Gamma-PGA polymerase gene recombinant strain as well as construction method and application thereof

A technology of gene recombination and construction method, which is applied in the field of synthetic biology, can solve problems such as the inability to accurately control the molecular weight of γ-PGA, and achieve the effect of precise regulation

Active Publication Date: 2021-01-05
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, it still cannot achieve precise control of the molecular weight of γ-PGA to meet different application requirements

Method used

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  • Gamma-PGA polymerase gene recombinant strain as well as construction method and application thereof
  • Gamma-PGA polymerase gene recombinant strain as well as construction method and application thereof
  • Gamma-PGA polymerase gene recombinant strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant strains that individually regulate the polymerase gene

[0037] Utilize NCBI to look up the gamma-PGA polymerase gene capB, capC, capA from B.licheniformis bacterial strain, design the amplification primer (such as table 1, sequence such as SEQ ID NO.1- Shown in SEQ ID NO.6), according to the construction method of the above-mentioned recombinant strains Cg 1 / 10BCA, Cg B1 / 10CA and Cg BC1 / 10A, PCR amplification, double digestion, connection, transformation and verification to construct recombinant plasmid pZMI (Ptac) -capB, pZMI(Ptac)-capC and pZMI(Ptac)-capA. Then, using the homologous enzymes Avr II and Nhe I, the two gene fragments to be connected in series were respectively subjected to double enzyme digestion, connection, and transformation of the two gene fragments to be connected in series, which were then transformed into C .glutamicum F343, carry out screening in the Kan culture medium that contains 25 μ g / L, pick transfor...

Embodiment 2

[0040] Embodiment 2: Recombinant bacterial strain Cg 1 / 10BCA, Cg B1 / 10CA and Cg BC1 / 10A shaking flask fermentation performance detection

[0041] The recombinant strains Cg 1BCA and Cg 10BCA, Cg B1CA and Cg B10CA, Cg BC1A and Cg BCA10A were subjected to shake flask fermentation according to the above Corynebacterium glutamicum transformation method (plasmid) method, and their fermentation characteristics were evaluated. The recombinant strains without RBS regulation C. glutamicum pZMI-capBCA (Cg BCA) was used as a control strain. The fermented liquid is sampled every 4h for the determination of bacterial growth, γ-PGA output and biomass. The results are as follows: figure 2 , 3 shown.

[0042] (1) Comparison of recombinant bacterial biomass

[0043] According to the intensity of BCD-RBS to compare the growth of each recombinant bacteria, by figure 2 It can be found that the recombinant bacteria Cg BC1A grows best, and its biomass reaches 12.36 in 24h, and the biomass of ...

Embodiment 3

[0054] Example 3: Molecular weight change of γ-PGA produced by recombinant strains Cg 1 / 10BCA, Cg B1 / 10CA and Cg BC1 / 10A The molecular weight of γ-PGA synthesized by fermentation of CgBCA10A was detected. First, draw a calibration curve using the molecular weight and peak retention time of the dextran standard, such as Figure 4 Get the functional relationship y=-0.739363x+17.3566 (R 2 =0.997567), and then calculate the molecular weight according to the peak time of the recombinant γ-PGA sample. like Figure 5 , and the maximum molecular weight of the control strain Cg BCA (36h, 3.5×10 3 Compared with kDa), the molecular weight of the recombinant strain Cg BC1A is greatly improved, and it can reach the maximum molecular weight of about 2.8×10 at 48h. 4 kDa; at the same time, the molecular weight of the recombinant strain Cg B10CA also increased significantly, reaching a maximum molecular weight of 1.4×10 at 36h 4 kDa.

[0055] Furthermore, from Figure 5 It can be seen ...

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Abstract

The invention discloses a gamma-PGA polymerase gene recombination strain as well as a construction method and application thereof, relates to a production process for synthesizing gamma-polyglutamic acid from a sugar raw material through one-step fermentation, and belongs to the field of synthetic biology and fermentation engineering. According to the invention, a gene cluster capBCA of gamma-polyglutamic acid synthetase of naturally produced bacillus licheniformis is cloned into corynebacterium glutamicum F343 with high yield of glutamic acid for exogenous expression, and on the basis, the molecular weight of gamma-PGA produced by recombinant strains Cg 1/10BCA, Cg B1/10CA and Cg BC1/10A obtained by regulating and controlling the expression level of each gene of a synthetase gene clusterby utilizing RBS regulating and controlling elements with different strengths is 295.47-28018 kDa. According to the method, an exogenous synthesis path of polyglutamic acid is successfully constructed, the purpose of reasonably controlling the molecular weight of gamma-PGA is achieved, raw materials and process control cost are saved, economic benefits are improved, the application range of polyglutamic acid is expanded, and the method has very good industrial application value and prospect.

Description

technical field [0001] The invention relates to the application of synthetic biology technology, in particular to a γ-PGA polymerase gene recombinant strain and a construction method and application technology thereof. The molecular weight is regulated by regulating γ-polyglutamic acid synthase, and belongs to synthetic biology and fermentation engineering. field. Background technique [0002] γ-Polyglutamic acid (γ-PGA) is a biopolymer polymerized from L-glutamic acid and D-glutamic acid monomers. It has properties such as thickening ability, and has excellent absorption and binding ability to heavy metal ions. In recent years, γ-PGA has been widely used in food, cosmetics, biomedicine, environmental protection and other fields. The molecular weight of polyglutamic acid is an important factor that limits its application. For example, the molecular weight of polyglutamic acid used in the field of water treatment needs to be about 150-200kDa, while the polyglutamic acid of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N1/21C12N15/52C12P13/02A23L29/269A61K8/88A61K47/34A61Q19/00C12R1/15
CPCC12N15/77C12N15/52C12N9/93C12Y603/02C12P13/02A23L29/269A61K47/34A61K8/88A61Q19/00C12N2830/20A61K2800/10A61K2800/48
Inventor 许正宏徐国强曹蓉段艳婷王籍阅张晓娟张晓梅史劲松
Owner JIANGNAN UNIV
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