Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine

A Corynebacterium bacilli, metabolic modification technology, applied in the biological field, can solve the problems of low production capacity of L-arginine, long fermentation cycle, etc.

Inactive Publication Date: 2014-09-24
JIANGNAN UNIV
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  • Abstract
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Problems solved by technology

Compared with industrially produced L-arginine production strains, this strain still has the disadvantages of low L-arginine production capaci

Method used

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  • Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine
  • Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine
  • Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Releasing the Feedback Inhibition of L-Arginine on NAGK in SYPA5-5

[0046] (1) Recombinant plasmid pK18mobsacB- arg B H268N build

[0047] Obtained from NCBI arg BD sequence, design related primers, use SYPA5-5 genome as template, and use arg B-F / H268N-R, H268N-F / arg D-R are primers, respectively amplified to obtain H268N-5' / H268N-3' homologous fragments, using homologous fragments as templates, and arg B-F / arg D-R is the primer, amplified to obtain the H268N mutation point arg BD fragment, about 1863 bp. Ligate the mutant fragment into pK18mob sac B plasmid, obtain the recombinant plasmid pK18mob sac B- arg B H268N , the result is as figure 1 (A) shown.

[0048] (2) Construction of anti-feedback inhibition strain H-7

[0049] pK18mob sac B- arg B H268N The recombinant plasmid was transformed into SYPA5-5 by electrotransformation. After two times of homologous recombination, 12 transformants were selected for the second reco...

Embodiment 2

[0050] Example 2. Research on the fermentation characteristics of recombinant strain H-7

[0051] Shake flask fermentation was carried out on the starting strain SYPH5-8 and the recombinant strain H-7, and samples were taken every 8 h. The results were as follows: figure 2 shown. Depend on figure 2 (A) It can be seen that the biomass of the recombinant strain is lower than that of the starting strain throughout the fermentation process. Depend on figure 2 (B) It can be seen that the L-arginine production of the recombinant strain H-7 is 26.5 g / L, which is 13.4% lower than that of the original strain (30.6 g / L). Such as figure 2 As shown in (C, D), the recombinant strain accumulated a large amount of L-citrulline and L-ornithine, and the accumulation of L-citrulline reached 7.82 g / L, which was 4.0 higher than that of the original strain (1.56 g / L). times, the accumulation of L-ornithine reached 2.97 g / L, which was 2.7 times higher than that of the starting strain (0...

Embodiment 3

[0052] Example 3. Transcription analysis of genes related to L-arginine synthesis in recombinant strain H-7

[0053] against the recombinant strain H-7 arg The transcription of each gene in the gene cluster was analyzed, and the results were as follows: image 3 shown. arg There was no significant change in the transcript levels of related genes in the CJBDF transcription unit, but arg The transcript levels of G decreased by 4.3 times, respectively, arg The transcript level of H decreased by 2.6 times. arg G-encoded arginine succinate synthase (ASS) and arg The enzyme activity of H-encoded spermine succinate lyase (ASL) was also decreased. The above results show that the SYPA5-5 genome on arg The precise mutation of the H268N site of the B gene releases the feedback inhibition effect of L-arginine on NAGK, which can lead to arg The transcriptional level of GH decreased, which in turn caused the enzyme activities of ASS and ASL to decrease, which may be the main ...

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Abstract

The invention provides a metabolic transformation strategy for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine, and belongs to the biological technical field. Histidine at a site 268 in N-acetyl glutamatekinase, NAGK, argB of SYPA-5 is mutated into asparaginate, so that feedback inhibition effect of the L-arginine on NAGK can be effectively relived; a mutation site is precisely introduced to a corresponding position of SYPA5-5 genome, so that yield of the obtained feedback inhibition-preventing bacterial strain (H-7)L-arginine is lowered, and metabolic intermediate (L-ornithine and L-citrulline) is largely accumulated. Transcriptional analysis is carried out on an arg gene cluster of h-7 to find that arg GH transcriptional level is lowered. Arg GH reinforced expression is carried out in H-7, and production capacity of the obtained recombinant bacterial strain H-7-GH, L-arginine is improved by 99.0% in comparison with SYPA5-5. Preliminary optimization is carried out on a fermentation culture medium of H-7-GH, so that yield of the L-arginine is 45.1g/L, which is improved by 49.8% in comparison with that before optimization, and improved by 1.13 times in comparison with SYPA5-5. Feedback inhibition of the L-arginine on NAGK is relieved, and metabolic transformation strategy of the arg GH gene is expressed in a strengthened manner at the same time, so that production capacity of the L-arginine of the SYPA 5-5 is effectively improved.

Description

technical field [0001] The present invention uses Corynebacterium blunt tooth ( Corynebacterium crenatum ) SYPA5-5 is the starting strain, and the rate-limiting enzyme of its synthesis pathway—N-acetylglutamate kinase (N-acetylglutamate kinase, NAGK, arg B) Feedback inhibition while enhancing expression arg The metabolic transformation strategy of GH gene can effectively improve the L-arginine production capacity of the bacterial strain, and belongs to the field of biotechnology. Background technique [0002] L-Arginine (L-Arginine) is a semi-essential amino acid and an important intermediate metabolite of the urea cycle. It is widely used in the fields of medicine, food and chemical industry because of its various 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] At present, the demand for L-arginine in th...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12P13/10C12R1/15
Inventor 许正宏赵芹芹罗玉常窦文芳张晓梅耿燕史劲松饶志明
Owner JIANGNAN UNIV
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