination” refer to strains artificially changed by biological means, which have one or more changes compared to the initial strain before the change, such as gene deletion, amplification or mutation, so as to have a changed biological property such as improved production performance.
  • the initial strain can be a natural strain or other recombinant strain to which the recombinant is to be performed.
  • aldehyde dehydrogenase as used herein can be oxidized aliphatic and aromatic aldehydes to become corresponding acids in the presence of NAD + .
  • the aldehyde dehydrogenase encoded by the endogenous ald gene of Corynebacterium glutamicum as used herein can efficiently catalyze the oxidation of 3-hydroxypropionic acid.
  • glycosylaldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to produce 1,3-bisphosphoglycerate.
  • diol dehydratase can catalyze the dehydration of 1,2-diol.
  • the diol dehydratase encoded by the pduCDEGH gene of the present invention can catalyze glycerol to produce 3-hydroxypropanal.
  • 3-phosphate dehydrogenase catalyzes the production of 3-phosphoglycerol from dihydroxyacetone phosphate.
  • glycerol 3-phosphatase catalyzes the production of glycerol from glycerol 3-phosphate.
  • proteins with the same function refer to proteins that are capable of catalyzing the same reaction and may have different structures and different origins.
  • heterologous . . . gene refers to a gene from a different species that did not originate from a common ancestor during evolution.
  • Gene expression refers to the transfer of a DNA region operably linked to an appropriate regulatory region, particularly a promoter, into The RNA can be translated into biologically active proteins or peptides.
  • endogenous ... gene refers to a cell's or organism's own gene, or a gene that is identical to a cell's or organism's own gene.
  • the expressions "a gene ... is upregulated” and "upregulated expression of" refer to an increase in expression of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more compared to a reference having such expression (e.g., an initial strain or a wild-type strain).
  • the expressions "the expression of" is downregulated” and “downregulate the expression of" mean that the expression is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable, compared to a reference (e.g., an initial strain or a wild-type strain) having such expression.
  • a reference e.g., an initial strain or a wild-type strain
  • the expressions "having enhanced expression" and “enhanced expression of" refer to an increase in expression of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more compared to a reference having such expression (e.g., an initial strain or a wild-type strain).
  • overexpression refers to an increase in the expression level of a gene relative to the level before genetic manipulation, such as an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more.
  • Methods for overexpressing genes are well known in the art, such as, but not limited to, using strong promoters, increasing gene copy number, enhancers, etc.
  • Increasing gene copy number can be, for example, but not limited to, achieved by introducing one or more copies of an exogenous gene or endogenous gene, such as, for example, by an expression vector or by integration into a genome.
  • enhanced ... enzyme/protein and “enhancing ... enzyme/protein” refer to a higher level and/or activity of a certain enzyme/protein expressed in a cell or tissue relative to the level before genetic manipulation, such as an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more.
  • a variety of ways to enhance proteins are known in the art, including but not limited to overexpression, mutants with increased activity, etc.
  • gene enhancement refers to increasing the expression level of a gene (when it is a protein coding gene) or the regulatory performance (when it is a regulatory element) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more.
  • Various gene enhancement methods are known in the art, including but not limited to the use of strong promoters, increasing gene copy number, enhancers, etc.
  • a gene is weakened and “gene weakening” refer to making the expression level of a gene
  • the attenuation of a gene is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable.
  • a variety of ways of weakening a gene are known in the art, including, for example, inhibiting gene expression such as knocking down (e.g., using small interfering RNA), using a weak promoter (when the gene is a polypeptide encoding gene), etc.; knocking out a gene, deleting part or all of a gene sequence; mutating certain sites in a gene, such as a coding sequence, to reduce gene expression or regulatory activity or the activity of an expression product, etc.
  • Attenuated ... enzyme/protein means that the level of a certain enzyme/protein expressed in a cell or tissue is lower, for example, reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable, relative to the level before genetic manipulation.
  • the terms “homology” and “identity” are used interchangeably and refer to the degree of invariance of a nucleotide sequence, which can be detected by comparing the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide. Sequence homology can be determined by a standard alignment algorithm program using a default gap penalty established by each supplier. Substantially homologous nucleic acid molecules typically hybridize full-length nucleic acids or at least or at least about 70%, 80% or 90% of the full-length nucleic acid molecule of interest under moderate stringency conditions or under highly stringent conditions.
  • the present invention also encompasses nucleic acid molecules containing degenerate codons replacing codons in hybridizing nucleic acid molecules. Whether any two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" nucleotide sequences can be determined using known computer algorithms such as BLASTN, FASTA, DNAStar and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison WI, USA).
  • the 3-phosphate dehydrogenase gene gpd (the gene sequence is shown in SEQ ID NO: 4) and the glycerol 3-phosphatase gene gpp (the gene sequence is shown in SEQ ID NO: 5) were artificially synthesized.
  • the gpd gene fragment was used as a template and gpd-F (5′-attaagcttgcatgcctgcactttaagaaggagatataccaatggctgctgctgatag-3′) and gpd-R (5′-ggtatatctccttctttaaagtaatcttcatgtagatctaa-3′) were used as primers for PCR to obtain a gpd-1 fragment of about 1.26 kb and PCR purification was performed.
  • the gpp gene fragment was used as a template and gpp-F (5′-attaagcttgcatgcctgcactttaagaaggagatataccaatgggattgactactaaacc-3′) and gpp-R (5′-ggtatatctccttctttaaagttaccatttcaacagatcgt-3′) were used as primers for PCR to obtain a gpp-1 fragment of about 0.8 kb and PCR purification.
  • the pXMJ19 plasmid (purchased from Addgene) was double-digested with PstI and XbaI, and the gpd-1 fragment and gpp-1 fragment obtained by the above purification were ligated to pXMJ19 in one step using the Gibson Assembly Kit (NEB).
  • the obtained recombinant plasmid was named pXMJ-gpd-gpp.
  • PCR was performed with pdu-F (5′-ggatccccgggtaccgagctctttaagaaggagatataccatgagatcgaaagatttga-3′) and pdu-R (5′-caaaacagccaagctgaattttaagcatggcgatcccgaa-3′) as primers to obtain a 5.1 kb pduCDEGH operon fragment including diol dehydratase and its activator (sequence shown in SEQ ID NO:3) and PCR product purification was performed.
  • the pXMJ-gpd-gpp plasmid was double-digested with KpnI and EcoRI, and the pduCDEGH fragment obtained by the above purification was ligated to pXMJ-gpd-gpp in one step using the Gibson Assembly Kit (NEB), and the obtained recombinant plasmid was named pXMJ-gpd-gpp-pduCDEGH.
