Strain for producing L-leucine and method for producing L-leucine

A technology of leucine and strain, applied in the field of bioengineering

Active Publication Date: 2017-05-31
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing leucine biosynthesis strains and methods have gradually been unable to meet the increasing market demand. Therefore, using bioengineering methods to further develop strains and production methods that can produce a large amount of leucine has become a research hotspot.

Method used

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  • Strain for producing L-leucine and method for producing L-leucine
  • Strain for producing L-leucine and method for producing L-leucine
  • Strain for producing L-leucine and method for producing L-leucine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Preparation has the mutant strain of leucine analogue 4-azaleucine resistance

[0073] The ATCC13869 strain was subjected to conventional mutagenesis treatment with ultraviolet 15W, 30cm, 20 minutes, and then with nitrosoguanidine (0.5mg / mL, 33°C, 30 minutes), and then coated with 1g / mL L's 4-azoleucine agar plate basic medium (glucose 20g / L, (NH4) 2 SO 4 2.0g / L, MgSO 4 ·7H 2 O 0.4g / L, CaCl 2 2H 2 O0.01g / L, FeSO 4 ·7H 2 O 0.02g / L, Na 2 HPO 4 12H 2 O 1.5g / L, Biotin 0.02mg / L, Vitamin B10.02mg / L, ZnSO4 0.01g / L, MnSO 4 0.01g / L, KH 2 PO 4 1.5g / L, agar 18g / L, pH 7.0-7.3), after standing at 33°C for 2-6 days, select the most resistant mutant strain MHZ-1200-1 to 4-azoleucine.

[0074] The obtained MHZ-1200-1 was activated on the brain heart infusion solid medium, and cultured at 33°C for 16-20h; scraped off a ring of bacteria from the plate, and inoculated it in 30mL seed medium (glucose 20g / L , urea 5g / L, yeast powder 10g / L, MgSO 4 ·7H 2 O 1.0g...

Embodiment 2

[0078] Example 2: Recombinant plasmid pK18mobsacB-leuA G561D Construction and introduction of leuA in ATCC13869 strain G561D mutation

[0079] Using the ATCC13869 genome as a template, use the leuA-1f / leuA-1r primer pair for PCR amplification to obtain the upstream fragment leuA-up; use the ATCC13869 genome as a template for PCR amplification with the leuA-2f / leuA-2r primer pair to obtain Downstream fragment leuA-dn. Using the mixture of leuA-up and leuA-dn fragments as a template, the leuA-1f / leuA-2r primer pair was used for PCR amplification to obtain leuA G561D Mutation target fragment. leuA G561D Mutation target fragment and pK18mobsacB vector were double digested with XbaI and SalI. The two digested products were ligated with T4 DNA Ligase for 1 h, and the ligated product was transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-leuA G561D .

[0080] According to the method in C.glutamicum Handbook, Charpter 23, ATCC13869 competen...

Embodiment 3

[0084] Example 3: Recombinant plasmid pK18mobsacB-ilvB G235S Construction and introduction of ilvB in ATCC13869 strain G235S mutation

[0085] Using the ATCC13869 genome as a template, the ilvB-1f / ilvB-1r primer pair was used for PCR amplification to obtain the upstream fragment ilvB-up; the ATCC13869 genome was used as a template, and the ilvB-2f / ilvB-2r primer pair was used for PCR amplification to obtain the downstream fragment Fragment ilvB-dn. Using the mixture of ilvB-up and ilvB-dn fragments as a template, the ilvB-1f / ilvB-2r primer pair was used for PCR amplification to obtain ilvB G235S Mutation target fragment. ilvB G561D Mutation target fragment and pK18mobsacB vector were double digested with XbaI and SalI. The two digested products were ligated with T4 DNA Ligase for 1 h, and the ligated product was transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-ilvB G235S .

[0086] According to the method in C.glutamicum Handbook,...

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Abstract

The invention relates to the technical field of biological engineering, in particular to a strain for producing L-leucine and a method for producing the L-leucine. According to the method, corynebacterium glutamicum is induced by using ultraviolet rays and nitrosoguanidine, then key mutants leuAG561D and ilvBG235S which are beneficial to generation of the L-leucine can be generated, research shows that under mutation conditions of leuAG561D and/or ilvBG235S, feedback inhibition of synthesis routes of the L-leucine can be released, the yield of the L-leucine can be greatly increased, a strain which can generate a great deal of the L-leucine can be obtained, the preservation number of the strain is CGMCC NO.13408, the strain is capable of achieving efficient accumulation of the L-leucine in the fermentation process, and the L-leucine can be up to 4.7g/L.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a strain for producing L-leucine and a method for producing L-leucine. Background technique [0002] L-leucine is α-amino-γ-methylpentanoic acid or α-aminoisocaproic acid, which belongs to branched-chain amino acids and is one of the eight essential amino acids that the human body must rely on external sources. L-leucine is the left-handed form of leucine, which is widely used in medicine, food, cosmetics, and feed industries. [0003] Extraction, chemical synthesis, enzyme catalysis and microbial fermentation are commonly used in the production of L-leucine. Among them, the microbial fermentation method has become the main method for the production of L-leucine due to its advantages of environmental protection, mild conditions, and stable quality. In the L-leucine production industry, Japanese companies play a leading role, especially the Japanese Ajinomoto Company, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P13/06C12R1/15
CPCC12N9/1022C12N9/1025C12P13/06C12Y202/01006C12Y203/03013
Inventor 常静胡丹程江红刁刘洋毛贤军
Owner MEIHUA BIOTECH LANGFANG CO LTD
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