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A metabolically modified Bacillus subtilis biotransformation cell and its preparation method and application

A technology of Bacillus subtilis and biotransformation, which is applied in the field of genetic engineering metabolic transformation and microorganisms, can solve problems such as accumulation of by-products, side reactions, degradation of substrates or products, etc., so as to increase the conversion rate, increase the effective conversion rate, and improve the conversion rate. efficiency effect

Active Publication Date: 2020-01-21
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] Whole-cell catalysis has been rapidly applied in industry due to the advantages of diversity and ease of operation, but there are also the following problems: the permeability of the substrate across the membrane affects the final conversion rate; side reactions lead to substrate or product degradation; problems with bypass reactions and by-product accumulation
However, there are few reports on the whole-cell transformation experiments using recombinantly transformed Bacillus

Method used

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  • A metabolically modified Bacillus subtilis biotransformation cell and its preparation method and application
  • A metabolically modified Bacillus subtilis biotransformation cell and its preparation method and application
  • A metabolically modified Bacillus subtilis biotransformation cell and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of recombinant Bacillus subtilis:

[0038]The gene sequence of L-glutamic acid oxidase (LGOX) derived from Streptomyces sp.X-119-6 was synthesized through codon optimization (the amino acid sequence of L-glutamic acid oxidase is shown in SEQ ID No.1. shown; the gene sequence of the L-glutamic acid oxidase is shown in SEQ ID No.2), and BamH Ⅰ and EcoR Ⅰ restriction sites were added to the 5' end and 3' end respectively, and the above-mentioned enzyme was synthesized by the company The LGOX gene was cloned into the pUC57 vector to obtain the recombinant plasmid pUC57-LGOX. Then, the above-mentioned LGOX gene was connected to the pHY-Bs.xyl inducible expression vector through molecular cloning to obtain a recombinant plasmid containing the L-glutamic acid oxidase gene. Plasmid pHY-Bs.xyl-LGOX, verified by double digestion with BamH Ⅰ and EcoR Ⅰ ( figure 1 ) to obtain target fragments of corresponding size.

[0039] Transform the inducible expressio...

Embodiment 2

[0040] Example 2 Knockout of gene glnA and integration of target gene

[0041] (1) Using primers GlnA-F and GlnA-R as upstream and downstream primers, amplify the gene glnA (glutamine synthetase) from Bacillus subtilis WB600 by PCR, and connect it to the pMDT19-simple plasmid vector by T-A cloning , to construct recombinant plasmids, respectively marked as pMD-glnA, select two suitable restriction sites Nco Ⅰ and Pst Ⅰ ​​in the above-mentioned amplified gene, respectively, after double digestion, remove the 818bp part of the glnA gene fragment, and recover from the gel Obtain a fragment of 3210bp, that is, use the remaining part as a homology arm;

[0042] (2) Using the B. subtilis WB600 genome as a template, P43-Nco Ⅰ-F, P43-R-LGOX as upstream and downstream primers to amplify the P43 promoter with a size of 426bp, using pUC57-LGOX as a template, P43-LGOX-F , LGOX-Pst Ⅰ-R as upstream and downstream primers to amplify the LGOX gene, using the amplified LGOX and P43 as templat...

Embodiment 3

[0049] Construction of embodiment 3 recombinant strain WB604

[0050] The expression plasmid pHY-Bs.xyl-LGOX was transformed into WB603 according to the transformation method, and the recombinant bacterium WB604 was constructed. The specific implementation method is as follows:

[0051] Medium: Bacillus subtilis B. subtilis WB600 transformation medium:

[0052] SP Ⅰ-a solution (g·L -1 ): (NH 4 ) 2 SO 4 4,K 2 HPO 4 ·3H 2 O 28, KH 2 PO 4 12. Sodium citrate dihydrate 2;

[0053] SP Ⅰ-b solution (g·L -1 ): MgSO 4 ·7H 2 O 0.4;

[0054] 500g·L -1 glucose solution;

[0055] 100×CAYE solution (g·L -1 ): casamino acids 20, yeast powder 100;

[0056] CaCl 2 Solution: 50mmol·L -1 ;

[0057] MgCl 2 Solution: 250mmol·L -1 ;

[0058] 100×EGTA solution: Weigh 3.8g of EGTA (ethylene glycol ditetraacetic acid) and dissolve it in 1L of deionized water, adjust the pH to 8.0 with NaOH, filter and sterilize, and store at -20°C for later use;

[0059] Sterilize the reagent...

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Abstract

The invention discloses a metabolism transformation Bacillus subtilis biotransformed cell. The preparation method comprises: carrying out recombinant over-expression to obtain a L-glutamic acid oxidase, carrying out metabolism engineering transformation on the endogenous glutamic acid decomposition pathway of Bacillus subtilis, and finally transforming the glutamic acid transport pathway of Bacillus subtilis to prepare the Bacillus subtilis biotransformed cell, ie., the recombinant Bacillus subtilis. According to the present invention, the experimental basis is provided for the construction and the improvement of the whole cell transformation system involved in cellular endogenous metabolism, and the reference is provided for the production of alpha-ketoglutaric acid through the efficient whole cell transformation of L-glutamic acid with Bacillus subtilis.

Description

technical field [0001] The invention relates to the field of genetic engineering metabolic modification and microorganism technology, in particular to a recombinant bacillus subtilis containing L-glutamic acid oxidase gene and a preparation method thereof. Background technique [0002] Biotransformation refers to the process of using biological methods to synthesize organic compounds, that is, the process of using whole cells or extracted enzymes as catalysts to convert substrates into target products. [0003] Biotransformation actually utilizes active enzymes. Therefore, the catalyzed reaction has the following characteristics: specificity of reaction, stereoselectivity, and less demanding reaction conditions. Compared with traditional chemical synthesis methods, biocatalytic The method can be carried out under the conditions of low pollution, low energy consumption, and high specificity. It can replace multi-step chemical reactions into one-step enzyme-catalyzed reactions...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12P7/50C12R1/125
CPCC07K14/32C12N9/0022C12N9/93C12N15/75C12N2800/22C12P7/50C12Y104/03011C12Y603/01002
Inventor 石贵阳李由然陈稳吴志勇张梁丁重阳顾正华
Owner JIANGNAN UNIV
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