  • the plasmid pXMJ-gpd-gpp-pduCDEGH was introduced into Corynebacterium glutamicum ATCC 13032 by electroporation, and the recombinant bacteria were screened on LB plates containing 10 mg/L chloramphenicol and named Cg/pXMJ-gpd-gpp-pduCDEGH.
  • aldehyde dehydrogenase gene ald in Corynebacterium glutamicum, and the production of 3-hydroxypropionic acid can be significantly increased by enhancing the expression of ald.
  • Corynebacterium glutamicum ATCC13032 The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and ald-F (5′-acagctatgacatgattacgtataagaaggagatatacaatgactgtctacgcaaatcc-3′) and ald-R (5′-tagaggatccccgggtaccgagtcttgtcaggccaaccca-3′) were used as primers for PCR to obtain an ald gene fragment of about 1.2 kb and PCR product purification.
  • Plasmid pEC-K18mob2 (purchased from Addgene) was double-digested with EcoRI and SacI, and the ald fragment obtained by the above purification was connected to pEC-K18mob2 using the Gibson Assembly kit (NEB). The obtained recombinant plasmid was named pEC-ald.
  • pEC-ald was transferred into Corynebacterium glutamicum Cg/pXMJ-gpd-gpp-pduCDEGH by electroporation, and the recombinant bacteria were screened on LB plates containing 50 mg/L kanamycin and named Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald.
  • the strain Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald and the control strain Cg/pXMJ-gpd-gpp-pduCDEGH were inoculated into the fermentation medium for culture.
  • the fermentation temperature was 30°C and the rotation speed was 200 rpm.
  • the OD 600 of the bacteria reached 2-3, 0.1 mM IPTG was added for induction, and the fermentation was continued for 48 hours.
  • the strain Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald could produce 35.2 g/L of 3-hydroxypropionic acid, while the control strain Cg/pXMJ-gpd-gpp-pduCDEGH only produced 10.1 g/L of 3-hydroxypropionic acid. This indicates that upregulating the expression of ald can significantly increase the yield of 3-hydroxypropionic acid.
  • the production of 3-hydroxypropionic acid can also be significantly improved by up-regulating the expression of ald on the Corynebacterium glutamicum genome.
  • the promoter of the ald gene of Corynebacterium glutamicum ATCC13032 was replaced with the strong promoter tac (5′-ttgacaattaatcatcggctcgtataatg-3′) to construct the recombinant strain Cg-ald.
  • the recombinant strain obtained by electrotransferring plasmid pXMJ-gpd-gpp-pduCDEGH into strain Cg-ald was named Cg-ald/pXMJ-gpd-gpp-pduCDEGH.
  • the production of 3-hydroxypropionic acid of strain Cg-ald/pXMJ-gpd-gpp-pduCDEGH reached 30.4g/L, which was significantly higher than the control strain (10.1g/L).
  • strain Cg-ald The construction process of strain Cg-ald is as follows: using the genome of Corynebacterium glutamicum ATCC13032 as a template, PCR was performed with ald-up-F1 (5′-tcatcagcgatggactcatgaacaa-3′) and ald-up-R1 (5′-ttgacaattaatcatcggctcgtataatgcttttgaaaggctttcggcg-3′) as primers to obtain the gene ald-up1 fragment about 1.0 kb upstream of ald and purify the PCR product.
  • ald-up-F1 5′-tcatcagcgatggactcatgaacaa-3′
  • ald-up-R1 5′-ttgacaattaatcatcggctcgtataatgctttttgaaaggctttcggcg-3′
  • Corynebacterium glutamicum ATCC13032 was used as a template, and ald-down-F1 (5′-cattatacgagccgatgattaattgtcaattattacccctgttcgggtg-3′) and ald-down-R1 (5′-atcaccgtgcgtatcccaac-3′) were used as primers for PCR to obtain the gene ald-down1 fragment of about 1.0 kb downstream of gapA and purify the PCR product.
  • ald-down-F1 5′-cattatacgagccgatgattaattgtcaattattacccctgttcgggtg-3′
  • ald-down-R1 5′-atcaccgtgcgtatcccaac-3′
  • the plasmid pK18mobsacB (purchased from Addgene) was double-digested with EcoRI and XbaI, and the ald-down1 fragment and ald-up1 fragment obtained by the above purification were connected to pK18mobsacB using the Gibson Assembly kit (NEB).
  • the obtained recombinant plasmid was named pK18-ald.
  • pK18-ald was transformed into Corynebacterium glutamicum ATCC 13032 by electroporation, and the correct recombinant strain was obtained by secondary screening and sequencing, named Cg-ald.
  • gapA was weakened by replacing ATG with the rare start codon GTG.
  • the genome of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR was performed with gapA-up-F1 (5′-acagctatgacatgattacgcgatgtcggtggaaaccagt-3′) and gapA-up-R1 (5′-gagacacaacgtgaccattcgtgttggtattaacgg-3′) as primers to obtain the gapA-up1 gene fragment of about 1.0 kb upstream of gapA and PCR product purification was performed.
  • gapA-down-F1 (5′-gaatggtcacgttgtgtctcctctaaagattgtaggaaatg-3′) and gapA-down-R1 (5′-tgcatgcctgcaggtcgacttcgcggcgaaaacgaagat-3′) were used as primers for PCR, and the gapA-down1 gene fragment of about 1.0 kb downstream of gapA was obtained and the PCR product was purified.
  • the plasmid pK18mobsacB (purchased from Addgene) was double-digested with EcoRI and XbaI, and the gapA-down1 fragment and gapA-up1 fragment obtained by the above purification were connected to pK18mobsacB using the Gibson Assembly Kit (NEB), and the obtained recombinant plasmid was named pK18-gapA1.
  • pK18-gapA1 was transformed into Corynebacterium glutamicum ATCC 13032 by electroporation, and a recombinant strain in which the gapA start codon ATG was correctly replaced with GTG was obtained by secondary screening and sequencing.
  • the plasmids pEC-ald and pXMJ-gpd-gpp-pduCDEGH were transferred into Corynebacterium glutamicum Cg-GTG by electroporation, and the obtained recombinant strain was named Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald.
  • strain Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald and the control strain Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald were inoculated into the fermentation medium for culture, and the culture conditions were exactly the same as in Example 2.
  • the strain Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald could produce 47.2 g/L of 3-hydroxypropionic acid, while the control strain Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald only produced 35.2 g/L of 3-hydroxypropionic acid. This indicates that downregulating the expression of gapA can significantly increase the yield of 3-hydroxypropionic acid.
  • strains Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald, Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald and control strain Cg/pXMJ-gpd-gpp-pduCDEGH were inoculated into sucrose fermentation medium for culture. Except for replacing 100 g/L glucose in the medium with 100 g/L sucrose, the other conditions remained unchanged and the culture conditions were exactly the same as in Example 2.
  • the strain Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald can produce 45.4 g/L of 3-hydroxypropionic acid
  • the strain Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald produces 33.1 g/L of 3-hydroxypropionic acid
  • the control strain Cg/pXMJ-gpd-gpp-pduCDEGH only produces 7.3 g/L of 3-hydroxypropionic acid.
  • Concentrated phosphoric acid solution was added to the fermentation broth containing 3-hydroxypropionic acid in Example 3, the pH value of the solution was adjusted to 1-2, the solution was heated to 140°C for 2 hours, and the concentration of the product in the fermentation broth was detected. The results showed that more than 90% of 3-hydroxypropionic acid was converted into acrylic acid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了发酵生产3-羟基丙酸和丙烯酸的方法。本发明提供一种重组谷氨酸棒杆菌，该微生物自身的醛脱氢酶基因ald被上调、甘油醛-3-磷酸脱氢酶gapA的表达被下调，同时表达异源的二醇脱水酶基因pduCDEGH、表达异源的3-磷酸脱氢酶gpd和甘油3-磷酸酶gpp。将该重组微生物在摇瓶或发酵罐中以葡萄糖等有机碳源为底物进行发酵培养获得3-羟基丙酸。将发酵培养液中的3-羟基丙酸进行酸化以及加热脱水进一步获得丙烯酸。本发明的重组微生物可以利用廉价的葡萄糖、蔗糖、糖蜜等为原料高效生产3-羟基丙酸和丙烯酸，生产工艺绿色安全简单，具有良好的市场应用前景。

