Corynebacterium glutamicum for high yield of L-glutamine as well as construction method and application thereof

A technology of Corynebacterium glutamicum and glutamine, applied in the field of bioengineering, can solve the problems of low L-Gln conversion rate, poor fermentation performance, can not meet large-scale industrial production, etc., and achieves improved yield and conversion rate, high Conversion rate, effect of broad industrial application prospects

Pending Publication Date: 2020-04-03
新疆梅花氨基酸有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Corynebacterium glutamicum for high yield of L-glutamine as well as construction method and application thereof
  • Corynebacterium glutamicum for high yield of L-glutamine as well as construction method and application thereof
  • Corynebacterium glutamicum for high yield of L-glutamine as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Recombinant plasmids pK18mobsacB-ΔodhA, pK18mobsacB-ΔyggB, pK18mobsacB-glnA Y405F build

[0050] Obtain the nucleotide sequence of Corynebacterium glutamicum ATCC13032 odhA gene in NCBI GenBank database, design (knock out this gene ORF sequence 1-500 500bp nucleotide sequence) to introduce base deletion to make odhA gene For inactivation, four primers were synthesized based on the base sequence and the selected deletion position (as shown in Table 1). Using Phusion ultra-fidelity polymerase (New England BioLabs), using A1 / A2, A3 / A4 as primers, and using the genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template to prepare the upstream and downstream homology arms of the odhA base deletion site Fragment, wherein the odhA upstream homology arm sequence is shown in SEQ ID NO.16, and the odhA downstream homology arm sequence is shown in SEQ ID NO.17). The PCR program was denaturation at 98°C for 10s, renaturation at 50°C for 20s, extension at 72°C f...

Embodiment 2

[0055] Embodiment 2: construct the bacterial strain that odhA gene disrupts from Corynebacterium glutamicum species ATCC 13032 bacterial strain

[0056] Preparation of Competent Corynebacterium glutamicum: transfer Corynebacterium glutamicum species ATCC13032 from the -80°C refrigerator to a BHI plate and culture at 30°C; pick a single colony and transfer it to a 5ml BHI liquid medium test tube medium, 200rpm, 30°C for 12h; inoculate 1% inoculum into 100ml BHIS liquid medium (3.7% brain heart extract powder, 9.1% sorbitol), 200rpm, 30°C until the OD600 reaches 1.5; 4°C, Centrifuge at 6000rpm for 20min, collect the bacteria, discard the supernatant; suspend in TGBuffer (1mM Tris, 10% glycerol (v / v), pH7.5), centrifuge at 4°C for 20min at 6000rpm, discard the supernatant, and repeat ; suspend with 10% glycerol, centrifuge at 4°C, 6000rpm for 20min, discard the supernatant, and repeat again; add 1ml of 10% glycerol to suspend the bacteria, and pack. Store the competent cells in ...

Embodiment 3

[0059] Embodiment 3: construct the bacterial strain that yggB gene destroys from ATCC 13032 bacterial strain

[0060] Preparation of Competent Corynebacterium glutamicum: transfer Corynebacterium glutamicum species ATCC13032 from the -80°C refrigerator to a BHI plate, and culture at 30°C; pick a single colony and transfer to 5ml BHI liquid medium ( 3.7% brain heart extract powder) test tube, 200rpm, 30 ℃ culture 12h; Inoculate in 100ml BHIS liquid culture medium (3.7% brain heart extract powder, 9.1% sorbitol) by 1% inoculum amount, 200rpm, 30 ℃ cultivate to OD 600 reached 1.5; centrifuge at 6000rpm at 4°C for 20min, collect the bacteria, discard the supernatant; suspend with TG Buffer (1mM Tris, 10% glycerol (v / v), pH7.5) and centrifuge at 4°C at 6000rpm for 20min , discard the supernatant, and repeat; suspend with 10% glycerol and centrifuge at 4°C, 6000rpm for 20min, discard the supernatant, and repeat; add 1ml of 10% glycerol to suspend the cells and aliquot. Store the co...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses corynebacterium glutamicum for high yield of L-glutamine as well as a construction method and an application of corynebacterium glutamicum. The activity of alpha-ketoglutarate dehydrogenase and/or glutamic acid exporter protein in cells of the corynebacterium glutamicum for highly producing L-glutamine is lost, and adenylation modification of glutamine synthetase is removed. According to the corynebacterium glutamicum for highly producing L-glutamine disclosed by the invention, L-glutamine can be accumulated in a culture medium or cells, so that the yield of L-glutamine is increased to a great extent. The experiments show that the corynebacterium glutamicum disclosed by the invention is an L-glutamine high-yieldstrain; compared with an unmodified strain, the strain has the advantages that the capacity of producing L-glutamine is enhanced, L-glutamine can be effectively accumulated, the yield of L-glutamine is increased, the conversion rate is relatively high, a foundation is laid for industrial production of L-glutamine, and the strain has a wide industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yield L-glutamine Corynebacterium glutamicum and its construction method and application. Background technique [0002] L-glutamine (L-Glutamine), the scientific name is 2-amino-5-carboxypentanamide, the molecular formula is C 5 h 10 N 2 o 3 , with a molecular weight of 146. L-Glutamine is an indispensable conditional essential amino acid in the process of nitrogen source metabolism in the human body. It plays an important role in maintaining muscle metabolism, cell differentiation and growth, repairing tissue trauma, and neutralizing toxins in the body. [0003] L-glutamine widely exists in nature, for example, it is contained in pumpkin and sunflower seedlings in a free state. Although glutamine can be extracted from natural products, the production methods of L-Gln mainly include chemical synthesis, enzymatic synthesis and fermentation. The chem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/14C12R1/15
CPCC12N15/77C12P13/14C12N9/0008C12Y102/04002C07K14/34C12N9/93C12Y603/01002
Inventor 王成胡丹吴涛
Owner 新疆梅花氨基酸有限责任公司
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