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Genetically engineered bacterium for efficiently producing (2S, 3S)-2, 3-butanediol, construction method and application thereof

A technology of genetically engineered bacteria and butanediol, applied in genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve control difficulties, high implementation costs, low optical purity of 2,3-butanediol, etc. problem, to achieve the effect of increasing product concentration, reducing accumulation, and improving substrate conversion rate

Pending Publication Date: 2022-02-22
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stereoisomer-selective production of chemically synthesized 2,3-butanediol is complex, difficult to control, and costly to implement
Furthermore, the optical purity of 2,3-butanediol produced by the chemical route is quite low
Therefore, the biotechnology route has become the preferred method for chiral 2,3-butanediol, but most natural microbial fermentation will produce two different configurations of 2,3-butanediol and the 2,3-butanediol produced by natural microorganisms The proportion of (2S,3S)-2,3-butanediol in the diol is usually small, for example, Klebsiella and Enterobacter strains mainly produce (2S,3S)-2,3-butanediol Alcohol and meso-2,3-butanediol, Bacillus mainly produces (2R,3R)-2,3-butanediol and meso-2,3-butanediol, no single configuration 2,3-butanediol has been found so far Butanediol's natural microorganisms

Method used

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  • Genetically engineered bacterium for efficiently producing (2S, 3S)-2, 3-butanediol, construction method and application thereof
  • Genetically engineered bacterium for efficiently producing (2S, 3S)-2, 3-butanediol, construction method and application thereof
  • Genetically engineered bacterium for efficiently producing (2S, 3S)-2, 3-butanediol, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Construction of recombinant plasmid pET28a-KbudC / pETDuet-KbudC:

[0059] The 2,3-butanediol dehydrogenase gene KbudC of Klebsiella pneumoniae was cloned into the pET28a plasmid and the pETDuet plasmid to construct the pET28a-KbudC plasmid and pETDuet-KbudC, and the map of the pET28a-KbudC plasmid was as follows figure 1 As shown in A, the map of pETDuet-KbudC plasmid is as follows figure 1 D shows. The specific operation steps are as follows:

[0060]Klebsiella pneumoniae genome was used as a template for PCR amplification, and bioinformatics software was used to design the amplification primers of the gene KbudC sequence, and the gene KbudC sequence was amplified, and the primer sequences were as follows:

[0061] Primer1: 5′-ATGGGTCGCGGATCCGAATTCATGAAAAAAGTCGCACTTGTTACC-3′ (contains EcoRI restriction site),

[0062] Primer2: 5′-GCAAGCTTGTCGACGGAGCTCTTAGTTAAATACCATCCCGCCG-3′ (containing SacI restriction site);

[0063] The PCR amplification system was pr...

Embodiment 2

[0066] Example 2: Construction of recombinant plasmid pET28a-GbudC / pETDuet-GbudC:

[0067] The 2,3-butanediol dehydrogenase gene GbudC of Corynebacterium glutamicum was cloned into the pET28a plasmid and the pETDuet plasmid, and the pET28a-GbudC plasmid and pETDuet-GbudC were constructed. The map of the pET28a-GbudC plasmid is as follows figure 1 As shown in B, the map of pETDuet-GbudC plasmid is as follows figure 1 Shown in E. The specific operation steps are as follows:

[0068] The genome of Corynebacterium glutamicum was used as a template for PCR amplification, and the amplification primers of the gene GbudC sequence were designed by using bioinformatics software, and the gene GbudC sequence was amplified, and the primer sequences were as follows:

[0069] Primer3: 5′-GCAAGCTTGTCGACGGAGCTCATGAGCAAAGTTGCAATGGTTACC-3′ (contains EcoRI restriction site),

[0070] Primer4: 5'-GCAAGCTTGTCGACGGAGCTCTTAGTTGTAGAGCATGCCGCC-3' (containing SacI restriction site);

[0071] The PCR...

Embodiment 3

[0074] Embodiment 3: Construction of plasmid pET28a-LbudC

[0075] The 2,3-butanediol dehydrogenase gene sequence of Staphylococcus aureus was searched at NCBI, which was synthesized by Shanghai Sangon with codon optimization and then ligated into pET28a to obtain the pET28a-LbudC plasmid. The map of the pET28a-LbudC plasmid is as follows figure 1 C shown. Use the pET28a-LbudC plasmid as a template for PCR verification, and the primer sequences are as follows

[0076] Primer5: 5′-ATGGGTCGCGGATCCGAATTCATGAACCTGAAAGATGCGAAAAT-3′

[0077] Primer6: 5′-GCAAGCTTGTCGACGGAGCTCTTACTGCGCCGCCCACGG-3′

[0078] The obtained PCR product was analyzed and detected by 1% agarose gel electrophoresis, and an electrophoresis band with a size of about 711 bp was obtained. Transform the pET28a-LbudC plasmid into E.coli DH5α competent cells by electroporation, spread on solid LB solid medium containing 50 μg / mL kanamycin and culture overnight at 37°C, pick positive recombinants at -80°C Preserva...

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Abstract

The invention relates to a genetically engineered bacterium for efficiently producing (2S, 3S)-2, 3-butanediol, a construction method and application thereof. According to the invention, based on escherichia coli, a new genetically engineered bacterium is constructed, and simultaneously expresses 2, 3-butanediol dehydrogenase in staphylococcus aureus and klebsiella pneumoniae, or simultaneously expresses 2, 3-butanediol dehydrogenase in staphylococcus aureus and corynebacterium glutamicum, or simultaneously expresses 2, 3-butanediol dehydrogenase in klebsiella pneumoniae and corynebacterium glutamicum; and through the synergistic effect of the double enzymes, accumulation of diacetyl in cells is reduced, and conversion of the diacetyl into the (2S, 3S)-2, 3-butanediol is enhanced, so that the concentration of a substrate is increased, and the conversion rate of the substrate is increased so as to increase the yield of the (2S, 3S)-2, 3-butanediol, wherein the yield reaches 7.85 g / L.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for efficiently producing (2S,3S)-2,3-butanediol, its construction method and application. Background technique [0002] 2,3-Butanediol has the molecular formula (CH 3 CHOH) 2 It is a colorless and odorless liquid or crystal at room temperature. 2,3-butanediol has two chiral carbon atoms, so it has three stereoisomers, namely meso-2,3-butanediol, (2S,3S)-2,3-butanediol and ( 2R,3R)-2,3-Butanediol. Moreover, the three stereoisomers of 2,3-butanediol have different properties due to the presence of different chiral groups, which makes optically pure 2,3-butanediol highly sought after in asymmetric synthesis. focus on. [0003] 2,3-Butanediol has a wide range of applications in aviation fuel, medicine, food, cosmetics and many other aspects. The optically active 2,3-butanediol has good application prospects in the asymmetric synthesis...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/66C12N15/53C12P7/18C12R1/19
CPCC12N9/0006C12N15/70C12P7/18C12Y101/01004C12N2800/22Y02E50/10
Inventor 马春玲邵明宇王瑞明李丕武苏静汪俊卿
Owner QILU UNIV OF TECH
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