Description

发酵生产3-羟基丙酸和丙烯酸的方法 技术领域
本发明属于基因工程和生物发酵技术领域，具体地说，涉及一种发酵生产3-羟基丙酸和丙烯酸的方法。
背景技术
3-羟基丙酸是一种重要的平台化合物，是美国能源部公布的12种高附加值生物基平台化学品之一。以3-羟基丙酸(3-HP)作为单体，可以合成高性能的生物可降解材料聚3-羟基丙酸(PHP)。同时，3-羟基丙酸可以进一步用于合成丙烯酸、1,3-丙二醇、丙二酸、丙二胺等。3-羟基丙酸还可以作为食品或饲料的添加剂和防腐剂。
丙烯酸是一种具有广泛应用的化学品，是合成聚丙烯酸(盐)的关键原料。聚丙烯酸具有极强的吸水能力，被广泛用于婴儿尿布、卫生巾、失禁产品等。目前，丙烯酸的生产主要依赖于化学法，通常是通过丙烯的分段氧化获得，该过程容易产生大量杂质，在制备卫生制品以及创伤材料中这些杂质会影响产品的安全性。同时该过程依赖于化石资源，生产过程需要高温高压，产生大量的污染物和二氧化碳排放。因此，开发绿色、安全的丙烯酸生产技术，实现从廉价的生物质原料直接生产丙烯酸具有重要的工业应用潜力。
目前，利用生物法生产3-羟基丙酸已有较多报道，但这些方法通常是利用克雷伯氏菌等微生物发酵甘油生产3-羟基丙酸，近年来甘油价格高涨，使得该路线难以有充分的经济竞争力。也有文献通过丙二酰-CoA路线或者β-丙氨酸路线合成3-羟基丙酸，但这些路线所获得的最终3-羟基丙酸浓度低，难以满足工业生产的需求。
发明内容
本发明的目的是提供一种发酵生产3-羟基丙酸和丙烯酸的方法。
本发明构思如下：通过在谷氨酸棒杆菌中引入甘油和3-羟基丙酸的合成途径，实现从各种廉价的碳源如葡萄糖，蔗糖，糖蜜等到3-羟基丙酸的高效转化。进一步地，发明人发现谷氨酸棒杆菌内源的ald基因编码的醛脱氢酶可以高效地催化3-羟基丙醛的氧化产生3-羟基丙酸，通过上调ald的表达，同时下调甘油醛-3-磷酸脱氢酶gapA的表达，可以实现高产量的3-羟基丙酸的生产。本发明还提供了将含有3-羟基丙酸的发酵液直接转化产生丙烯酸的方法。
为了实现本发明目的，第一方面，本发明提供一种重组谷氨酸棒杆菌，所述重组谷氨酸棒杆菌中的醛脱氢酶基因ald被强化，且甘油醛-3-磷酸脱氢酶基因gapA被弱化。
本发明中，基因强化的方式可选自以下1)～4)，或任选的组合：
1)通过导入具有所述基因的质粒；
2)通过增加微生物基因组中所述基因的拷贝数；
3)通过改变微生物基因组中所述基因的启动子序列；
4)通过将强启动子与所述基因可操作地连接。
本发明中，基因弱化的方式可选自以下a)～c)，或任选的组合：
a)通过将弱启动子与所述基因可操作地连接；
b)采用稀有密码子；
c)采用反义RNA。
进一步地，所述重组谷氨酸棒杆菌还过表达了二醇脱水酶基因pduCDEGH。
进一步地，所述重组谷氨酸棒杆菌还过表达了3-磷酸脱氢酶基因gpd和甘油3-磷酸酶基因gpp。
本发明中，
基因ald为：
A1)SEQ ID NO:1所示的核苷酸序列；
A2)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
A3)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
A4)与A1)、A2)或A3)的核苷酸序列具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且表达相同功能蛋白质的核苷酸序列。
基因gapA为：
B1)SEQ ID NO:2所示的核苷酸序列；
B2)SEQ ID NO:2所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
B3)在严格条件下与SEQ ID NO:2所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
B4)与B1)、B2)或B3)的核苷酸序列具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且表达相同功能蛋白质的核苷酸序列。
基因pduCDEGH为：
C1)SEQ ID NO:3所示的核苷酸序列；
C2)SEQ ID NO:3所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
C3)在严格条件下与SEQ ID NO:3所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
C4)与C1)、C2)或C3)的核苷酸序列具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且表达相同功能蛋白质的核苷酸序列。
基因gpd为：
D1)SEQ ID NO:4所示的核苷酸序列；
D2)SEQ ID NO:4所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
D3)在严格条件下与SEQ ID NO:4所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
D4)与D1)、D2)或D3)的核苷酸序列具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且表达相同功能蛋白质的核苷酸序列。
基因gpp为：
E1)SEQ ID NO:5所示的核苷酸序列；
E2)SEQ ID NO:5所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
E3)在严格条件下与SEQ ID NO:5所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
E4)与E1)、E2)或E3)的核苷酸序列具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且表达相同功能蛋白质的核苷酸序列。
在一个实施方式中，本发明提供了一种重组谷氨酸棒杆菌，所述重组谷氨酸棒杆菌具有增强的例如过表达的醛脱氢酶，和弱化的甘油醛-3-磷酸脱氢酶。
在一个实施方式中，所述醛脱氢酶包含SEQ ID NO:1所示的核苷酸序列编码的氨基酸序列或与其具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有相同功能的氨基酸序列。
在一个实施方式中，编码所述甘油醛-3-磷酸脱氢酶的基因是包含SEQ ID NO:2所 示的核苷酸序列的基因或其同源基因。
在一个实施方式中，所述重组谷氨酸棒杆菌还具有增强的例如过表达的二醇脱水酶、3-磷酸脱氢酶和/或甘油3-磷酸酶。
