Transformant of coryneform bacterium and method for producing valine by using same

一种棒状细菌、转化体的技术，应用在缬氨酸生产领域，能够解决缬氨酸生产率不充分等问题，达到高效制造的效果

Active Publication Date: 2014-07-23
RES INST OF INNOVATIVE TECH FOR THE EARTH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the productivity of valine in the methods of Patent Documents 1 and 2 is not practically sufficient.

Method used

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  • Transformant of coryneform bacterium and method for producing valine by using same
  • Transformant of coryneform bacterium and method for producing valine by using same
  • Transformant of coryneform bacterium and method for producing valine by using same

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preparation example Construction

[0141] As the method for preparing the reaction solution under reducing conditions, known methods can be used without limitation. For example, an aqueous solution for the reaction liquid can be used instead of distilled water or the like as a liquid medium for the reaction liquid, and the preparation method for the aqueous solution for the reaction liquid can refer to the method for preparing a culture liquid for absolute anaerobic microorganisms such as sulfuric acid reducing microorganisms (Pfennig, N et.al. (1981): The dissimilatory sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria, Ed.by Starr, M.P.et.al.p.926-940, Berlin, Springer Verlag.) or " Agrochemical Experiment Book Volume 3, edited by Agrochemical Classroom, Faculty of Agriculture, Kyoto University, 26th printing in 1990, published by Sangyo Shoshu Co., Ltd., etc. to obtain an aqueous solution under desired reducing conditions.

[0142] Specifically, an a...

Embodiment 1

[0150] Cloning of embodiment 1 mutant acetohydroxy acid synthase gene

[0151] (1) Acquisition of valine analogue-resistant strains

[0152] Corynebacterium glutamicum R strain was exposed to 300 μg / ml of N-methyl-N'-nitro-N-nitrosoguanidine at 30°C for 1 hour, and then placed in medium A [Will (NH 2 ) 2 CO2g, (NH 4 ) 2 SO 4 7g, KH 2 PO 4 0.5g, K 2 HPO 4 0.5g, MgSO 4 ·7H 2 O0.5g, 0.06% (w / v) Fe 2 SO 4 ·7H 2 O+0.042% (w / v) MnSO 4 2H 2 01ml, 0.02% (w / v) biotin solution 1ml, 0.01% (w / v) thiamine solution 2ml, yeast extract 2g, vitamin determination casamino acid 7g are dissolved in distilled water 1L] and cultivated to about 10 9 cells / ml. The bacterial cells were recovered, washed with a minimal medium (BT medium), and spread on a BT plate medium containing 4% glucose and 4% DL-α-aminobutyric acid as a valine analogue. Cultured at 30°C for 5 days.

[0153] The mutant strain grown on the plate medium containing a valine analogue was inoculated into a test t...

Embodiment 2

[0161] Cloning and expression of embodiment 2 valine production gene

[0162] (1) Extract chromosomal DNA from microorganisms

[0163] The operation of extracting chromosomal DNA from Corynebacterium glutamicum (Corynebacterium glutamicum) R (FERM P-18976) is carried out as follows: in A medium [Will (NH 2 ) 2 CO2g, (NH 4 ) 2 SO 4 7g, KH 2 PO 4 0.5g, K 2 HPO 4 0.5g, MgSO 4 ·7H 2 O0.5g, 0.06% (w / v) Fe 2 SO 4 ·7H 2 O+0.042% (w / v) MnSO 4 2H 2 01 ml, 0.02% (w / v) biotin solution 1 ml, 0.01% (w / v) thiamine solution 2 ml, yeast extract 2 g, vitamin assay casamino acids 7 g were dissolved in 1 L of distilled water] to reach a final concentration of 4 Add 50% (w / v) glucose solution as a carbon source in the manner of %, inoculate the bacterial strain with a platinum loop, shake and cultivate it to the logarithmic growth phase at 33°C, and use the DNA genome extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit (manufactured by Amersham Co.) was ...

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Abstract

This transformant is obtained by introducing, to a host coryneform bacterium, at least one of the DNAs (a), (b), and (c): (a) a DNA, or an analog thereof, obtained by introducing, to a DNA that encodes an acetohydroxy acid synthase derived from Corynebacterium glutamicum, a mutation that mutates the No. 156 glycine in the amino acid sequence encoded by this DNA to glutamic acid (G156E); (b) a DNA, or an analog thereof, obtained by introducing, to a DNA that encodes an acetohydroxy acid isomeroreductase derived from Corynebacterium glutamicum, mutations that mutate the No. 34 serine in the amino acid sequence encoded by this DNA to glycine (S34G), mutate the No. 48 leucine to glutamic acid (L48E), and mutate the No. 49 arginine to phenylalanine (R49F); and (c) a DNA, or an analog thereof, that encodes a leucine dehydrogenase derived from Lysinibacillus sphaericus.

Description

technical field [0001] The invention relates to valine production technology. More specifically, it relates to a transformant of a coryneform bacterium subjected to a specific genetic manipulation for conferring a valine production function, and a method for producing highly efficient valine using the transformant. Background technique [0002] In the context of global warming and the depletion of fossil resources, it has been recognized that chemical manufacturing using renewable resources as raw materials and biofuel manufacturing as a new industry biorefining have become an important strategy for realizing a low-carbon society, and focused on Widespread concern. [0003] Valine, one of the essential amino acids, is useful as a component of medicines, foods, cosmetics, livestock feed additives, and the like. Conventionally, valine was produced by fermentation and hydrolysis of protein. [0004] However, compared with the production of lactic acid and ethanol, the produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12P13/08C12R1/15
CPCC12P13/08C12N9/0006C12N9/0016C12N9/1022C12Y101/01086C12Y104/01009C12Y202/01006C12N15/77Y02P20/52
Inventor 汤川英明乾将行
Owner RES INST OF INNOVATIVE TECH FOR THE EARTH
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