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Corynebacterium glutamicum strain capable of fermenting xylose to produce succinic acid and application

A technology of Corynebacterium glutamicum, succinic acid, applied in fermentation, microorganism-based methods, bacteria, etc., can solve the problems of negative strain, influence, unclear genomic background, etc., and achieve the effect of increasing productivity

Active Publication Date: 2019-12-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the evolution process is uncertain. Along with favorable mutations, some irrelevant mutations may also occur. These mutations will cause the genome background of the strain to be unclear, affecting subsequent genetic engineering operations, and some mutations may even affect the strain. Some traits have negative effects, limiting the strain's future application prospects
Therefore, no matter by genetic engineering means or evolutionary engineering method, the improved strains have obvious deficiencies.

Method used

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  • Corynebacterium glutamicum strain capable of fermenting xylose to produce succinic acid and application
  • Corynebacterium glutamicum strain capable of fermenting xylose to produce succinic acid and application
  • Corynebacterium glutamicum strain capable of fermenting xylose to produce succinic acid and application

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Experimental program
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Effect test

Embodiment 1

[0026] Construction of Recombinant Bacteria CGL8 from Corynebacterium glutamicum

[0027] (1) Construction of the Corynebacterium glutamicum non-scarring operation technology and tool vector pD-sacB involved in the present invention and knockout in Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032

[0028] The pta-ackA gene operon of the by-product acetate production pathway, the actA gene of the by-product acetate production pathway, the ldh gene of the by-product lactic acid production pathway, and the start codon of the phosphoenolpyruvate carboxylase ppc gene of the anaplerotic pathway on chromosome Insert P sod Strong promoter, insertion of P before the start codon of the anaplerotic pathway pyruvate carboxylase pyc gene on chromosome sod Strong promoter, pentose phosphate pathway transketoaldase tkt gene on chromosome Insert P before the start codon sod Strong promoter, pentose phosphate pathway transketolase tal gene on chromosome Insert P before the ...

Embodiment 2

[0036] Example 2: Laboratory adaptive evolution using xylose as the sole carbon source based on the recombinant strain CGL11

[0037] The growth ability of Corynebacterium glutamicum using xylose was improved by continuous subculture of Corynebacterium glutamicum CGL11 in basic salt medium CGXII with xylose as the only carbon source.

[0038] The specific operation is as follows: the CGL11 strain preserved at -80°C was activated overnight at 30°C and 220rpm in a test tube BHI medium containing 10g L -1 Glucose CGIII medium (filling volume 50mL / 500mL Erlenmeyer flask) was cultured for 12h. Collect the cells by centrifugation at 5000 rpm at 4°C and wash the cells twice with CGXII. Transplant the suspended cells into a cell containing 20g L -1 Xylose, 1gL -1 In the CGXII medium of yeast extract (filling volume 25mL / 250mL Erlenmeyer flask), the initial OD 600 is 1. Monitor OD for 12h and 24h 600 , every 24h transferred to fresh containing 20g L -1 Xylose in the CGXII medium...

Embodiment 3

[0043] Embodiment 3: under aerobic condition, take xylose as the growth curve of sole carbon source

[0044] Activate the starting strains CGL11 and Cev-18-5 respectively into 5mL BHI test tubes at 220rpm, activate overnight at 30°C, and transfer to 20g L -1 Glucose CGIII medium, 220rpm, 30°C for 12h. Collect the cells by centrifugation at 4°C and 5000rpm, wash the cells twice with CGXII, and transfer to cells containing 20g L -1 In the CGXII medium of xylose (filling volume 50mL / 500mL Erlenmeyer flask), take samples every 2h for the first 12h to measure OD 600 , the growth curve as image 3 shown. Centrifuge at 12000rpm for 5min to take the supernatant as a deposit. Compared with the starting strain CGL11, the maximum specific growth rate is 0.15h -1 In comparison, the maximum specific growth rate of the evolved strain Cev-18-5 was 0.39h -1 , 160% higher than the starting strain. The xylose consumption rate of the strains was as follows Figure 4 As shown, the average...

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Abstract

The invention discloses a corynebacterium glutamicum strain capable of fermenting xylose to produce succinic acid and application. The orynebacterium glutamicum strain capable of fermenting xylose toproduce succinic acid is classified and named as Corynebacterium glutamicum Cev-18-5 and is preserved in China General Microbiological Culture Collection Center, and the preservation number of the corynebacterium glutamicum strain is CGMCC No.15040. The corynebacterium glutamicum strain CGMCC No.15040 capable of fermenting xylose to produce succinic acid can grow rapidly in a basic salt culture medium which takes xylose as a sole carbon source, xylose is efficiently utilized, and no plasmid or inducer needs to be applied. The yield of succinic acid under an anaerobic condition is also increased, so that the corynebacterium glutamicum strain CGMCC No.15040 is a good platform strain which utilizes lignocellulose-based biomass for producing succinic acid.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, in particular to a Corynebacterium glutamicum strain for fermenting xylose to produce succinic acid and its use. Background technique [0002] As a renewable biomass resource that widely exists in nature, lignocellulose has broad application prospects. And xylose is the second sugar in lignocellulose hydrolyzate, second only to glucose. Therefore, effective utilization of xylose is one of the key factors for better and efficient utilization of lignocellulose. However, only a few of the currently known microorganisms have a good ability to utilize xylose, and many important industrial microorganisms, including Corynebacterium glutamicum, do not have a good ability to utilize xylose, or even cannot utilize xylose at all. [0003] C. glutamicum can utilize a broad spectrum of sugars, such as hexoses and disaccharides, but not certain pentoses. Corynebacterium glutamicum ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/46C12R1/15
CPCC12P7/46C12R2001/15C12N1/205
Inventor 王智文孙曦毛雨丰陈聪陈涛
Owner TIANJIN UNIV
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