在一个实施方式中，所述二醇脱水酶包含SEQ ID NO:3所示的核苷酸序列编码的氨基酸序列或与其具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有相同功能的氨基酸序列。
在一个实施方式中，所述3-磷酸脱氢酶包含SEQ ID NO:4所示的核苷酸序列编码的氨基酸序列或与其具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有相同功能的氨基酸序列。
在一个实施方式中，所述甘油3-磷酸酶包含SEQ ID NO:5所示的核苷酸序列编码的氨基酸序列或与其具有90％以上例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有相同功能的氨基酸序列。
在本发明的一个具体实施方式中，所述重组谷氨酸棒杆菌是通过在谷氨酸棒杆菌中过表达基因ald、pduCDEGH、gpd和gpp，并将基因gapA的起始密码子由ATG替换成GTG构建得到的。
第二方面，本发明提供所述重组谷氨酸棒杆菌的构建方法，可采用常规的基因工程方法对上述基因进行改造或修饰。
在一个实施方式中，所述方法包括在谷氨酸棒杆菌中增强例如过表达本文所述醛脱氢酶和弱化本文所述甘油醛-3-磷酸脱氢酶。
在一个实施方式中，所述方法进一步包括增强例如过表达本文所述二醇脱水酶、3-磷酸脱氢酶和/或甘油3-磷酸酶。
第三方面，本发明提供所述重组谷氨酸棒杆菌在发酵生产3-羟基丙酸中的应用。
进一步地，利用所述重组谷氨酸棒杆菌，以廉价碳源为原料发酵生产3-羟基丙酸。
所述廉价碳源可选自糖蜜、蔗糖、葡萄糖、纤维二糖等中的至少一种。
第四方面，本发明提供丙烯酸的生产方法，发酵培养所述重组谷氨酸棒杆菌，发酵产生的3-羟基丙酸经过酸化、加热脱水得到丙烯酸。
在一个实施方式中，本发明提供了丙烯酸的生产方法，包括发酵培养本文所述重组谷氨酸棒杆菌的步骤。一个实施方式中，所述方法进一步包括酸化和/或加热脱水步骤。
进一步地，所述方法包括：向含有3-羟基丙酸的发酵液中加入浓磷酸溶液，调pH值至1-2，将发酵液加热到100-140℃反应1-5h(优选140℃反应2h)。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供一种重组谷氨酸棒杆菌，该微生物自身的醛脱氢酶基因ald被上调、甘油醛-3-磷酸脱氢酶gapA的表达被下调，同时表达异源的二醇脱水酶基因pduCDEGH、表达异源的3-磷酸脱氢酶gpd和甘油3-磷酸酶gpp。将该重组微生物在摇瓶或发酵罐中以葡萄糖等有机碳源为底物进行发酵培养获得3-羟基丙酸。将发酵培养液中的3-羟基丙酸进行酸化以及加热脱水进一步获得丙烯酸。本发明的重组微生物可以利用廉价的葡萄糖、蔗糖、糖蜜等为原料高效生产3-羟基丙酸和丙烯酸，生产工艺绿色安全简单，具有良好的市场应用前景。
具体实施方式
除非另有定义，本文所用的技术和科学术语具有本领域技术人员通常理解的含义。例如参见，Singleton et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed.,J.Wiley&Sons(New York,NY 1994)和Sambrook et al.,MOLECULAR CLONING,A LABORATORY MANUAL,Cold Springs Harbor Press(Cold Springs Harbor,NY 1989)。
如本文所用的“改造”和“重组”是指通过生物学手段人工改变的菌株，其与改变前的初始菌株相比具有一或多个改变，例如基因缺失、扩增或突变，从而具有改变的生物学性质例如改良的生产性能。如本文所用的初始菌株可以是对其要进行所述重组的天然菌株或其它重组菌株。
如本文所用的“醛脱氢酶”可在NAD+存在下氧化脂肪族和芳香族的醛变成相应的酸。如本文所用的谷氨酸棒杆菌内源的ald基因编码的醛脱氢酶可以高效地催化3-羟基丙醛的氧化产生3-羟基丙酸。
如本文所用的“甘油醛-3-磷酸脱氢酶”可催化甘油醛-3-磷酸的氧化磷酸化生成1,3-双磷酸甘油酸。
如本文所用的“二醇脱水酶”可催化1,2-二醇脱水。本发明的由pduCDEGH基因编码的二醇脱水酶可将甘油催化生成3-羟基丙醛。
如本文所用的“3-磷酸脱氢酶”可催化磷酸二羟丙酮生成3-磷酸甘油。
如本文所用的“甘油3-磷酸酶”可催化3-磷酸甘油生成甘油。
如本文所用的“相同功能蛋白质”是指能够催化相同反应的蛋白质，可以具有不同结构和不同来源。
如本文所用的“异源……基因”是指来自不同物种的、在进化过程中不源于共同祖先的基因。
如本文所用的表述“基因”和“核苷酸序列”可互换使用，是指核苷酸链，包含DNA和RNA。“基因的表达”是指将与适当调节区特别是启动子可操作地连接的DNA区域转 录成具有生物学活性的RNA以及RNA能够被翻译成生物学活性蛋白或肽。
如本文所用的“内源的……基因”是指细胞或生物体自身的基因，或与细胞或生物体自身的基因相同的基因。
如本文所用的表述“基因……被上调”和“上调……的表达”是指与具有该表达的参照(例如初始菌株或野生型菌株)相比，表达增加至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。
如本文所用的表述“……的表达被下调”和“下调……的表达”是指与具有该表达的参照(例如初始菌株或野生型菌株)相比，表达减少至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到。
如本文所用的表述“具有增强表达的……”和“增强……的表达”是指与具有该表达的参照(例如初始菌株或野生型菌株)相比，表达增加至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。
如本文所用，“过表达”是指相对于遗传操作前的水平，基因的表达水平是升高的，例如升高至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。过表达基因的方法是本领域熟知的，例如包括但不限于使用强启动子、增加基因拷贝数、增强子等。增加基因拷贝数可以例如但不限于通过引入一或多个拷贝的外源基因或内源基因实现，例如通过表达载体或整合进基因组中。
如本文所用，“增强的……酶/蛋白质”和“增强……酶/蛋白质”是指相对于遗传操作前的水平，在细胞或组织中表达的某种酶/蛋白质的水平和/或活性更高，例如升高至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。本领域已知多种增强蛋白的方式，包括但不限于过表达、增加活性的突变体等。
