Method for preparing L-alanine by using lactic acid and strain

A lactic acid bacteria, alanine dehydrogenase technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria and other directions, can solve the problems of difficult industrial production, complex fermentation broth composition, high price of malic acid, and achieve production Simple process, good prospects for industrial application, and the effect of high NAD content

Active Publication Date: 2019-10-08
卓虹超源生物科技(郑州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is also a method of co-immobilizing malic acid and L-alanine dehydrogenase with malic acid as a substrate, but the price of malic acid itself is very high, and it is difficult to realize industrial production
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  • Method for preparing L-alanine by using lactic acid and strain
  • Method for preparing L-alanine by using lactic acid and strain

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Embodiment 1

[0056] Using the method described in the literature Large scale validation of an efficient CRISPR / Cas-based multigene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1): 68, the corresponding gene on the Escherichia coli BL21 (DE3) genome was increased The moderate expression intensity constitutive promoter (PG) in front of the 3-phosphate glyceraldehyde dehydrogenase gene (gpdA) of Escherichia coli, the sequence is shown in SEQ ID NO:22.

[0057] When enhancing the expression of the gene lldP, use the Escherichia coli BL21 (DE3) genome as a template, and use primers lldP-FF / lldP-FR, lldP-gpdA-F / lldP-gpdA-R, lldP-RF / lldP-RR to amplify The upstream, promoter, and downstream sequences were fused with lldP-FF and lldP-RR as primers to form an expression cassette containing a gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including lldP ​​sgRNA) were transferred into Escherichia coli BL21 (DE3), Cas9 / sgRNA induced a double-strand break in...

Embodiment 2

[0066] Construction of recombinant Escherichia coli: Firstly, the genes encoding lactate dehydrogenase and L-alanine dehydrogenase were respectively connected to the double gene expression plasmid pETDuet-1. Various double-gene co-expression recombinant plasmids were obtained, and the plasmids were transformed into Escherichia coli BL21 (DE3), and positive transformants were obtained by screening with ampicillin plate, that is, recombinant Escherichia coli was obtained.

[0067] Induced expression was performed as in Example 1. After the induced expression was completed, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes.

[0068] The collected cells were analyzed for transformation, and the results are shown in Table 1. The whole cell transformation system is: cell wet weight 100g / L, L-lactic acid 50g / L, ammonium chloride 100g / L, pH 8.0, temperature 35°C, shaker speed 250 rpm; transformation time 5 hours.

[0069] Table 3 L-lactate dehydrogenase a...

Embodiment 5

[0078] According to the method for example 1, the medium expression strength constitutive promoter (PG) before the 3-phosphoglyceraldehyde dehydrogenase gene (gpdA) of Escherichia coli before icsA and / or nadA gene in Escherichia coli BLP is increased, and sequence is as SEQ ID NO :22. The plasmid is then reintroduced.

[0079]When enhancing the expression of the gene icsA, use the Escherichia coli BY genome as a template, and use primers icsA-FF / icsA-FR, icsA-gpdA-F / icsA-gpdA-R, icsA-RF / icsA-RR to amplify the upstream, starting sub, downstream sequences, and icsA-FF and icsA-RR primers were fused into an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including icsAsgRNA) were transferred into Escherichia coli BY, Cas9 / sgRNA induced a double-strand break in the host at the icsA gene locus, and the recombinase Red integrated the gpdA promoter into the front of the icsA gene, and sequenced verify.

[0080] When enhancing the...

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Abstract

The invention discloses an engineering bacteria and an application in preparation of L-alanine, which belongs to the technical field of bioengineering. The recombinant Escherichia coli of the presentinvention simultaneously express exogenous L-lactate dehydrogenase and L-alanine dehydrogenase, and enhances the expression of a lactate transporter gene and a NAD synthase gene on the basis of the host Escherichia coli. The method constructs the double-enzyme co-expressed engineering bacteria on the basis of engineering Escherichia coli transport and coenzyme synthesis system, and realizes efficient preparation of L-alanine by using cheap raw materials.

Description

technical field [0001] The invention relates to a method and strain for producing L-alanine by using lactic acid, and belongs to the technical field of bioengineering. Background technique [0002] L-alanine is an important amino acid, widely used in chemical industry, food, medicine and other fields. [0003] At present, the production of L-alanine mainly includes chemical method, enzymatic conversion method and fermentation method. The enzymatic conversion method is mainly produced through the decarboxylation of aspartic acid, but the price of aspartic acid itself is relatively high. There is also a method of co-immobilizing malic acid and L-alanine dehydrogenase with malic acid as a substrate, but the price of malic acid itself is very high, and it is difficult to realize industrial production. The production of L-alanine by genetically engineered bacteria using glucose as raw material for fermentation has the problems of low sugar-acid conversion rate and complex compo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/0006C12N9/0016C12P13/06C12Y101/01027C12Y104/01001
Inventor 蔡宇杰梁鑫鑫蒋静丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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