Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

High-yield fumaric acid Rhizopus delemar and application thereof

A technology of Rhizopus oryzae and Rhizopus oryzae, applied in the directions of application, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc., can solve the problem of unclear details of carbon metabolism flow, unstable mitosis of recombinant cells, genetic Renovate problems such as weak technology to achieve the effect of promoting excessive accumulation

Inactive Publication Date: 2012-07-11
JIANGNAN UNIV
View PDF2 Cites 6 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • High-yield fumaric acid Rhizopus delemar and application thereof
  • High-yield fumaric acid Rhizopus delemar and application thereof
  • High-yield fumaric acid Rhizopus delemar and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of embodiment 1 germination spore

[0027] Wash the spores from the plate with sterile normal saline, filter through 6 layers of lens paper, and then wash with sterile normal saline 3 times, inoculate the washed spores in 30mL YEPD liquid medium, culture at 37°C, 200rpm for 4h to germinate the spores During the period, samples were taken every 1h to observe the spore morphology once. The 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 10 minutes, wash once with 10 mL of pre-cooled EB buffer, suspend the spores with 5 mL of pre-cooled EB buffer, aliquot them, and put them on ice for later use.

Embodiment 2

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

[0029] Using Rhizopus oryzae pdcA non-translated promoter and terminator fragments as templates, PCR primers pdcProFPac I, pdcProR, pdcTerF, pdcTerRSalI (see Table 1 for details) were designed based on the R. oryzae AF282846 and AF282847 sequences published by NCBI, and thus amplified The pdcA promoter fragment with a length of 1219bp, and the terminator fragment with a length of 943bp, 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 Saccharomyces cerevisiae pyruvate carboxylase gene 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 amp...

Embodiment 3

[0036] Embodiment 3 fermentation produces the method for fumaric acid

[0037] Use Rhizopus oryzae R.delemar-pRS303H-PC as the starting strain, inoculate it into the spore-forming medium, and cultivate it at 30°C for 5-7 days until the conidia mature; wash the spores with sterile water, and dilute to 10 6 Individual / mL; transfer the spore suspension to the seed medium at a volume ratio of 4%, and cultivate at 30°C and 200rpm for 30h; cultivate the seeds before transfer with a volume ratio of 10% to the fermentation medium, and cultivate at 30°C and 200rpm for 72h.

[0038] Compared with the control bacteria, the recombinant bacteria: (1) the output of fumaric acid in the control bacteria was 46.87g / L, and the output of fumaric acid in the recombinant bacteria could reach 55.92g / L, which was 1.19 times that of the control bacteria; (2) after the fermentation, the control bacteria The body weight is 7.58g / L, and the weight of the recombinant bacteria is 7.41g / L, which is almost ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses high-yield fumaric acid Rhizopus delemar and the application thereof, and belongs to the field of genetic engineering. The high-yield fumaric acid Rhizopus delemar is characterized in that by adopting a metabolic engineering means, a gene ScPYC1 from encoded pyruvate carboxylase in Saccharomyces cerevisiae is excessively expressed in a strain Rhizopus delemar NRRL1526 forproducing fumaric acid by adopting a fermentation method, and then a recombinant strain R.delemar-pRS303H-PC of which the pyruvate carboxylase activity is improved is obtained, wherein the pyruvate carboxylase activity of the recombinant strain R.delemar-pRS303H-PC is improved by 5.4 times (reaching 4.59U / mg protein); and glucose is used as the only carbon source, and the yield of the fumaric acid reaches 55.92g / L after the fermentation is conducted for 72 hours, which is 1.19 times of the original strain. The high-yield fumaric acid Rhizopus delemar has wide 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/15C12N15/60C12N15/63C12P7/46C12R1/845
Inventor 陈坚周正雄周景文堵国成
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com