Corynebacterium fermentative production method of l-lysine and its promoter modification

A technology of lysine and corynebacterium, which is applied in the field of amino acid fermentation, can solve the problem of less promoter improvement and achieve the effect of increasing yield

Active Publication Date: 2017-07-18
HEILONGJIANG EPPEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few improvements to the promoters of rod-shaped bacteria of the genus Corynebacterium in the prior art, and most of them still retain wild-type promoters, such as Corynebacterium glutamicum ATCC 13869 strains (see GenBank: 2531528 to 2531972 of CP016335.1 ), CP strain (see GenBank: CP012194.1 No. 2575805 to 2576249), ZL-6 strain (see GenBank: CP004062.1 No. 2560677 to 2561121), Brevibacterium flavum ZL-1 ((see GenBank :|CP004046.1 from 2569176 to 2569620)

Method used

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  • Corynebacterium fermentative production method of l-lysine and its promoter modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Promoter of the present invention

[0056] According to the polynucleotide sequence designed by the inventor, such as the promoter shown in SEQ ID NO: 1, the corresponding polynucleotide was entrusted to be synthesized and connected to the pMD19-T vector. The new vector obtained was T-EP5, which was sequenced (commissioned Shanghai Yingjun Company sequencing), the sequencing results are as follows:

[0057] GTAACCCGAG GTTAAGTGTA TTTTAGGTGA ACAAATTTCA GCTTCGGGTA GAAGACcTTCGATGCGCTTC AGAGCTTCTA TTGGGAAATC TGAtACCACT TGATTAAATA GCCTACCCCC GAATTGGGGGATTGGTCATT TTTTGCTGTG AAGGTAGTTT TGATGCATAT GACCTGCGTT TATAAAGAAA GTAAACGTGATCAGATCGA TATAAAAGAA ACAGTTTGTA CTCAGGTTTG AAGCATTTTC TCCGATTCGC CTGGCAAAAATCTCAATTGT CGCTTACAGT TTTTCTCAAC

[0058] GACAGGCTGC TAAGCTGCTA GTTCGGTGGC CTAGTGAGTG GCGTTTACTT GGATAAAAGTAATCCCATGT CGTGATCAGC CATTTTGGGT TGTTTCCATA GCAATCCAAA GGTTTCGTCT TTCGATACCTATTCAAGGAG CCTTCGCCTC T

[0059] The sequencing result contained the promoter shown in ...

Embodiment 2

[0060] Example 2 Regulation of EP5 promoter ddh Gene expression construct experiments

[0061] Primers were designed according to the sequence of the above-mentioned EP5 and the genome sequence of Corynebacterium glutamicum ATCC13032 announced on the NCBI, for inserting the EP5 fragment into ddh The front end of the gene start codon ATG allows EP5 to drive ddh For gene expression, specific primers were designed as follows:

[0062] P15: 5' GCTCTAGACGTAGCCAACGAAGTAATC 3' (xba1)

[0063] P16: 5' CTATTCAAGG AGCCTTCGCC TCTATGACCA ACATCCGCGT AGCTATC 3'

[0064] P17: CTAAAATACA CTTAACCTCG GGTTACGTTC TTGTAATCCT CCAAAATTG

[0065] P18: CAATTTTGGA GGATTACAAG AACGTAACCC GAGGTTAAGT GTATTTTAG

[0066] P19: 5' CTAAAATACA CTTAACCTCG GGTTACATGA TGATTCAGGG ACATCT 3'

[0067] P20: 5' CGGAATTCTTTCGGGCGGCAATATAG 3' (EcoR1)

[0068] Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was carried out with primers P15 / P16 and P19 / P20 respectively to obtain a 700bp ...

Embodiment 3

[0070] Example 3 EP5 promoter regulation lysC Gene expression construct experiments

[0071] Primers were designed according to the sequence of the above-mentioned EP5 and the genome sequence of Corynebacterium glutamicum ATCC13032 announced on the NCBI, for inserting the EP5 fragment into lysC The front end of the gene start codon CTG enables EP5 to drive the expression of the lysC gene. The specific primers are designed as follows:

[0072] P3: 5'CGGAATTCCCGCAAGCAGCCACATTC 3' (EcoR1)

[0073] P4: 5' CTAAAATACA CTTAACCTCG GGTTACCTTT GTGCACCTTT CGATCTAC3'

[0074] P5: 5'GTAGATCGAA AGGTGCACAA AGGTAACCCG AGGTTAAGTG TATTTTAG3'

[0075] P6: 5' CATATTTCTG TACGACCAGG GCCAGAGAGG CGAAGGCTCC TTGAATAG3'

[0076] P7:5'CTATTCAAGG AGCCTTCGCC TCTCTGGCCC TGGTCGTACA GAAATATG3'

[0077] P8: 5'CCCAAGCTTGTGGTGCCGTCTTTCTACAG 3' (Hind3)

[0078]Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed with primers P3 / P4 and P7 / P8 respectively to obtain a 740...

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Abstract

The invention provides a method for fermenting to produce L-lysine. The method includes replacing the promoter of one or more genes of the chromosome of the coryneform bacterium with the EP5 promoter, and modifying L-lysine produced by the bacterial fermentation. In addition, the invention also provides methods and applications derived from the method, as well as bacteria and promoters that can be used in these methods and applications.

Description

technical field [0001] The present invention belongs to the field of amino acid fermentation, in particular, the present invention relates to methods and applications of fermentative production of L-lysine, and bacteria and promoters that can be used in these methods and applications. Background technique [0002] Production of L-lysine by fermentation of L-lysine-producing bacteria (eg, rod-shaped bacteria of the genus Corynebacterium, especially Corynebacterium glutamicum) has been industrially applied. These bacteria can be bacteria isolated from nature, bacteria obtained through mutagenesis or genetic engineering, or both. [0003] The bacteria of the genus Corynebacterium through genetic engineering are mainly achieved by increasing or decreasing the enzyme activity or expression level related to the L-lysine metabolic pathway. For example, Chinese patent CN1017906B discloses a method for producing L-lysine, including using Corynebacterium bacterium containing recombin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/77C12N15/67C12N15/113C12N1/21C12P13/08C12R1/15
CPCC12N15/113C12N15/67C12N15/77C12P13/08
Inventor 孟刚魏爱英马风勇贾慧萍马吉银
Owner HEILONGJIANG EPPEN BIOTECH CO LTD
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