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Construction method and application of gene engineering strain for producing (R,R)-2,3-butanediol

A technology of genetically engineered strains and construction methods, applied in the production of highly optically pure -2,3-butanediol, the field of producing highly optically pure -2,3-butanediol genetically engineered strains, can solve the problem The optical purity does not meet the requirements, the output of butanediol is low, and the production capacity is insufficient, etc., to achieve the effect of releasing transcription inhibition, high production efficiency, and easy operation

Active Publication Date: 2017-09-26
南宁邦尔克生物技术有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ability of strains to synthesize (R,R)-2,3-butanediol can be significantly improved through metabolic engineering and synthetic biology methods, but the problem of high cost of carbon and nitrogen sources has not yet been solved
However, in the current method of producing (R,R)-2,3-butanediol by fermentative production of cheap carbon sources, the yield of (R,R)-2,3-butanediol is low due to the insufficient production capacity of the strain, or Optical purity does not meet the requirements

Method used

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  • Construction method and application of gene engineering strain for producing (R,R)-2,3-butanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of the genetically engineered strain GXASR1 for the production of (R,R)-2,3-butanediol:

[0033] The genome sequence of Paenibacillus polymyxa DSM 365 was analyzed, and the (R,R)-2,3-butanediol dehydrogenase gene bdh of the strain was obtained, and it was combined with the α-acetolactate synthase gene budB, The nucleotide sequence of the α-acetolactate decarboxylase gene budA was codon-optimized, and the nucleotide sequence TAAGGAGGATATACA containing the ribosome binding site was added in front of each gene, and then the gene cluster budB-budA- The length of bdh's nucleotide sequence is 3574 bases, and the nucleotide sequence is as described in SEQ ID NO.1. The gene cluster budB-budA-bdh was inserted behind the promoter of the plasmid pTrc99A by double enzyme digestion and ligation to obtain the polycistronic recombinant plasmid pTrc99A-budB-budA-bdh, and then the recombinant plasmid pTrc99A-budB-budA- The bdh was introduced into the host strain E.coli MG1...

Embodiment 2

[0035] The construction of the genetically engineered strain GXASR2 producing (R,R)-2,3-butanediol:

[0036] The genome sequence of Paenibacillus polymyxa DSM 365 was analyzed, and the nucleotide sequences of the α-acetolactate synthase gene alsS and α-acetolactate decarboxylase gene alsD of the strain were obtained. Codon optimization was performed on the nucleotide sequences of alsS, alsD and the (R,R)-2,3-butanediol dehydrogenase gene bdh from Paenibacillus polymyxa, and a ribosome binding site was added in front of each gene The nucleotide sequence TAAGGAGGATATACA, and then use artificial synthesis method to obtain the gene cluster alsS-alsD-bdh, its nucleotide sequence length is 3562 bases, the nucleotide sequence is as described in SEQ ID NO.2. Then insert the gene cluster alsS-alsD-bdh behind the promoter of the plasmid pTrc99A to obtain the polycistronic recombinant plasmid pTrc99A-alsS-alsD-bdh, which is introduced into the host strain E.coli MG1655 to obtain the (R,R...

Embodiment 3

[0038] Construction of the genetically engineered strain GXASR3 for the production of (R,R)-2,3-butanediol:

[0039] Through analysis, it was found that the main by-products of the fermentation of engineering strains GXASR1 and GXASR2 were meso-2,3-butanediol, succinic acid, formic acid, ethanol and acetic acid, and the key genes of the synthesis pathway were dar, frdABCD, pflB, adhE and pta . Using the principle that the Red recombination system derived from Escherichia coli λ phage can efficiently mediate homologous recombination events in bacteria, first replace the above target gene with an antibiotic resistance gene with FRT sites on both sides, and then induce exogenous temperature The sensitive plasmid expresses FLP recombinase to delete the antibiotic resistance gene to achieve the purpose of knocking out the target gene. The specific steps are as follows:

[0040] Transform the pKD46 plasmid into host cells to prepare electroporation-competent cells; use primers to c...

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Abstract

The invention discloses a construction method and application of a gene engineering strain for producing (R,R)-2,3-butanediol. The method comprises the following steps of carrying out codon optimization on nucleotide sequences of an alpha-acetolactic acid synthetase gene, an alpha-acetolactic acid decarboxylase gene and an R,R-2,3-butanediol dehydrogenase gene, and obtaining a gene cluster containing the three genes by utilizing an artificial synthesis method; inserting the gene cluster into an expression vector, so as to obtain a polycistronic recombinant plasmid; introducing the polycistronic recombinant plasmid into a host bacterium E. coli, and knocking out a key gene of the synthetic route of a main by-product, so as to obtain the gene engineering strain for producing (R,R)-2,3-butanediol. Raw materials used by the engineering strain provided by the construction method are wide in sources and low in costs; the strain has no pathogenicity; the strain is high in yield and high in production efficiency for the (R,R)-2,3-butanediol, has the highest yield which can reach 93.5g / L and the optical purity which can reach 99 percent or above. According to the construction method, non-grain cassava meal and a low-cost nitrogen source are also utilized as fermentation raw materials for producing the (R,R)-2,3-butanediol; the production cost is lowered.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the construction of a genetically engineered bacterial strain for producing highly optically pure (R,R)-2,3-butanediol, and its use in the production of high Application of optically pure (R,R)-2,3-butanediol. Background technique [0002] 2,3-butanediol (2,3-butanediol) is a platform compound, which has wide application value in chemical industry, food, energy, medicine and other fields. Its microbial fermentation production is an important topic of modern biochemical industry. The 2,3-butanediol molecule contains two chiral carbon atoms, so there are three optical isomers, namely (2R,3R)-2,3-butanediol, (2S,3S)-2, 3-butanediol and meso-2,3-butanediol. In addition to the function of mixed configuration 2,3-butanediol, single-configuration (R,R)-2,3-butanediol is also an important precursor for the synthesis of chiral reagents and chiral ligands. It has special applications in the...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/54C12N15/53C12N15/60C12N1/21C12P7/18
CPCC12N9/0006C12N9/1022C12N9/88C12N15/70C12N2800/22C12P7/18C12Y101/01004C12Y202/01006C12Y401/01005
Inventor 陈先锐黄日波谢能中黄艳燕韦航李检秀王青艳李晓明
Owner 南宁邦尔克生物技术有限责任公司
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