L-histidine production method and special recombinant bacteria

一种重组菌、组氨酸的技术，应用在氨基酸发酵领域，能够解决菌株生长和葡萄糖代谢能力下降、菌株葡萄糖代谢能力减弱、L-组氨酸产量下降等问题，达到便于工业化控制、易于过程和成本控制、发酵周期短的效果

Active Publication Date: 2015-08-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] By inactivating the 6-phosphate glucose isomerase in the glycolytic pathway, the carbon metabolic flow can be directed to the pentose phosphate pathway, but it will lead to the growth of the strain and the weakening of glucose metabolism, which is not conducive to the application of the strain in fermentation production
Previous research results confirmed that knocking out the gene pgi encoding 6-phosphate glucose isomerase led to a severe decline in the growth and glucose metabolism of the strain, and at the same time the production of L-histidine also decreased
In addition, it was also found that increasing the expression of glucose-6-phosphate dehydrogenase alone has a poor effect on increasing the production of L-histidine

Method used

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  • L-histidine production method and special recombinant bacteria
  • L-histidine production method and special recombinant bacteria
  • L-histidine production method and special recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, the acquisition of L-histidine chassis engineering bacteria CG160

[0063] 1. The promoter of the L-histidine synthesis operon in the wild-type Corynebacterium glutamicum ATCC13032 was replaced by a strong promoter P glyA

[0064] According to the hisEG operon of Corynebacterium glutamicum ATCC13032 in Genbank and its upstream and downstream sequences and P glyA The primers were designed separately for the promoter sequence.

[0065] Using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, using P1 and P2 as primers to PCR amplify the upstream homology arm of the hisEG operon promoter; using P3 and P4 as primers to amplify the promoter P glyA ; Use P5 and P6 as primers to amplify the homology arm downstream of the hisEG promoter. Then use the purified PCR product as a template, use P1 and P6 as primers, and use overlap extension PCR technology (SOE) to amplify to obtain a PCR product of 1920bp, which is P glyA and replaced promoter P hisE...

Embodiment 2

[0123] Embodiment 2, the construction of the recombinant bacterium CG171 containing the high-production L-histidine of plasmid

[0124] 1. Construction of L-histidine primary engineering bacteria CG176

[0125] The genomic DNA of strain CG160 was used as a template, and the prsA gene (992bp) and hisG were amplified by PCR using P28 / P29 and P30 / P31 as primers, respectively fbr (860bp). The two genes were joined using overlap extension PCR to amplify hisG fbr and the prsA gene as a template, using P28 and P31 as primers for PCR amplification, and the PCR product of 1852bp is prsA-hisG fbr fragment (sequence 3).

[0126] Wherein, the nucleotides 1-992 from the 5' end of sequence 3 are prsA, and the nucleotides 993-1852 from the 5' end of sequence 3 are hisG fbr (hisG gene with three point mutations).

[0127] After the above PCR product was double-digested with Xba I and Sma I, it was ligated with the Corynebacterium glutamicum-Escherichia coli shuttle expression plasmid pXM...

Embodiment 3

[0165] Example 3, Construction of plasmid-free L-histidine high-yielding recombinant strain CG324

[0166] According to the prsA gene of Corynebacterium glutamicum ATCC13032 in Genbank and its upstream and downstream sequences and P sod The primers were designed separately for the promoter sequence.

[0167] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the upstream homology arm of the prsA promoter was amplified with P48 and P49 as primers; the prsA promoter was amplified with P50 and P51 as primers sod Promoter; use P52 and P53 as primers to amplify the downstream homology arm of prsA promoter. Then, using the purified PCR product as a template, using P48 and P53 as primers, and using overlap extension PCR (SOE) to amplify, a 1455bp PCR product was obtained, which was a P sod and replaced promoter P prsA A fragment of the upstream and downstream homology arms of (SEQ ID NO: 9).

[0168] Wherein, the 1-655th nucleotide from the 5' end of seq...

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Abstract

Provided is a recombinant strain producing L-amino acids, a constructing method therefor and a method for producing L-amino acids. Compared with the starting strains, the recombinant strain producing L-amino acids has decreased expression of 6-phosphoglucose isomerase Pgi and increased expression of 6-phosphogluconate dehydrogenase Zwf-OpcA. The starting strain can accumulate objective amino acids. Fermentation culturing of the recombinant strain can increase the yield of L-amino acids significantly. Also provided is a novel method for increasing the fermentation yield of L-amino acids, capable of being be used in bacterial fermentation of L-amino acids.

Description

technical field [0001] The invention relates to the field of amino acid fermentation, in particular, the invention relates to a method for producing L-histidine and special recombinant bacteria thereof. Background technique [0002] L-histidine is the ninth essential amino acid for humans and animals. It participates in important physiological processes such as body growth and development, anti-oxidation, and immune regulation. It is an important medicinal amino acid and can be used for the treatment of heart disease, anemia, and gastrointestinal ulcers. infusion preparations. At present, the production of L-histidine mainly adopts the protein hydrolysis extraction method using pig (cattle) blood meal as raw material. The production cost of L-histidine is high and expensive. The production of L-histidine by microbial fermentation has not yet been applied on a large scale industrially. The biosynthesis of L-histidine has the characteristics of competing with nucleotide syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/24C12R1/15
Inventor 温廷益商秀玲张芸刘树文梁勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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