Construction and application method for L-ornithine synthesis bacteria

A technology of ornithine and arginine, applied in the biological field, can solve the problems of restricting industrial application, low yield, carrying foreign plasmids, etc.

Inactive Publication Date: 2014-09-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned L-ornithine production strains still have problems such as low yield or carrying foreign plasmids, which limit their industrial application

Method used

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  • Construction and application method for L-ornithine synthesis bacteria
  • Construction and application method for L-ornithine synthesis bacteria
  • Construction and application method for L-ornithine synthesis bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1. Recombinant strain SYPA5-5△ arg Construction of F

[0027] (1) Recombinant knockout plasmid pK18mob sac B- arg Construction of F

[0028] Using SYPA5-5 genomic DNA as template, using F1 / F2, F3 / F4 as primers (Table 1), amplified arg F The upstream fragment is 517 bp, and the downstream fragment is 450 bp, obtained by crossover PCR amplification arg Deletion fragment in F gene (△ arg F) 950 bp ( figure 1 A). will get △ arg F linked into pK18mob sac B plasmid obtained recombinant plasmid pK18mob sac B△ arg F ( figure 1 B).

[0029] Table 1 Primers used in this study

[0030]

[0031] (2) Recombinant strain SYPA5-5△ arg F acquisition, screening and verification

[0032] The recombinant plasmid pK18mob sac B△ arg F was electroporated into SYPA5-5 competent cells, after two homologous recombination, the obtained transformants were extracted genome, PCR amplified and sent to Shanghai Sangong for sequencing. The results showed that ...

Embodiment 2

[0033] Example 2. Recombinant strain SYPA5-5△ arg Study on L-Ornithine Produced by Fermentation of F

[0034] (1) Effects of different concentrations of L-arginine on the recombinant strain SYPA5-5△ arg Effect of F Fermentation on Production of L-Ornithine

[0035] Add 0-5 g / L of L-arginine to the fermentation medium, and shake the flask for 96 h of fermentation, the results are as follows: figure 2 Shown in A: When 0-0.5 g / L L-arginine was added, the biomass of the bacteria and the output of L-ornithine increased with the increase of the concentration of L-arginine; as the concentration continued to increase, The biomass of bacteria and the output of L-ornithine decreased slightly. The effect of adding 0-0.5 g / L L-arginine was further studied ( figure 2 B), when the concentration of L-arginine is 0.3 g / L, the recombinant strain SYPA5-5△ arg The biomass of F and the yield of L-ornithine reached the highest value, which were 24.4 g / L and 21.3 g / L, respectively, which ...

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Abstract

The invention relates to construction and an application method for L-ornithine synthesis bacteria. An argF gene coding ornithine carbamoyltransferase (OTC) in corynebacterium crenatum SYPA5-5 is knocked out, and research on fermenting on an obtained recombination bacterial strain for producing L-ornithine is performed, and the technical scheme belongs to the technical field of biology. Firstly, an argF gene deletion segment (delta argF) is constructed and linked to pK18mobsacB for obtaining a recombinant plasmid pK18mobsacB delta argF, the recombinant plasmid pK18mobsacB delta argF is electrically transferred into SYPA5-5, two times of homologous recombination and screening are performed, so that a recombination strain SYPA5-5 delta argF is obtained. Secondly, influences of different nutrition conditions on growth of the recombination strain and accumulation of L-ornithine are investigated, addition of 0.3 g / L of L-arginine is capable of satisfying requirements on growth of SYPA5-5 delta argF and accumulation of L-ornithine; by utilizing a saccharic raw material as a raw material and fermenting for 96 h, the output of L-ornithine reaches 21.5 g / L, the saccharic acid conversion efficiency is 0.17 g (L-ornithine) / g (glucose).

Description

technical field [0001] Knockout Corynebacterium blunt tooth ( Corynebacterium crenatum ) SYPA5-5 encoding ornithine acetyltransferase (Ornithine carbamoyltransferase, OTC) arg F gene, and the research on the obtained recombinant strain to produce L-ornithine by fermentation, which belongs to the field of biotechnology. Background technique [0002] L-ornithine is a non-protein amino acid and an important intermediate metabolite of the urea cycle. It is widely used in the fields of food, medicine and chemical industry because of its important physiological functions. Microbial fermentation has the advantages of energy saving, environmental protection, easy extraction and separation, etc., and has important industrial application value. [0003] The key to realizing amino acid production by microbial fermentation lies in the selection of high-yield strains. As a technical means of rational breeding, metabolic engineering is widely used in the transformation of industrial m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N1/21C12P13/10C12R1/15
Inventor 许正宏窦文芳史劲松赵芹芹罗玉常张晓梅耿燕刘雪
Owner JIANGNAN UNIV
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