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A recombinant bacterium with improved pyruvate production intensity and its application

A technology of recombinant bacteria and pyruvic acid is applied in the field of fermentation engineering to achieve the effects of increasing yield, increasing production intensity and improving production intensity

Active Publication Date: 2019-01-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few literature reports on how to strengthen pyruvate out of cells

Method used

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  • A recombinant bacterium with improved pyruvate production intensity and its application
  • A recombinant bacterium with improved pyruvate production intensity and its application
  • A recombinant bacterium with improved pyruvate production intensity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Amplification of Saccharomyces cerevisiae MPC1, codon-optimized synthesis of signal peptide Shrew1p and construction of expression plasmid

[0033] Using the Saccharomyces cerevisiae genome as a template, the target gene MPC1 fragment was amplified by PCR, and a specific fragment of about 400bp was obtained by agarose gel electrophoresis. The target gene MPC1 was digested by restriction endonucleases EcoR I and Xam I, concentrated After purification, the MPC1 gene was directional cloned into the expression plasmid pY26 to obtain the recombinant yeast expression plasmid pY26-MPC1, which was transformed into competent cells JM109 and spread on LB plates containing ampicillin for amplification.

[0034] For the signal peptide sequence from Drosophila cells, after appropriate codon optimization and the artificially synthesized sequence such as Shrew1p of SEQ ID NO.1, it was digested with BamH I and Xma I double enzymes, concentrated and purified, and combined with...

Embodiment 2

[0035] Embodiment 2: Construction and identification of recombinant bacteria

[0036] Since the above-mentioned recombinant plasmid pY26-Shrew1p-MPC1 carries the Ura3 gene, the recombinant yeast plasmid pY26-Shrew1p-MPC1) was transformed into the recipient strain TgU - (that is, the T.glabrata CCTCC M202019 that has deleted the Ura gene), obtain the recombinant bacteria TgU that grows normally on the basal medium without uracil - -pY26-Shrew1p-MPC1.

Embodiment 3

[0037] Embodiment 3: Recombinant bacterium and control bacterium control fermentation experiment

[0038] Pick recombinant TgU - (pY26-Shrew1p-MPC1) and TgU containing empty plasmid pY26 - The single colony of the above-mentioned activated culture was activated and cultivated on the seed medium for 18-24h, and the above-mentioned activated cultured seed solution was inoculated into the fermentation medium with an inoculation amount of 10%, and the fermentation culture was carried out at 30°C and 220rpm. The fermentation results are shown in Table 1. The dry cell weight, glucose consumption rate, pyruvate production and pyruvate production intensity of the recombinant bacteria are relative to the control bacteria (TgU - (pY26)) increased by 58.3%, 68.1%, 154.4% and 152.6%, respectively. At the same time, with the control bacteria TgU - Compared with (pY26-MPC1) (without Shrew1p signal peptide), the glucose consumption rate and pyruvate production intensity of the recombinant...

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Abstract

The invention belongs to the technical field of fermentation engineering and discloses a recombinant bacterium with a function of pyruvate production intensity improvement and application of the recombinant bacterium with the function of pyruvate production intensity improvement. By targeting expression of a mitochondrion pyruvate carrier protein to a cell membrane in a torulopsis glabrata strain, cell outgoing rate of pyruvate is increased while a feedback inhibition effect of the pyruvate on glycolytic pathway key enzymes is weakened, and consequently pyruvate yield is increased, and production intensity is improved. By means of genetic engineering technology, a signal peptide sequence Shrew1p and MPC1 derived from saccharomyces cerevisiae are positioned and integrated to a pY26 expression vector to construct a saccharomyces cerevisiae recombinant expression plasmid pY26-Shrew1p-MPC1 which is electrophoretically transferred into a recipient bacterium TgU to obtain an engineering strain TgU-(pY26-Shrew1p-MPC1) high in pyruvate yield. Compared with a reference strain TgU-(pY26), the engineering strain TgU-(pY26-Shrew1p-MPC1) has the advantage that dry cell weight, glucose consumption rate, pyruvate yield and pyruvate production intensity are increased by 58.3%, 68.1%, 154.4% and 152.6% respectively.

Description

technical field [0001] The invention relates to a recombinant bacterium with improved pyruvate production intensity and application thereof, belonging to the technical field of fermentation engineering. Background technique [0002] Pyruvate (pyruvate) is an intermediate product of biological metabolism, which is in the middle link of each metabolic pathway, such as: it is the end product of glycolysis; it is converted into acetyl-CoA, the starting material of TCA cycle through dehydrogenation and decarboxylation; in pyruvate Under the action of carboxylase, oxaloacetate, an intermediate product of the TCA cycle, is generated; alanine is generated through transamination, and it is also one of the very important ketoacids. Since pyruvate is a synthetic precursor of various substances, the demand for pyruvate is increasing day by day, and it is widely used in daily chemical, food, medical, agricultural and other industries. [0003] The production methods of pyruvate mainly i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N1/16C12P7/40C12R1/72
Inventor 周景文陈坚罗正山堵国成刘松方芳
Owner JIANGNAN UNIV
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