Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum

A technology of glutamic acid rods, construction methods, applied in the field of genetic engineering

Inactive Publication Date: 2021-03-09
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There has been no report on the xylose pathway construction of Corynebacterium glutamicum to co-utilize glucose and xylose in lignocellulose to produce glutamate, so a co-fermentation of gluco...

Method used

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  • Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum
  • Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum
  • Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0047] Example 1: The xylAB expression cassette was integrated into the ldhA gene locus of C. glutamicum

[0048] First construct the integrated plasmid of xylAB, and the specific construction method is as follows: use the genome of C. glutamicum as a template, use Peftu-F (as shown in the Peftu-F sequence in the sequence listing) and Peftu-R (as shown in the Peftu-F in the sequence listing) Shown in the R sequence) primers are amplified by PCR to obtain the Peftu promoter (as shown in the Peftu sequence in the sequence listing); with the genome of E.coliBL21 as a template, use xylAB_BL21-F (such as xylAB_BL21-F in the sequence listing sequence) and xylAB_BL21-R (as shown in the xylAB_BL21-R sequence in the sequence listing) primers are amplified by PCR to obtain xylAB_BL21 (as shown in the xylAB_BL21 sequence in the sequence listing) fragment; with Peftu and xylAB_BL21 as templates, Utilize Peftu-F and xylAB_BL21-R primer, obtain Peftu_xylAB_BL21 fusion fragment by the mode o...

Embodiment example 2

[0056] Example 2: Modification of glutamate secretion channel protein

[0057] First construct a knockout plasmid with 330 bases at the carbon end of MscCG, the specific construction method is as follows: use the genome of C. glutamicum as a template, use MscCG-up-F (as shown in the MscCG-up-F sequence in the sequence listing) and MscCG-up-R (as shown in the MscCG-up-R sequence in the sequence listing) primers amplify the MscCG-up fragment (as shown in the MscCG-up sequence in the sequence listing) by PCR (operation with Example 1) with the genome of C.glutamicum as a template, using MscCG-down-F (as shown in the MscCG-down-F sequence in the sequence listing) and MscCG-down-R (as shown in the MscCG-down-R in the sequence listing sequence shown) primers are amplified by the method of PCR (operation with Example 1) to obtain the MscCG-down fragment (as shown in the MscCG-down sequence in the sequence listing); with MscCG-up and MscCG-down as templates, using -up-F and MscCG-dow...

Embodiment example 3

[0058] Example 3: Weakening the expression of α-ketoglutarate dehydrogenase

[0059] The original RBS sequence replacing odhA is firstly constructed to be an integrated plasmid with an RBS sequence whose transcription initiation strength is 0.1. The specific construction method is as follows: using the genome of C. glutamicum as a template, using odhA-up-F (such as odhA-up-F in the sequence listing shown in the up-F sequence) and odhA-up-R (as shown in the odhA-up-R sequence in the sequence listing) primers are amplified by the method of PCR (operation with Example 1) to obtain the odhA-up fragment (as shown in the sequence odhA-up sequence shown in the list); with the genome of C.glutamicum as a template, using odhA-down-F (as shown in the odhA-down-F sequence in the sequence listing) and odhA-down-R (as shown in the sequence Shown in the odhA-down-R sequence in the list) primers are amplified by the method for PCR (operation with embodiment 1) to obtain the odhA-down fragmen...

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Abstract

The invention belongs to the field of gene engineering, and discloses a construction method of a synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum. The construction method comprises the following steps of (1) expressing heterogenous pentose transport protein in the corynebacterium glutamicum; (2) expressing heterogenous xylose isomerase and xylulokinasein the corynebacterium glutamicum; (3) generating alpha-ketoglutarate from generated 5-xylulosephosphate through a phosphopentose pathway and a glycolysis pathway, and carrying out weakened expressionon alpha-ketoglutarate dehydrogenase, so that more alpha-ketoglutarate flows to the synthesis of glutamic acid; and (4) modifying glutamic acid secretory protein to ensure that the corynebacterium glutamicum does not respond to external biotin pressure any more, and continuously secreting the glutamic acid out of cells. According to the constructed synthetic path from the xylose to the glutamic acid, the corynebacterium glutamicum can utilize the xylose to produce the glutamic acid in a conventional synthetic culture medium, and the synthesis from the xylose to the glutamic acid can also be conducted in a high-biotin environment such as lignocellulose.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for constructing a synthetic pathway of using xylose to produce glutamic acid in Corynebacterium glutamicum and construction of an engineering strain capable of producing glutamic acid from xylose in a high-biotin environment. its application. Background technique [0002] Glutamic acid is an important industrial amino acid, which is widely used in the fields of food and feed, and is also used as a monomer to produce biopolymer materials such as polyglutamic acid. In 2013, the annual output of glutamic acid has reached 3 million tons, indicating that its demand is huge. At present, glutamic acid is mainly produced through microbial fermentation, but the raw materials used in this method are mostly grain crops such as starch, which has caused the problem of "competing with others for food". Therefore, it is imperative to seek cheaper and widely sourced raw materials. L...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N15/90C12N15/61C12N15/54C12N15/53C12N15/31C12N1/21C12P13/18C12P13/14C12R1/15
CPCC12N15/77C12N15/902C12N15/52C12N9/92C12N9/1205C12N9/0008C07K14/245C12P13/18C12P13/14C12Y503/01005C12Y207/01017C12Y102/04002
Inventor 鲍杰金慈
Owner EAST CHINA UNIV OF SCI & TECH
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