如本文所用的表述“基因……被强化”和“基因强化”是指使得基因的表达水平(作为蛋白编码基因时)或调控性能(作为调节元件时)增加至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。本领域已知多种基因增强的方式，包括但不限于使用强启动子、增加基因拷贝数、增强子等。
如本文所用的表述“基因……被弱化”和“基因弱化”是指使得基因的表达水平(作 为蛋白编码基因时)或调控性能(作为调节元件时)降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到。本领域已知多种基因弱化的方式，包括例如抑制基因表达如敲低(例如使用小干扰RNA)、使用弱启动子(基因是多肽编码基因时)等；基因敲除、缺失部分或全部基因序列；突变基因中某些位点例如编码序列以降低基因表达或调控活性或表达产物的活性等。
如本文所用，“弱化的……酶/蛋白”是指相对于遗传操作前的水平，在细胞或组织中表达的某种酶/蛋白质的水平更低，例如降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到。
如本文所用，术语“同源性”和“相同性”可互换使用，是指核苷酸序列无变化的程度，可通过比对多核苷酸与参考多核苷酸之间的相同核苷酸碱基的数目而检测。序列同源性可以通过标准排列对比算法程序使用由每个供应商制定的默认缺口罚分确定。基本同源的核酸分子典型在中等严格条件下或者在高度严格条件下杂交全长核酸或者至少或至少约70％、80％或90％全长的感兴趣的核酸分子。本发明还涵盖了含有简并密码子代替杂交核酸分子中密码子的核酸分子。任两个核酸分子是否具有至少80％、85％、90％、95％、96％、97％、98％或99％“相同的”核苷酸序列可以使用已知的计算机算法确定，如BLASTN、FASTA、DNAStar及Gap(University of Wisconsin Genetics Computer Group(UWG),Madison WI,USA)。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1 3-羟基丙醛合成模块的构建
人工合成3-磷酸脱氢酶基因gpd(基因序列如SEQ ID NO:4所示)和甘油3-磷酸酶基因gpp(基因序列如SEQ ID NO:5所示)。以gpd基因片段为模板以gpd-F(5′-attaagcttgcatgcctgcactttaagaaggagatataccaatggctgctgctgctgatag-3′)和gpd-R(5′-ggtatatctccttcttaaagtaatcttcatgtagatctaa-3′)为引物进行PCR，获得约1.26kb的gpd-1片段并进行PCR纯化。以gpp基因片段为模板以gpp-F(5′-attaagcttgcatgcctgcactttaagaaggagatataccaatgggattgactactaaacc-3′)和gpp-R(5′-ggtatatctccttcttaaagttaccatttcaacagatcgt-3′)为引物进行PCR，获得约0.8kb的gpp-1片段并进行PCR纯化。将pXMJ19质粒(购自Addgene)用PstI和XbaI进行双酶切，利用Gibson Assembly试剂盒(NEB)将上述纯化获得的gpd-1片段和gpp-1片段一步连接到pXMJ19 上，获得的重组质粒命名为pXMJ-gpd-gpp。
以克雷伯氏肺炎杆菌DSM 2026的基因组为模板，以pdu-F(5′-ggatccccgggtaccgagctctttaagaaggagatataccatgagatcgaaaagatttga-3′)和pdu-R(5′-caaaacagccaagctgaattttaagcatggcgatcccgaa-3′)为引物进行PCR，获得约5.1kb包括二醇脱水酶及其激活因子的pduCDEGH操纵子片段(序列如SEQ ID NO:3所示)并进行PCR产物纯化。将pXMJ-gpd-gpp质粒用KpnI和EcoRI进行双酶切，利用Gibson Assembly试剂盒(NEB)将上述纯化获得的pduCDEGH片段一步连接到pXMJ-gpd-gpp上，获得的重组质粒命名为pXMJ-gpd-gpp-pduCDEGH。
通过电转化将质粒pXMJ-gpd-gpp-pduCDEGH转入到谷氨酸棒杆菌ATCC 13032中，在含10mg/L的氯霉素LB平板上筛选获得重组菌，命名为Cg/pXMJ-gpd-gpp-pduCDEGH。
实施例2具有增强表达的醛脱氢酶基因ald的重组菌株构建
在谷氨酸棒杆菌中存在醛脱氢酶基因ald，通过增强ald的表达可以显著提高3-羟基丙酸的产量。
以谷氨酸棒杆菌ATCC13032的基因组为模板，以ald-F(5′-acagctatgacatgattacgtataagaaggagatatacaatgactgtctacgcaaatcc-3′)和ald-R(5′-tagaggatccccgggtaccgagtcttgtcaggccaaccca-3′)为引物进行PCR，获得约1.2kb的ald基因片段并进行PCR产物纯化。将质粒pEC-K18mob2(购自Addgene)用EcoRI和SacI进行双酶切，利用Gibson Assembly试剂盒(NEB)将上述纯化获得的ald片段连接到pEC-K18mob2上，获得的重组质粒命名为pEC-ald。通过电转法将pEC-ald转入到谷氨酸棒杆菌Cg/pXMJ-gpd-gpp-pduCDEGH中，在含50mg/L的卡那霉素LB平板上筛选获得重组菌，命名为Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald。
将菌株Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald和对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH接种到发酵培养基中进行培养，发酵温度为30℃，转速为200rpm，当菌体OD600达到2-3时加入0.1mM的IPTG进行诱导，并继续发酵48h。
发酵培养基配方(g/L)：葡萄糖100，(NH4)2SO4 10，K2HPO4 0.5，MgSO4 0.5，FeSO4 0.01，MnSO4 0.01，硫胺素0.005，β-丙氨酸0.01，烟酸0.005，玉米浆10，生物素0.001，盐酸硫胺素0.001，氯霉素0.005和卡那霉素0.05。
