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Construction and application of high-yield L-valine engineering bacteria

A technology of heterogeneous and recombinant bacteria, applied in the biological field, can solve problems such as unknown genetic background, limited yield, heavy workload, etc., and achieve the effect of reducing the probability of bacterial infection and increasing yield

Active Publication Date: 2015-04-29
FUJIAN NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional strain breeding is mainly based on the fact that there are strains capable of producing L-valine in nature, combined with repeated mutagenesis and breeding, and improving the yield of L-valine through optimized culture, but the amino acid that can be accumulated by this method is limited. The level is limited, the method of mutation breeding is blind, the workload is heavy, the genetic background is unknown, it is difficult to increase the yield through further mutagenesis or transformation, so the yield is greatly limited
Moreover, the strains obtained by mutagenesis are generally weak in anti-miscellaneous bacteria, which is very easy to cause bacterial contamination, and is not conducive to industrial scale-up production.

Method used

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  • Construction and application of high-yield L-valine engineering bacteria
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  • Construction and application of high-yield L-valine engineering bacteria

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Experimental program
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Embodiment 1

[0035] Embodiment 1, the construction of high-yielding L-valine engineering bacteria

[0036] 1. Recombinant vector pZ8-1BN * C build

[0037] 1. pMDBN * Construction of C vector

[0038] 1) According to the genome information and gene function annotation of Corynebacterium glutamicum ATCC13032 published in NCBI, combined with the analysis of the metabolic pathway of L-valine, primers BNC-F and BNC-R were designed, and the genome of Brevibacterium flavum 20160 was used as the Template, PCR amplification of the ilvBN gene and ilvC gene connected in series in the gene, as well as the ribosome binding site sequence (RBS) carried by them, to obtain the amplified product - the ilvBNC gene fragment.

[0039] BNC-F:5'- GAATTCAGTAAAGGAGCCAGAAAGTCGTG-3';

[0040] BNC-R:5'- GGATCC GTACAAAGTGCACAGCAGGTAGC-3'.

[0041] Primer BNC-F has an EcoRI restriction site (underlined sequence), and primer BNC-R has a BamHI restriction site (underlined sequence).

[0042] PCR amplification...

Embodiment 2

[0087] Embodiment 2, the application of Brevibacterium flavum MDV-07 in the production of L-valine

[0088] 1. Preparation of culture medium

[0089] Seed culture medium (pH=6.5-7.5): take 100g glucose, 5g corn steep liquor, 100g soybean cake powder enzymolysis solution, 3g yeast extract, 5g (NH 4 ) 2 SO 4 , 0.2g MgSO 4 ·7H 2 O, 0.3g KH 2 PO 4 ·3H 2 O, 0.03g kanamycin monosulfate and 10g calcium carbonate are dissolved in deionized water and settled to 1L;

[0090] The preparation method of soybean cake powder enzymatic hydrolysis solution is: grind 100g soybean powder, add 400mL deionized water and mix thoroughly, adjust the pH value to 6.5-7.0, add 1% final concentration of neutral protease (purchased from Shanghai Jiahe Biotechnology Co., Ltd. Technology Co., Ltd.), enzymolysis at 60°C for 3 hours to prepare soybean cake powder enzymatic hydrolysis solution. Sterilize at 125°C for 25 minutes.

[0091] Fermentation medium (pH=6.5-7.5): take 120g glucose, 20g corn s...

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Abstract

The invention discloses construction and an application of high-yield L-valine engineering bacteria and provides a method for preparing recombinant bacteria. The method comprises steps as follows: an acetohydroxyacid synthase mutant coding DNA module, an acetohydroxyacid isomerism reductase coding DNA module, a branched chain amino acid aminotransferase coding DNA module and a dihydroxy acid dehydrase DNA molecule are introduced into target bacteria and then the recombination bacteria are obtained. An experiment proves that when Brevibacterium flavum MDV-07 is used for fermenting and producing L-valine, the yield of L-valine and a conversion rate can be greatly increased, and great production application value is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to the construction of a high-yielding L-valine engineering bacterium and a method for using the strain to ferment and produce L-valine. Background technique [0002] L-valine (L-valine) is one of the eight essential amino acids for the human body. It is also a branched chain amino acid. It has significant functions in the metabolic process of the human body and other animals. It can be used in the pharmaceutical industry, food industry and feed industry etc. [0003] In the field of medicine, L-valine is the raw material of compound amino acid infusion and amino acid injection. The compound amino acid infusion prepared by it is used in the treatment of blood-brain barrier, hepatic coma, chronic liver cirrhosis and renal failure, and congenital metabolic defects. It is widely used in dietary therapy, treatment of sepsis and postoperative diabetic patients, treatment of accelerating surgi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P13/08C12R1/13
Inventor 黄建忠黄钦耿吴伟斌翁雪清曾邦定黄祥峰施巧琴吴松刚
Owner FUJIAN NORMAL UNIV
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