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Genetic engineering compound bacteria and application thereof in biosynthesis of PGA (phenylglyoxylic acid)

A technology for acetophenone acid and biosynthesis, which is applied in the directions of genetic engineering, application, plant genetic improvement, etc., can solve the problems of increasing separation cost and high cost of mandelic acid

Pending Publication Date: 2018-09-07
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high cost of splitting mandelic acid itself, it is unprofitable for manufacturers to synthesize acetophenone acid with D-mandelic acid as a substrate; if racemic mandelic acid is used as a substrate for synthesis, the theoretical conversion rate is only 50%, the remaining L-mandelic acid also increases the cost of subsequent separation

Method used

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  • Genetic engineering compound bacteria and application thereof in biosynthesis of PGA (phenylglyoxylic acid)
  • Genetic engineering compound bacteria and application thereof in biosynthesis of PGA (phenylglyoxylic acid)
  • Genetic engineering compound bacteria and application thereof in biosynthesis of PGA (phenylglyoxylic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Gene mining of genes encoding L-lactate dehydrogenase:

[0053] Using the highly active L-LDH (OJF74586) derived from Lactobacillus casei as a probe, BLAST analysis was performed based on the non-redundant database, and a series of genome information sources without expression identification and putative genes were found from the query results. L-lactate dehydrogenase sequence. Construct and analyze the phylogenetic tree of these sequences, and then select 1 to 2 representative gene sequences from each branch. For the target gene that is easy to obtain from the strain, the target gene can be obtained by PCR amplification using the genome as a template. The obtained gene was codon-optimized for whole-gene synthesis. After comparative analysis, the Lactobacillus helveticus genome was selected as a potential L-lactate dehydrogenase for further research.

[0054] Taking the codon usage frequency of E.coli K12 strain as a reference, the OPTIMIZER server was used to optimize...

Embodiment 2

[0056] Gene mining of the gene encoding mandelate racemase:

[0057]Using the highly active PpMR derived from Pseudomonas putida as a probe, BLAST analysis was performed based on the non-redundant database, and a series of putative racemases that were not expressed and identified and were found from the query results were found sequence. Construct and analyze the phylogenetic tree of these sequences, and then select 1 to 2 representative gene sequences from each branch. For the target gene that is easy to obtain from the strain, the target gene can be obtained by PCR amplification using the genome as a template. The obtained gene was codon-optimized for whole gene synthesis, and Herbaspirillum rubrisubalbicans genome source was selected as a potential mandelate racemase for further research after comparative analysis.

[0058] Taking the codon usage frequency of E.coli K12 strain as a reference, the OPTIMIZER server was used to optimize the codon of the potential HrMR gene se...

Embodiment 3

[0060] Construction and expression analysis of genetic engineering bacteria producing D-mandelate dehydrogenase (LhDMDH) and L-lactate dehydrogenase (LhLDH):

[0061] Step 1. Construction of a recombinant plasmid: link the coding genes of D-mandelate dehydrogenase (LhDMDH) and L-lactate dehydrogenase (LhLDH) to the pETDuet1 plasmid respectively to obtain the recombinant plasmid pETDuet1-LhDMDH-LhLMDH;

[0062] Step 2: Transform the recombinant plasmid into host cells: transform the recombinant plasmid pETDuet1-LhDMDH-LhLMDH into Escherichia coli BL21 (purchased from Invitrogen) competent cells by heat shock, add 0.4mL LB liquid medium, and heat at 37°C, 220rpm After incubation for 1 hour under high temperature, spread on LB solid plates containing 50 μg / mL ampicillin, and incubate at 37°C for 12-16 hours to obtain monoclonal colonies;

[0063] Step 3. Screening and identification of recombinant bacteria: Pick single-clonal colonies into 4 mL of LB liquid medium containing 50 μ...

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Abstract

The invention discloses genetic engineering compound bacteria and an application thereof in biosynthesis of PGA (phenylglyoxylic acid). The genetic engineering compound bacteria contain a novel L-lactate dehydrogenase gene LhLDH, an existing D-mandelate dehydrogenase gene and encoded genes thereof as well as a novel mandelate racemase gene HrMR, the genetic engineering compound bacteria are utilized for production and application of PGA synthesized through whole-cell catalysis of racemic mandelic acid, the production process is simple, the conditions are mild, the efficiency is high and the cost is low. The genetic engineering compound bacteria have the benefits as follows: the constructed genetic engineering compound bacteria can realize high yield of D-mandelate dehydrogenase, L-lactatedehydrogenase and mandelate racemase; on the basis of whole-cell catalysis, the genetic engineering compound bacteria can efficiently oxidize racemic mandelic acid for production of PGA without addition of coenzymes. Through the genetic engineering compound bacteria, the conversion rate of racemic mandelic acid can be 96% or above, the purity of PGA can be 99% or above, and the genetic engineeringcompound bacteria have good industrial prospects.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a genetically engineered compound bacterium and its application in the biosynthesis of acetophenone. Background technique [0002] Phenylglyoxylic acid (PGA), also known as benzoylformic acid, belongs to α-keto acid compounds and is an important synthetic building block. A variety of important pharmaceutical intermediates can be synthesized using acetophenone as raw materials . Because acetophenone acid is easily oxidized, decarboxylated and decarbonylated, its synthesis is relatively difficult. The reported synthetic methods mainly include benzoyl nitrile hydrolysis, styrene oxidation, mandelic acid oxidation, Friedel-Crafts acylation and Biocatalytic synthesis, wherein mandelate dehydrogenase plays an important role in the biosynthesis of acetophenone. Through the unremitting efforts of many scientific researchers, the catalytic activity of D-mandelate dehydrogenase ha...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12N9/90C12N15/61C12P7/40
CPCC12N9/0006C12N9/90C12N15/70C12P7/40C12Y101/01027C12Y501/02002
Inventor 唐存多史红玲焦铸锦史鸿飞姚伦广阚云超
Owner NANYANG NORMAL UNIV
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