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Inositol-3-phosphate synthase mutant and application of inositol-3-phosphate synthase mutant in construction of corynebacterium glutamicum with high yield of glutamine

A technology of Corynebacterium glutamicum and phosphate synthase, applied in the field of bioengineering, can solve problems such as poor fermentation performance, unsatisfactory glutamine conversion rate, etc., and achieve the effect of enhancing the ability to produce glutamine

Active Publication Date: 2021-08-03
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 the strains producing glutamine is still poor at present, the conversion rate of glutamine is not ideal, and the demand for glutamine is extremely high in industry, and the existing strains cannot meet the needs of large-scale industrial production at all.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The isolation of embodiment 1 mutant strain Q8 and the performance of producing glutamine

[0039] It is known that Corynebacterium glutamicum MHZ-0513-3 (strain preservation number is CGMCC No. 13405) can be used as a glutamine-producing bacterium to carry out glutamine fermentation, isolate, purify and rejuvenate it.

[0040] The activation steps are as follows:

[0041]The MHZ-0513-3 strain in the glycerol tube was activated on the slant medium, cultured at 33°C for 24 hours, then cultured overnight at 33°C in the brain heart infusion liquid test tube, diluted 10 5 , 10 6 , 10 7 Spread on a slant medium plate, and select different strains Q1-Q12 from the plate for shake-flask fermentation. It was unexpectedly found that the glutamine content of a strain Q8 increased from 28.7g / L to 30.9g / L.

[0042] The medium formula is as follows:

[0043] Slant medium: brain heart infusion 37g / L, agar 1.8%, sterilized at 0.1MPa at 121°C for 20min;

[0044] The seed medium is...

Embodiment 2

[0049] Example 2 Sequencing found point mutations in the nucleic acid sequence of inositol-3-phosphate synthase

[0050] In view of the obvious increase in the glutamine content of the Q8 strain, the genes related to the glutamine synthesis and metabolic pathways were sequenced, but these genes were all consistent with MHZ-0513-3, so the strain MHZ-0513-3 was sequenced. Whole genome sequencing, the sequencing results show that the 84th amino acid of inositol-3-phosphate synthase is mutated from serine (TCC) to alanine (GCC), its amino acid sequence is shown in SED ID NO: 1, and its nucleotide sequence is shown in SED ID NO: 2 (including the upstream homology arm and the outer identification primer part).

[0051] It is known that inositol-3-phosphate synthase is related to the synthesis of cell wall and AcCys-GlcN-Ins (MSH), and inactivation will reduce the survival rate of bacteria under oxidative stress conditions. It is speculated that point mutations may lead to increased ...

Embodiment 3

[0052] Example 3 Recombinant plasmid pK18-ino-1 S84A Construction and introduction of point mutations in the MHZ-0513-3 strain

[0053] Using Phusion ultra-fidelity polymerase (New England BioLabs), UP-1F / UP-1R, DN-2F / -DN-2R as a primer pair, using the genome of Corynebacterium glutamicum MHZ-0513-3 as a template, prepare For the recombinant fragment, the PCR program was denatured at 98°C for 10s, annealed at 50°C for 20s, extended at 72°C for 15s, and cycled 30 times. -2R is a primer pair, and the upper and lower homology arms are used as templates to prepare recombinant fragments. The PCR program is denaturation at 98°C for 10s, annealing at 50°C for 20s, extension at 72°C for 30s, and 30 cycles. The recovery kit (Tiangen) was purified, and then digested with XbaI / PstI. At the same time, pK18-mobsacB was digested with XbaI / PstI, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and transformed into Trans1T1 competent cells ( TransGen Biot...

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Abstract

The invention relates to the field of bioengineering, in particular to an inositol-3-phosphate synthase mutant and application of the inositol-3-phosphate synthase mutant in construction of corynebacterium glutamicum for high yield of glutamine. It is found that after 84th-site serine of inositol-3-phosphate synthase is mutated into other amino acids, the glutamine production capacity of corynebacterium glutamicum can be enhanced in corynebacterium glutamicum. Compared with a wild strain without mutation, the corynebacterium glutamicum has the advantages that the glutamine production capacity of the corynebacterium glutamicum is enhanced, the glutamine yield is increased to 30.7 g / L from 28.5 g / L, the acid production is increased by 7.7%, and screening and construction of a glutamine high-yield strain are facilitated.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an inositol-3-phosphate synthase mutant and its application in constructing high glutamine-producing Corynebacterium glutamicum. Background technique [0002] Glutamine is a non-essential amino acid. The chemical name is 2-amino-4-carbamoylbutyric acid. Glutamine is an encoded amino acid in protein synthesis, which can promote protein synthesis and inhibit protein decomposition. It can be used to treat gastric and duodenal ulcers, and plays an important role in the pharmaceutical industry. [0003] At present, the most commonly used production method of glutamine is the fermentation method, and Corynebacterium glutamicum is mainly used as the production bacterium to ferment and produce glutamine. Corynebacterium glutamicum is a heterotrophic aerobic type, a Gram-positive bacterium, which has the characteristics of fast growth, non-pathogenicity, and weak degradation ability to its...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/77C12N1/21C12P13/14C12R1/15
CPCC12N9/90C12N15/77C12P13/14C12Y505/01004
Inventor 栾明月程江红吴涛李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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