发酵48小时后，利用高效液相色谱(HPLC)检测菌株的产物。菌株Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald可以生产35.2g/L的3-羟基丙酸，而对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH仅生产10.1g/L的3-羟基丙酸。说明上调ald的表达可以显著提高3-羟基丙酸的产量。
同样，通过上调谷氨酸棒杆菌基因组上ald的表达也可以显著提高3-羟基丙酸的产量。将谷氨酸棒杆菌ATCC13032的ald基因的启动子替换为强启动子tac(5′-ttgacaattaatcatcggctcgtataatg-3′)构建重组菌株Cg-ald。将质粒pXMJ-gpd-gpp-pduCDEGH电转入菌株Cg-ald获得的重组菌株命名为Cg-ald/pXMJ-gpd-gpp-pduCDEGH。在相同的发酵条件下，菌株Cg-ald/pXMJ-gpd-gpp-pduCDEGH的3-羟基丙酸的产量达到30.4g/L，显著高于对照菌株(10.1g/L)。
菌株Cg-ald的构建过程如下：以谷氨酸棒杆菌ATCC13032的基因组为模板，以ald-up-F1(5′-tcatcagcgatggactcatgaacaa-3′)和ald-up-R1(5′-ttgacaattaatcatcggctcgtataatgcttttgaaaggctttcggcg-3′)为引物进行PCR，获得ald上游约1.0kb的基因ald-up1片段并进行PCR产物纯化。以谷氨酸棒杆菌ATCC13032的基因组为模板，以ald-down-F1(5′-cattatacgagccgatgattaattgtcaattattacccctgttcgggtg-3′)和ald-down-R1(5′-atcaccgtgcgtatcccaac-3′)为引物进行PCR，获得gapA下游约1.0kb的基因ald-down1片段并进行PCR产物纯化。将质粒pK18mobsacB(购自Addgene)用EcoRI和XbaI进行双酶切，利用Gibson Assembly试剂盒(NEB)将上述纯化获得的ald-down1片段和ald-up1片段连接到pK18mobsacB上，获得的重组质粒命名为pK18-ald。将pK18-ald通过电转化转入到谷氨酸棒杆菌ATCC 13032中，通过二次筛选以及测序获得正确的重组菌株，命名为Cg-ald。
实施例3下调gapA基因的表达提高3-羟基丙酸的产量
在上述菌株的基础上，进一步发现下调甘油醛-3-磷酸脱氢酶gapA基因的表达，可以显著提高3-羟基丙酸的产量。
通过用稀有起始密码子GTG替代ATG以弱化gapA的表达。以谷氨酸棒杆菌ATCC13032的基因组为模板，以gapA-up-F1(5′-acagctatgacatgattacgcgatgtcggtggaaaccagt-3′)和gapA-up-R1(5′-gagacacaacgtgaccattcgtgttggtattaacgg-3′)为引物进行PCR，获得gapA上游约1.0kb的基因gapA-up1片段并进行PCR产物纯化。以谷氨酸棒杆菌ATCC13032的基因组为模板，以gapA-down-F1(5′-gaatggtcacgttgtgtctcctctaaagattgtaggaaatg-3′)和gapA-down-R1(5′-tgcatgcctgcaggtcgacttcgcggcgaaaacgaaagat-3′)为引物进行PCR，获得gapA下游约1.0kb的基因gapA-down1片段并进行PCR产物纯化。将质粒pK18mobsacB(购自Addgene)用EcoRI和XbaI进行双酶切，利用Gibson Assembly试剂盒(NEB)将上述纯化获得的gapA-down1片段和gapA-up1片段连接到pK18mobsacB上，获得的重组质粒命名为pK18-gapA1。将pK18-gapA1通过电转化转入到谷氨酸棒杆菌ATCC 13032中，通过二次筛选以及测序获得gapA起始密码子ATG被正确替换为GTG的重组菌株，命名为 Cg-GTG。通过电转法将质粒pEC-ald和pXMJ-gpd-gpp-pduCDEGH转入到谷氨酸棒杆菌Cg-GTG中，获得的重组菌株命名为Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald。
将菌株Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald和对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald接种到发酵培养基中进行培养，培养条件与实施例2完全相同。
发酵48小时后，利用高效液相色谱(HPLC)检测菌株的产物。菌株Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald可以生产47.2g/L的3-羟基丙酸，而对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald仅生产35.2g/L的3-羟基丙酸。说明下调gapA的表达可以显著提高3-羟基丙酸的产量。
同样地，将菌株Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald、Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald和对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH接种到蔗糖发酵培养基中进行培养，培养基中除100g/L的葡萄糖置换为100g/L蔗糖以外，其他保持不变，培养条件也与实施例2完全相同。菌株Cg-GTG/pXMJ-gpd-gpp-pduCDEGH/pEC-ald可以生产45.4g/L的3-羟基丙酸，菌株Cg/pXMJ-gpd-gpp-pduCDEGH/pEC-ald生产33.1g/L的3-羟基丙酸，而对照菌株Cg/pXMJ-gpd-gpp-pduCDEGH仅产生7.3g/L的3-羟基丙酸。
实施例4将发酵液中的3-羟基丙酸转化为丙烯酸
向实施例3的包含3-羟基丙酸的发酵液中加入浓磷酸溶液，调整溶液的pH值为1-2，将溶液加热到140℃反应2h，检测发酵液中产物的浓度。结果显示，90％以上的3-羟基丙酸被转化为丙烯酸。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (10)

