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Construction of coenzyme efficient regeneration system and application thereof

A technology of dehydrogenase and formate dehydrogenase is applied in the field of Candida boidinia formate dehydrogenase gene sequence synthesis and preparation of α-aminobutyric acid, which can solve the problem of increasing the production cost and price of α-aminobutyric acid. Expensive, low formate dehydrogenase enzyme activity, etc., to achieve important industrial application value, improve conversion efficiency, and reduce costs

Inactive Publication Date: 2016-01-13
ANHUI HUAHENG BIOTECH
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

In the conversion system using formate dehydrogenase as the coenzyme NADH cycle, the substrate formate or formate is catalyzed to generate CO 2 , the reaction does not produce any by-products, which is beneficial to the separation and extraction of α-aminobutyric acid, but formate dehydrogenase has problems such as low enzyme activity and high price, which makes the regeneration rate of NADH a limiting factor for α-aminobutyric acid. main factors of conversion
In addition, the production of α-aminobutyric acid with an enzyme system requires the cell disruption of the enzyme-producing recombinant bacteria. At the same time, due to the loss of cofactors in the transformation process, continuous exogenous addition is required, which further increases the concentration of α-aminobutyric acid. acid production cost

Method used

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  • Construction of coenzyme efficient regeneration system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Codon optimization of formate dehydrogenase

[0043] Upload the formate dehydrogenase gene sequence (fdh, GenBankAccessionNo.AJ011046) derived from Candida boidinii to http: / / www.jcat.de / to optimize the codon for the codon preference of Escherichia coli, and then send it to Shanghai Sangon Biotech Co., Ltd. performed gene synthesis. After the optimized gene fdh was compared with the original sequence by DNAMAN, it was found that the sequence homology was 80.37%.

Embodiment 2

[0044] Example 2: Construction and transformation of recombinant plasmid pET-28a-Bcldh / pET-28a-Rjpdh

[0045] [1] Genomic DNA of Bacillus cereus and Rhodococcus were used as templates.

[0046] [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.

[0047] PBcldhF: 5'-CGGGATCCATGACATTAGAAATCTTCG-3'(BamHI)

[0048] PBcldhR: 5'-CGAGCTCTTAGCGACGGCTAATAATATC-3'(SacI)

[0049] PRjpdhF: 5'-CGGGATCCATGACTCTCACCGCGGAAC-3' (BamHI)

[0050] PRjpdhR: 5'-CGAGCTCCTACCTGGCTGCAGCGATG-3'(SacI)

[0051] [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 3

[0054] Example 3: Construction and transformation of recombinant plasmid pET-28a-fdh+Bcldh / pET-28a-fdh+Rjpdh

[0055] [1] Synthetic codon-optimized formate dehydrogenase DNA was used as a template.

[0056] [2] Design fdh gene primers according to the synthesized codon-optimized formate dehydrogenase gene sequence and restriction sites on pET-28a and pET-duet plasmids. According to the codon-optimized fdh gene sequence, primers P1 and P2 of formate dehydrogenase gene and primers P3 and P4 co-expressed with L-amino acid dehydrogenase in pET-28a were designed.

[0057] P1: 5'-ACCGGGATCCATGAAAATCGTTCTGGTTCTG-3'(BamHI)

[0058] P2: 5'-CGCGTCGACTTATTTTTTGTCGTGTTTACC-3'(SalI)

[0059] P3: 5'-ACATGCATGCCGATCCCGCGAAATTAATAC-3'(SphI)

[0060] P4: 5'-GAAGATCTTTATTTTTTTGTCGTGTTTACC-3' (BglII)

[0061] [3] Use the synthesized formate dehydrogenase DNA as a template, and use P1 and P2 as primers to amplify the gene by PCR. PCR amplification system: template 2 μL, upstream and downstre...

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Abstract

The invention relates to a method for preparing alpha-aminobutyric acid by utilizing series connection amino acid dehydrogenase and codon optimization formate dehydrogenase recombinant escherichia coli. First, codon optimization is performed on a Candida boidinii formate dehydrogenase gene sequence according to the codon preference of the escherichia coli. After codon optimization on formate dehydrogenase, the expression quantity of formate dehydrogenase in the escherichia coli can be improved remarkably, the yield of alpha-aminobutyric acid is increased, and in the escherichia coli, co-expression formate dehydrogenase and L-amino acid dehydrogenase can promote circulation of cofactors in mycetome without needing the adding of any exogenous cofactors. In addition, the process of utilizing whole-cell transformation bulk chemical L-threonine to produce alpha-aminobutyric acid is simple and quick, and the cost is low. In a 5 L fermentation tank, the yield of alpha-aminobutyric acid obtained through the method can be 81.5 g / L, and an actually effective strategy is provided for industrial production of alpha-aminobutyric acid.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, in particular to a codon-optimized method for synthesizing the gene sequence of Candida boidinii formate dehydrogenase, which is connected in series with the amino acid dehydrogenase to the plasmid and placed in the large intestine Expression in bacillus, a method for efficiently preparing α-aminobutyric acid by using recombinant Escherichia coli for whole cell transformation. Background technique [0002] Unnatural α-amino acids are a large class of amino acids that are different from the 22 natural α-amino acids that can be synthesized by organisms themselves. They have important biological activities and physiological effects, and are widely used in compounds such as polypeptides, chiral drugs, and alkaloids. Synthesis. α-aminobutyric acid is an unnatural amino acid that inhibits the transmission of human nerve information. It can enhance the activity of glucose phosp...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/53C12N15/10C12P13/04
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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