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Corynebacterium glutamicum and its construction method and application

A technology of Corynebacterium glutamicum and a construction method, applied in the field of L-proline-producing Corynebacterium glutamicum and its construction, can solve the problem of poor fermentation performance, low L-proline conversion rate, and inability to meet large demands. problems such as large-scale industrial production, to achieve the effect of enhancing regeneration capacity and increasing output

Active Publication Date: 2017-10-03
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation performance of L-proline strains is still poor, and the conversion rate of L-proline is still low, which still cannot meet the needs of large-scale industrial production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of recombinant plasmid pK18mobsacB-gnd361 and introduction of point mutations in MHZ-0700

[0040] (1)pK18mobsacB-gnd 361 Plasmid construction

[0041] Using ATCC13870 genome as template, PCR amplification was performed with gnd-1f / gnd-1r primer pair to obtain the upstream fragment gnd 361 -up; Take ATCC13870 genome as template and use gnd-2f / gnd-2r primer pair (as shown in Table 1) to perform PCR amplification to obtain downstream fragment gnd 361 -dn. Gnd 361 -up, gnd 361 -dn mixture of two fragments as template, PCR amplification with gnd-1f / gnd-2r primer pair to obtain mutant gnd 361 Fragment. gnd 361 The fragment was double digested with EcoRI and BamHI, and pK18mobsacB was double digested with the same enzyme. The two digested products were ligated with T4DNA Ligase and transformed into Trans1T1 competent cells to obtain the recombinant plasmid pK18mobsacB-gnd 361 .

[0042] (2) Point mutations introduced in MHZ-0700

[0043] Competent cells of M...

Embodiment 2

[0046] Example 2: Fermentation of L-proline genetically engineered bacteria to produce L-proline

[0047] 1. Medium

[0048] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.2.

[0049] Seed medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, urea 0.1%, CaCO 3 0.5%, pH 7.2.

[0050] Fermentation medium: corn steep liquor 0.6%, glucose 12.0%, ammonium sulfate 3.7%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, CaCO 3 4%, VH 70μg / L, VB 1 · HCl 80μg / L, pH 7.2.

[0051] 2. MHZ-0701 shake flask fermentation to produce L-proline

[0052] (1) Seed culture: Pick MHZ-0700 and MHZ-0701 slant seeds 1 and connect them to a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium, and cultivate for 16-22 hours at 33°C and 220r / min;

[0053] (2) Fermentation culture: Inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of...

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PUM

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Abstract

The invention belongs to the field of microorganisms and particularly relates to Corynebacterium glutamicum and its construction method and application. Serine at the 361th site of a gnd gene for coding 6-phosphogluconate dehydrogenase in the cell of Corynebacterium glutamicum is mutated into phenylalanine. Corynebacterium glutamicum is used for point mutation of the gnd gene in the pentose phosphate pathway so that serine at the 361th site of 6-phosphogluconate dehydrogenase is replaced by phenylalanine and thus allosteric regulation of 6-phosphogluconate dehydrogenase is reduced, the ability of NADPH regeneration is improved, enough NADPH is supplied for proline synthesis and a proline yield is improved. An experiment result shows that the Corynebacterium glutamicum is an L-proline high-yield strain, effectively accumulates L-proline, improves an L-proline yield and lays the foundation for the industrial production of L-proline.

Description

Technical field [0001] The invention belongs to the field of microorganisms, and specifically relates to a Corynebacterium glutamicum and a construction method and application thereof, in particular to a Corynebacterium glutamicum that produces L-proline and a construction method and application thereof Background technique [0002] L-proline (L-proline), chemical name is L-pyrrolidone carboxylic acid, molecular formula is C 5 H 9 NO 2 , The relative molecular mass is 115. Proline is the basic unit of protein and is one of the 21 amino acids that make up human protein. Proline is widely used in medicine, food and flavoring, agriculture and organic chemistry. As an amino acid drug, proline is one of the raw materials for compound amino acid infusion, which is used for protein supplementation after malnutrition, protein deficiency, severe gastrointestinal diseases, burns and surgical operations. Proline can regulate the osmotic pressure of cells and stabilize the structure of pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/04C12R1/15
CPCC12N9/0006C12P13/04C12Y101/01044
Inventor 杨晟蒋宇钱峰慧杨俊杰程江红吴涛胡丹刁刘洋
Owner MEIHUA BIOTECH LANGFANG CO LTD
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