A kind of Corynebacterium glutamicum and its application

A technology of Corynebacterium glutamicum and polyhydroxyalkanoate, which is applied in the field of microorganisms and can solve problems such as synthetic limitations

Active Publication Date: 2019-05-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the Corynebacterium glutamicum currently used to synthesize PHB or PHBV accumulates more acetyl-CoA in the cell and only a small amount of propionyl-CoA, which has great restrictions on the synthesis of metabolites utilizing propionyl-CoA effect

Method used

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  • A kind of Corynebacterium glutamicum and its application
  • A kind of Corynebacterium glutamicum and its application
  • A kind of Corynebacterium glutamicum and its application

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Experimental program
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Embodiment 1

[0020] The identification of embodiment 1 bacterial strain

[0021] Corynebacterium glutamicum was isolated from Wuxi landfill plant. This strain grows well on LBG medium. The composition of LBG medium is 1% peptone, 0.5% yeast powder, 0.5% NaCl, pH 7.0-7.2. The colonies are round, beige, with neat edges. The 16S rDNA gene sequence of this Corynebacterium glutamicum strain was homologously compared with the bacterial 16S rDNA gene sequence in NCBI, and it was found that this strain had the closest similarity to the reported Corynebacterium glutamicum ATCC13869. The bacterium was preserved in the China Center for Type Culture Collection on June 1, 2016, and the preservation number is CCTCC NO: M 2016303.

Embodiment 2

[0022] Example 2 Extraction and determination of intracellular propionyl-CoA and acetyl-CoA of strain CCTCC NO: M 2016303 of the present invention

[0023] 1. Cell culture

[0024] After culturing with the seed medium for 18 hours, transfer to the fermentation medium according to 1% inoculation amount, and after culturing for 24 hours, take the fermentation sample;

[0025] 2. Extraction of Intracellular Propionyl-CoA and Acetyl-CoA

[0026] Take 1mL of mid-log phase fermentation broth, centrifuge at 10,000rpm, 0oC for 30s to collect the cells, wash the cells twice with ice-cold 0.9% saline, wash with 1mL of solution I (methanol: acetonitrile: water = 45:45:10, v / v / v) resuspend the cells, and add 0.1M formic acid to quench; then, the quenched bacterial suspension was sonicated for 5 minutes at 0oC, centrifuged to remove cell debris and other precipitates, and the supernatant was filtered to obtain propionyl-CoA and acetyl-CoA extract for liquid mass spectrometry.

[0027] 3...

Embodiment 3

[0035] Example 3 Application of bacterial strains of the present invention in PHBV synthesis

[0036] 1. Construction of recombinant engineering bacteria WM001 / pDXW-8-phaCAB

[0037] (1) Taking Eutropha rosenbergii genome NC_008313.1 as a template, primers phaCAB-F / phaCAB-R were used to amplify the phaCAB gene cluster, and the sequence of the primer phaCAB-F / phaCAB-R for amplifying the phaCAB gene cluster was

[0038] phaCAB-F: 5'-CTGGAATTCAGAAGGAGAATCAAATCATGGCGACCGG-3'

[0039] phaCAB-R: 5'-CCGCTCGAGAGGTCAGCCCATATGCAGG-3'

[0040] (2) Digest the PCR product with restriction endonucleases EcoRI and XhoI, carrier pDXW-8 (for the construction method of the carrier pDXW-8, refer to the application number 200910233618.1, and the name of the invention is a kind of Escherichia coli-coryneform bacteria shuttle type induction expression carrier pDXW -8 and the patent application for its construction method) were digested with EcoRI and XhoI, and after purification of the digested p...

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Abstract

The invention discloses a Corynebacterium glutamicum and application thereof, belonging to the field of microorganisms. The Corynebacterium glutamicum was preserved in the China Center for Type Culture Collection on June 1, 2016, and the preservation number is CCTCC NO: M 2016303. The Corynebacterium glutamicum can accumulate more propionyl-CoA in cells, which is 15.8 times higher than that of ATCC13869, and can be used for the production of related metabolites using propionyl-CoA as a substrate. After the polyhydroxyalkanoate synthesis gene is introduced, the Corynebacterium glutamicum can synthesize PHBV, but the synthetic product is PHB in ATCC13869. The invention solves the current situation of producing PHBV by adding propionate or propionate from an exogenous source, realizes the direct production of PHBV in Corynebacterium glutamicum without adding propionate or propionate from an exogenous source, and reduces production costs.

Description

technical field [0001] The invention relates to a Corynebacterium glutamicum and application thereof, belonging to the field of microorganisms. Background technique [0002] In recent years, polyhydroxyalkanoates, which can be obtained through intracellular accumulation by microorganisms, have received increasing attention from the scientific and commercial communities as a potential alternative to petrochemically derived plastics. At present, PHB and PHBV are mainly used for industrialization by using bacteria. Compared with PHB, PHBV has much improved material properties and higher added value. The synthesis of PHB requires cell metabolism to provide acetyl-CoA, while the synthesis of PHBV also requires a large amount of propionyl-CoA. Generally, less propionyl-CoA is accumulated in the cells of common bacterial strains, which is not enough to synthesize PHBV. It needs to add propionic acid to synthesize PHBV, which greatly increases the production cost. [0003] At pres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/32C12P7/62C12R1/15
CPCC12P7/625C12P19/32C12N1/205C12R2001/15
Inventor 王小元马文渐王建莉李烨王甜忆林琳孙康康
Owner JIANGNAN UNIV
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