  1. 重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌中的醛脱氢酶基因ald被强化，且甘油醛-3-磷酸脱氢酶基因gapA被弱化；
    其中，基因ald为：
    A1)SEQ ID NO:1所示的核苷酸序列；
    A2)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
    A3)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    A4)与A1)、A2)或A3)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列；
    基因gapA为：
    B1)SEQ ID NO:2所示的核苷酸序列；
    B2)SEQ ID NO:2所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
    B3)在严格条件下与SEQ ID NO:2所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    B4)与B1)、B2)或B3)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
  2. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，基因强化的方式选自以下1)～4)，或任选的组合：
    1)通过导入具有所述基因的质粒；
    2)通过增加微生物基因组中所述基因的拷贝数；
    3)通过改变微生物基因组中所述基因的启动子序列；
    4)通过将强启动子与所述基因可操作地连接。
  3. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，基因弱化的方式选自以下a)～c)，或任选的组合：
    a)通过将弱启动子与所述基因可操作地连接；
    b)采用稀有密码子；
    c)采用反义RNA。
  4. 根据权利要求1-3任一项所述的重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌还过表达了二醇脱水酶基因pduCDEGH，基因pduCDEGH为：
    C1)SEQ ID NO:3所示的核苷酸序列；
    C2)SEQ ID NO:3所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
    C3)在严格条件下与SEQ ID NO:3所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    C4)与C1)、C2)或C3)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
  5. 根据权利要求4所述的重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌还过表达了3-磷酸脱氢酶基因gpd和甘油3-磷酸酶基因gpp；
    基因gpd为：
    D1)SEQ ID NO:4所示的核苷酸序列；
    D2)SEQ ID NO:4所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
    D3)在严格条件下与SEQ ID NO:4所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    D4)与D1)、D2)或D3)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列；
    基因gpp为：
    E1)SEQ ID NO:5所示的核苷酸序列；
    E2)SEQ ID NO:5所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；
    E3)在严格条件下与SEQ ID NO:5所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    E4)与E1)、E2)或E3)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
  6. 根据权利要求5所述的重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌是通过在谷氨酸棒杆菌中过表达基因ald、pduCDEGH、gpd和gpp，并将基因gapA 的起始密码子由ATG替换成GTG构建得到的。
  7. 权利要求1-6任一项所述重组谷氨酸棒杆菌在发酵生产3-羟基丙酸中的应用。
  8. 根据权利要求7所述的应用，其特征在于，以廉价碳源为原料发酵生产3-羟基丙酸；
    所述廉价碳源选自糖蜜、蔗糖、葡萄糖、纤维二糖中的至少一种。
  9. 丙烯酸的生产方法，其特征在于，发酵培养权利要求1-6任一项所述重组谷氨酸棒杆菌，发酵产生的3-羟基丙酸经过酸化、加热脱水得到丙烯酸。
  10. 根据权利要求9所述的方法，其特征在于，所述方法包括：向含有3-羟基丙酸的发酵液中加入浓磷酸溶液，调pH值至1-2，将发酵液加热到100-140℃反应1-5h。
PCT/CN2023/114590 2022-09-26 2023-08-24 发酵生产3-羟基丙酸和丙烯酸的方法 WO2024066819A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211177464.0 2022-09-26
CN202211177464.0A CN116396914A (zh) 2022-09-26 2022-09-26 发酵生产3-羟基丙酸和丙烯酸的方法

