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Corynebacterium glutamicum engineering strain producing L-isoleucine

A technology of isoleucine glutamic acid and coryneform bacteria, applied in the field of genetic engineering and fermentation engineering, to achieve the effect of stable genetic performance

Active Publication Date: 2020-06-23
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report combining multiple strategies to transform Corynebacterium glutamicum to produce L-isoleucine

Method used

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  • Corynebacterium glutamicum engineering strain producing L-isoleucine
  • Corynebacterium glutamicum engineering strain producing L-isoleucine
  • Corynebacterium glutamicum engineering strain producing L-isoleucine

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Embodiment 1

[0042] The construction of embodiment 1 bacterial strain WM002

[0043] Use primers tac-F and tac-R to use pJYW-4 as a template (the nucleotide sequence / construction method of pJYW-4 is disclosed in the patent "A coryneform bacteria expression system that does not rely on antibiotics as selection pressure" publication number: CN 103834679B) , to amplify the promoter tac fragment (as shown in SEQ ID NO.1); using primers PilvA-U-F and PilvA-U-R to use the WM001 genome as a template (see Genome Extraction Kit of Beijing Tiangen Biochemical Technology Co., Ltd. for detailed steps), Amplify the upstream fragment of the ilvA natural promoter (as shown in SEQ ID NO.2); Utilize primers PilvA-D-F and PilvA-D-R with the WM001 genome as a template to amplify the downstream fragment of the ilvA natural promoter (as shown in SEQ ID shown in NO.3); using primers PilvA-U-F and PilvA-D-R, overlapping fragments were obtained by overlapping PCR, and BamHI and XhoI restriction sites were added a...

Embodiment 2

[0044] Construction of embodiment 2 bacterial strain WM003

[0045] Use primers alaTloxL-kan-F and alaTloxL-kan-R to pDTW-202 as a template to amplify the loxL-kan-loxR (as shown in SEQ ID NO.4) fragment, and add BamHI and XbaI digestion at both ends site; Utilize primers alaT-U-F and alaT-U-R to use WM001 genome as a template to amplify the upstream fragment of the knockout gene alaT (as shown in SEQ ID NO.5), and add BamHI and XhoI restriction sites at both ends point; Utilize primer alaT-D-F and alaT-D-R with WM001 genome as template, amplify the downstream fragment of the knockout gene alaT (as shown in SEQ ID NO.6), and add XbaI and EcoRI restriction sites at both ends . Then, the three fragments were treated with corresponding restriction enzymes, and the vector pBluescript II SK(+) was digested with EcoRI and XhoI at the same time, and then ligated together to obtain the correct knockout vector pYCW-△alaT. Then, starting from the strain WM002, the knockout vector pYCW...

Embodiment 3

[0046] Construction of Example 3 bacterial strains WM004 and WM005

[0047]Using the WM001 genome as a template, respectively amplify the upstream fragment of the knockout gene brnQ (as shown in SEQ ID NO.7) and the downstream fragment of the knockout gene brnQ (as shown in SEQ ID NO.8), the knockout gene alr The upstream fragment (as shown in SEQ ID NO.9) and the downstream fragment of the knockout gene alr (as shown in SEQ ID NO.10) are used to construct knockout vectors pYCW-△brnQ and pYCW-△alr (the construction method is the same as that of pYCW -△alaT is similar, the primers used are pYCW-△brnQ: brnQloxL-kan-F and brnQloxL-kan-R, brnQ-U-F and brnQ-U-R, brnQ-D-F and brnQ-D-R; pYCW-△alr: alrloxL -kan-F and alrloxL-kan-R, alr-U-F and alr-U-R, alr-D-F and alr-D-R). Starting from the strain WM003, the knockout vectors pYCW-△brnQ and pDTW-109 were successively transformed (after removing the genomic kan resistance, it will be removed in a 37°C warm bath to facilitate subsequen...

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Abstract

The invention relates to a corynebacterium glutamicum engineering strain producing L-isoleucine, and belongs to the technical field of genetic engineering and fermentation engineering. The strain is based on L-isoleucine industrial production strain WM001, and modifies a L-isoleucine anabolic pathway by gene knockout, overexpression and other means, a finally obtained strain WM005 / pYCW-1-ilvBN-ppnK can be stably inherited, antibiotics do not need to be added in the process of preparing L-isoleucine, the risk of producing drug-resistant strains is reduced, the prepared L-isoleucine is suitablefor production of various fields such as medical, cosmetics, and industry, and the situation that products are polluted due to antibiotics is avoided; and 32.1g / L of L-isoleucine is accumulated in fed-batch fermentation of a 5L fermentation cylinder, the conversion rate of sugar acid reaches 0.181g / g, and the fed-batch fermentation is suitable for the industrial production of L-isoleucine.

Description

technical field [0001] The invention constructs a L-isoleucine-producing Corynebacterium glutamicum engineering bacterium, which belongs to the technical field of genetic engineering and fermentation engineering. Background technique [0002] L-Isoleucine is one of the three branched-chain amino acids (BCAAs) essential to the human body and is used in various industrial applications to produce various products such as human nutritional enhancers, animal feed additives, medical product ingredients, and cosmetic ingredients . L-isoleucine is mainly produced by microbial fermentation, and Escherichia coli and Corynebacterium glutamicum are the main production strains. However, E. coli often synthesizes endotoxins, which can contaminate products. Therefore, Corynebacterium glutamicum becomes an ideal strain for producing L-isoleucine. [0003] In Corynebacterium glutamicum, L-isoleucine biosynthetic pathway consisting of ten reaction steps is complex due to the intertwined sy...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
CPCC07K14/34C12N15/77C12P13/06
Inventor 王小元张炎潮柳亚迪胡晓清
Owner JIANGNAN UNIV
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