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Strategy for efficiently coproducing alpha-aminobutyric acid and gluconic acid

A technology of aminobutyric acid and gluconic acid, which is applied in the field of microorganisms, can solve the problems of long fermentation time, strict control of fermentation conditions, and low energy consumption, and achieve important industrial application value and fast and efficient transformation process

Active Publication Date: 2016-01-20
ANHUI HUAHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of gluconic acid mainly includes microbial fermentation, electrolysis and catalytic oxidation. Among them, microbial fermentation is widely used because of its environmental friendliness and low energy consumption, but there are also problems such as long fermentation time and strict control of fermentation conditions.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction and transformation of recombinant plasmid pET-28a-Bcldh / pET-28a-Rjpdh

[0040] [1] Genomic DNA of Bacillus cereus and Rhodococcus were used as templates.

[0041] [2] Design ldh gene primers according to the L-alanine dehydrogenase gene sequence of Bacillus subtilis and the L-phenylalanine dehydrogenase gene sequence of Rhodococcus and the restriction site on pET-28a plasmid.

[0042] PBcldhF: 5'-CGGGATCCATGACATTAGAAATCTTCG-3'(BamHI)

[0043] PBcldhR: 5'-CGAGCTCTTAGCGACGGCTAATAATATC-3'(SacI)

[0044] PRjpdhF: 5'-CGGGATCCATGACTCTCACCGCGGAAC-3' (BamHI)

[0045] PRjpdhR: 5'-CGAGCTCCTACCTGGCTGCAGCGATG-3'(SacI)

[0046] [3] The DNA of Bacillus cereus and Rhodococcus was used as a template to amplify the gene by PCR. PCR amplification system: template 2 μL, upstream and downstream primers 0.5 μL, dNTPMix 4 μL, 10×ExTaqBuffer 5 μL, sterilized ddH 2 O37 μL, ExTaq DNA polymerase 1 μL. PCR reaction conditions: 94°C pre-denaturation, 5min, one cycle; ...

Embodiment 2

[0049] Example 2: Construction and transformation of recombinant plasmid pET-28a-Bsglcdh+Bcldh / pET-28a-Bsglcdh+Rjpdh

[0050] [1] Genomic DNA of Bacillus subtilis was used as template.

[0051] [2] Design glcdh gene primers and glcdh gene primers for tandem L-amino acid dehydrogenase according to the glucose dehydrogenase gene sequence of Bacillus subtilis and the restriction site on the pET-28a plasmid.

[0052] PBsglcdhF: 5'-CGGGATCCATGTATCCGGATTTAAAAGG-3'(BamHI)

[0053] PBsglcdhR: 5'-CCCAAGCTTTTAACCGCGGCCTGCCTGG-3'(HindIII)

[0054] P28aPromoterF: 5'-ACATGCATGCCGATCCCGCGAAATTAATAC-3'(SphI)

[0055] PBsglcdhRBglII: 5'-GAAGATCTTTAACCGCGGCCTGCCTGG-3'(BglII)

[0056] [3] Using chromosomal DNA as a template, the gene was amplified by PCR. PCR amplification system: template 2 μL, upstream and downstream primers 0.5 μL, dNTPMix 4 μL, 10×ExTaqBuffer 5 μL, sterilized ddH 2 O37 μL, ExTaq DNA polymerase 1 μL. PCR reaction conditions: 94°C pre-denaturation, 5min, one cycle; 94°C...

Embodiment 3

[0062] Example 3: Construction and Transformation of Recombinant Plasmid pET-duet-Ppglcdh+Bsadh / pET-duet-Ppglcdh+Scvdh

[0063] [1] Genomic DNA of Pseudomonas putida, Bacillus subtilis and Streptomyces coelicolor were used as templates.

[0064] [2] According to the glucose dehydrogenase gene sequence of Pseudomonas putida, the L-alanine dehydrogenase gene sequence of Bacillus subtilis, the valine dehydrogenase gene sequence of Streptomyces coelicolor and the pET-duet plasmid Gene primers were designed based on the enzyme cutting sites on the gene.

[0065] PPpglcdhF: 5'-CGGGATCCATGAGCACTGAAGGTGCGAACC-3' (BamHI)

[0066] PPpglcdhR: 5'-CCCAAGCTTTTACTCGGCTAATTTGTAAG-3'(HindIII)

[0067] PBsadhF: 5'-GGGGTACCATGATCATAGGGGTTCCT-3'(KpnI)

[0068] PBsadhR: 5'-CCGCTCGAGTTAAGCACCCGCCACAGATG-3'(XhoI)

[0069] PScvdhF: 5'-GGAATTCCATATGGTGACCGACGTAAACGG-3'(NdeI)

[0070] PScvdhR: 5'-CGAGCTCTCACGGCCGGGGACGGGCCT-3'(XhoI)

[0071] [3] Using the DNA of Pseudomonas putida, Bacillus subtili...

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Abstract

The invention relates to a method for coproducing alpha-aminobutyric acid and gluconic acid by constructing recombinant escherichia coli by virtue of series connection of glucose dehydrogenase and L-amino acid dehydrogenase. According to the method, recombinant co-expression vectors are constructed by virtue of glucose dehydrogenase genes and L-amino acid dehydrogenase genes and are transferred into escherichia coli of genetically engineered bacteria; meanwhile, recombinant escherichia coli which expresses L-amino acid dehydrogenase is constructed; by virtue of efficiently co-expressing glucose dehydrogenase and L-amino acid dehydrogenase into escherichia coli, the circulation of cofactors in mycetome can be promoted, and by virtue of a cyclic regeneration system of the cofactors, high-added-value alpha-aminobutyric acid and gluconic acid can be coproduced from cheap substrates, namely L-amino acid and glucose without adding any exogenous cofactor; the transfer process is simple and rapid, and the cost is low; the yields of alpha-aminobutyric acid and gluconic acid produced in a 5L fermentation tank by virtue of the method can respectively reach 102.8g / L and 196.8g / L, and a practical and effective strategy is provided for industrial production.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and specifically relates to a method for constructing glucose dehydrogenase and L-amino acid dehydrogenase in series on a plasmid and co-expressing them in Escherichia coli, and using the recombinant Escherichia coli to perform whole-cell transformation to efficiently prepare α-aminobutyric acid and gluconate methods. Background technique [0002] α-aminobutyric acid is a non-natural amino acid that can inhibit the transmission of human nerve information. It can enhance the activity of glucose phosphatase and promote the metabolism of brain cells. α-aminobutyric acid, as an important chemical raw material and pharmaceutical intermediate such as the synthetic precursor of the anti-tuberculosis drug ethambutol hydrochloride and the anti-epileptic drug levetiracetam, has a broad application market. α-aminobutyric acid is mainly prepared by chemical synthesis, enzymatic resolution and enzymat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P13/00C12P7/58
Inventor 饶志明周俊平杨套伟张蔡喆戚云龙郑俊贤张显徐美娟
Owner ANHUI HUAHENG BIOTECH
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