High-yield fumaric acid Rhizopus delemar engineering bacterium and application thereof

A technology of Rhizopus oryzae fusate and Rhizopus oryzae, applied to fungi, using vectors to introduce foreign genetic material, microorganisms, etc.

Inactive Publication Date: 2013-04-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene manipulation technology and genetic transformation technology for Rhizopus oryzae are relatively weak, and Rhizopus oryzae is a multinucleated cell, and the mitosis of recombinant cells is unstable
Therefore, the expression of exogenous genes targeting Rhizopus oryzae has not had a breakthrough until the past 10 years, and the details of carbon metabolism flow are still unclear

Method used

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  • High-yield fumaric acid Rhizopus delemar engineering bacterium and application thereof
  • High-yield fumaric acid Rhizopus delemar engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Wash the spores from the plate with sterile normal saline, filter them through 6 layers of lens paper and wash them with sterile normal saline 3 times, inoculate the washed spores in 30mL YEPD liquid medium, cultivate them at 37°C and 200rpm for 4h to make the spores During germination, samples were taken every 1 h to observe the spore morphology once. Germinated spores were collected by centrifugation at 8000 rpm at 4°C for 15 min, washed once with 20 mL of pre-cooled sterile saline, resuspended in 10 mL of YED medium, and incubated at 30°C at 150 rpm for 60 min. Collect the spores by centrifuging at 8000rpm at 4°C for 10min, wash once with 10mL pre-cooled EB buffer, suspend the spores with 5mL pre-cooled EB buffer, aliquot them, and put them on ice for later use.

[0028] Example 2 Fusion PCR method to construct the target fragment ScPYC1 and the construction of the expression plasmid

Embodiment 2

[0029] The pdcA promoter fragment with a length of 1219 bp and the terminator fragment with a length of 943 bp were obtained by chemical total synthesis method, and two restriction sites of PacI and SalI were added to the 5' ends of the pdcProF and pdcTerR fragments, respectively.

[0030] Using the NM 001180927.1 sequence published by NCBI, the PYC1 fragment of the coding region of the pyruvate carboxylase gene of Saccharomyces cerevisiae was synthesized by chemical total synthesis;

[0031] Under the action of pfu enzyme, through fusion PCR, the fusion fragment PYC1 of the three oligonucleotide chains of pdcAPro, pdcATer and PYC1 is amplified. Under the action of restriction enzymes PacI and SalI and DNA ligase, the fusion fragment PYC1 was connected to the previously constructed plasmid pRS303H (Taxis C, Knop M. System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques, 2006.40(1):73-78.) The transf...

Embodiment 3

[0033] After the recombinant plasmid pRS303H-PC was purified, R. delemar NRRL1526 was electroporated to germinate spores. The strains that can grow on the PDA+HygB medium were transferred to the PDA medium for spore production culture, and the spore suspension was coated on the PDA+HygB medium culture dish, which was continuously passaged for 3 times to obtain genetically stable recombinants. The results of chemical sequencing were consistent with expectations, indicating that pRS303H-PC had been successfully integrated into the R.delemar genome. The resulting recombinant strain was named R.delemar-pRS303H-PC. When the bacterium grows on the medium with glucose as the only carbon source, the activity of pyruvate carboxylase is 4.59U / mg protein, which is 5.4 times that of the original strain.

[0034] Embodiment 4 fermentation produces the method for fumaric acid

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Abstract

The invention discloses a high-yield fumaric acid Rhizopus delemar engineering bacterium and application thereof, and belongs to the field of genetic engineering. An encoded gene ScPYC1 of pyruvate carboxylase, which originates from saccharomyces cerevisiae, is overexpressed in a bacterial strain Rhizopus delemar NRRL1526 for producing the fumaric acid through a fermentation method, so that a recombinant bacterial strain R. delemar-pRS303H-PC, of which the activity of the pyruvate carboxylase is improved, is obtained, and the activity of the pyruvate carboxylase of the recombinant bacterial strain R. delemar-pRS303H-PC is improved by 5.4 times (reaches 4.59 U / mg protein); and glucose serves as the only carbon source, the output of the fumaric acid reaches 55.92 g / L and is 1.19 times of that of an original strain after the glucose is fermented for 72h, and the high-yield fumaric acid Rhizopus delemar engineering bacterium has a broad application prospect.

Description

technical field [0001] The present invention relates to a kind of rhizopus oryzae with high yield of fumaric acid, especially a method of replenishing tricarboxylic acid cycle by overexpressing ScPYC1 gene by means of metabolic engineering to improve the activity of intracellular pyruvate carboxylase, thereby regulating the flow of carbon metabolism from acetone to The acid enters the TCA reduction pathway to realize the method of excessive accumulation of fumaric acid. Background technique [0002] Fumaric acid (fumaric acid) is an important dicarboxylic acid, widely used in resin synthesis, food additives, feed additives, medicine, aviation materials and other industrial production. Rhizopus delemar, as one of the main microorganisms that accumulate a large amount of fumaric acid, is widely used in the production of organic acids, enzymes, antibodies, and low cholesterol. There are TCA oxidation pathways in R. delemar cells and TCA reduction pathways in the cytosol. Pyru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12P7/46C12R1/845
Inventor 陈坚周景文周正雄堵国成
Owner JIANGNAN UNIV
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