Publications (1)

Publication Number Publication Date
WO2024066819A1 true WO2024066819A1 (zh) 2024-04-04

Family

ID=87014773

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/114590 WO2024066819A1 (zh) 2022-09-26 2023-08-24 发酵生产3-羟基丙酸和丙烯酸的方法

Country Status (2)

Country Link
CN (1) CN116396914A (zh)
WO (1) WO2024066819A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116396914A (zh) * 2022-09-26 2023-07-07 北京绿色康成生物技术有限公司 发酵生产3-羟基丙酸和丙烯酸的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950529A (zh) * 2016-06-24 2016-09-21 清华大学 产3-羟基丙酸的重组谷氨酸棒杆菌、其构建方法及应用
CN116396914A (zh) * 2022-09-26 2023-07-07 北京绿色康成生物技术有限公司 发酵生产3-羟基丙酸和丙烯酸的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950529A (zh) * 2016-06-24 2016-09-21 清华大学 产3-羟基丙酸的重组谷氨酸棒杆菌、其构建方法及应用
CN116396914A (zh) * 2022-09-26 2023-07-07 北京绿色康成生物技术有限公司 发酵生产3-羟基丙酸和丙烯酸的方法

Also Published As

Publication number Publication date
CN116396914A (zh) 2023-07-07

Similar Documents

Publication Publication Date Title
US11225675B2 (en) D-lactate dehydrogenase, engineered strain containing D-lactate dehydrogenase and construction method and use of engineered strain
JP5254353B2 (ja) ２−デオキシ−シロ−イノソース（ｄｏｉ）生産細菌及びこれを用いた２−デオキシ−シロ−イノソース（ｄｏｉ）生産方法
KR102400332B1 (ko) 정제 화학약품의 개선된 생산을 위한 재조합 미생물
KR101576186B1 (ko) 에탄올 생산 경로가 봉쇄된 클루이베로마이세스 막시아누스 균주 및 이의 용도
KR102321146B1 (ko) 정밀 화학물질의 개선된 생산을 위한 재조합 미생물
CA2751280A1 (en) Novel microbial succinic acid producers and purification of succinic acid
KR101616171B1 (ko) 유기산 제조에서의 모나스쿠스의 용도
WO2005106005A1 (ja) ヒドロキシカルボン酸類の生産方法
CN108350040A (zh) 用于精细化学品的改进生产的重组微生物
JP2020526213A (ja) エクトイン産生酵母
WO2024066822A1 (zh) 发酵生产γ-丁内酯或1,4-丁二醇的方法
WO2024066819A1 (zh) 发酵生产3-羟基丙酸和丙烯酸的方法
KR101521045B1 (ko) 유기산을 생산하기 위한 재조합 미생물유기체
MXPA06012770A (es) Un metodo para producir acido succinico a partir de hidrolizados crudos.
KR20150121789A (ko) 2,3―부탄디올의 생성능이 증강된 재조합 미생물 및 이를 이용한 2,3―부탄디올의 생산 방법
CN112280723B (zh) 联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用
CN115058374B (zh) 一种利用丙酮酸合成乙偶姻的重组运动发酵单胞菌及其构建方法与应用
US10760100B2 (en) Polypeptide having ferredoxin-NADP+ reductase activity, polynucleotide encoding the same and uses thereof
WO2016030373A1 (en) Modified microorganism for improved production of fine chemicals on sucrose
WO2007099994A1 (ja) 新規カルボニル還元酵素、その遺伝子、ベクター、形質転換体、およびそれらを利用した光学活性アルコールの製造方法
WO2003078635A1 (fr) Gene favorisant la tolerance a l'acide acetique, bacterie d'acide acetique obtenue au moyen du gene, et procede de production de vinaigre au moyen de cette bacterie d'acide acetique
US9637761B2 (en) Recombinant microorganism metabolizing 3,6-anhydride-L-galactose and a use thereof
KR101743021B1 (ko) 재조합 미생물을 이용한 l형 2,3-부탄디올의 제조방법
CN113403315B (zh) 一种提升菌体生长和产生物酶潜力的基因表达盒
KR102611978B1 (ko) 숙신산 생산성이 향상된 미생물 및 이를 이용한 숙신산 생산 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23870063

Country of ref document: EP

Kind code of ref document: A1