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Construction of bacterial strain producing pyruvic acid recombination and method for improving production strength of pyruvic acid

A pyruvate, recombinant bacteria technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc.

Inactive Publication Date: 2008-08-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But for the regulation of NADH / NAD in microbial cells + There are few literature reports on strengthening the production intensity of target metabolites

Method used

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  • Construction of bacterial strain producing pyruvic acid recombination and method for improving production strength of pyruvic acid
  • Construction of bacterial strain producing pyruvic acid recombination and method for improving production strength of pyruvic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Amplification of Lactococcus lactis noxE gene and construction of expression plasmid

[0030] Using the genome of Lactococcus lactis NZ9000 as a template, the noxE fragment of the target gene was amplified by PCR, and a specific band of about 1.5 kb was obtained by agarose electrophoresis. The target gene noxE was digested by restriction endonucleases EcoR I and BamH I, concentrated and purified, and the noxE gene was directional cloned into the expression plasmid pYX212 to obtain the recombinant yeast expression plasmid. The recombinant yeast expression plasmid was transformed into competent cell JM109 and spread on LB plates containing ampicillin for amplification.

Embodiment 2

[0031] Example 2 Construction and Identification of Recombinant Bacteria

[0032] Due to the Ura3 gene on the recombinant plasmid, the recipient strain T.glabrata (Ura - ), to obtain the recombinant bacteria PdnoxE CCTCC NO: M208022 that can grow normally on the basal medium without adding uracil. In the enzyme activity assay, it was found that the specific activity of NADH oxidase in the recombinant bacteria was 34.8 U / mg protein, but it was difficult to detect the activity of NADH oxidase in the starting strain CCTCC NO: M202019.

Embodiment 3

[0033] Embodiment 3 Recombinant bacteria and control bacteria control fermentation experiment

[0034] As shown in the table below, the comparison between the two bacteria: (1) The glucose of the recombinant strain PdnoxE CCTCC NO: M 208022 was consumed in 36 hours, while the starting strain CCTCC NO: M202019 remained 5.34 g / L of glucose in the fermentation broth after 48 hours; (2) The glucose consumption rate of the recombinant bacteria was 2.57g / L / h, which was 44.9% (1.78g / L / h) higher than that of the starting strain; (3) the pyruvate output was 31.4g / L when the recombinant bacteria fermented for 36h, and the starting The pyruvate output of the strain was only 28.3g / L, and the pyruvate output of the starting strain increased to 37.3g / L when the fermentation was continued for 48 hours, but the pyruvate output of the recombinant strain did not increase with the prolongation of the fermentation time; (4) the recombinant strain The production intensity of pyruvate in this metho...

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Abstract

The invention relates to a construction of a recombinant bacterium for producing pyruvate and a method for improving the production strength of pyruvate by using the recombinant bacterium, which pertains to the technical filed of auxiliary factor metabolic regulation and control strategic optimized fermentation process. The method regulates and controls the carbon metabolic flow to strengthen the production strength of the pyruvate by changing an auxiliary factor NADH and adopts molecular approach to allow the NADH oxidase noxE gene which is coded into water and is derived from L. lactis to be over expressed in an industrial bacterial strain Torulosis glabrata CCTCC NO: M 202019 for producing the pyruvate by the fermentation method, so as to obtain an NADH oxidase over expressed recombinant bacterium PdnoxE CCTCC NO: M 208022; compared with the starting bacterial strain, the bacterial dry weight, the glucose consumption rate and the pyruvate production strength thereof are respectively improved by 168 percent, 44.9 percent and 12 percent. The method has universally applicable significance for the improvement of a plurality of important fermentation products (such as, organic acids and amino acids etc.).

Description

technical field [0001] The invention relates to the construction of a pyruvate-producing recombinant bacterium and the method for using it to increase the production intensity of pyruvate, which relates to a method of enhancing the production intensity of pyruvate by regulating carbon metabolism flow through the cofactor NADH, and belongs to the technical field of cofactor metabolism regulation strategy optimization fermentation process. By studying the effect of the cofactor NADH on the direction of metabolic flow and the corresponding metabolic flux, the intensity of pyruvate production can be enhanced. Background technique [0002] Pyruvate, also known as 2-Oxopropanic acid, α-Ketopropionic acid or Acetylformic acid, is a colorless to pale yellow liquid with acetic acid aroma and pleasant Sour, is one of the most important α-oxocarboxylic acids. [0003] Pyruvate is an important intermediate metabolite of living organisms. Since it is widely used in chemical, pharmaceuti...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/81C12P7/40C12R1/645
Inventor 陈坚董志姚刘立明堵国成
Owner JIANGNAN UNIV
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