Genetically engineered bacterium for producing free fatty acid under assistance of formate

A technology of free fatty acid and genetically engineered bacteria, applied in the field of genetically engineered bacteria, can solve the problems of high energy consumption and difficult to obtain precursors, etc.

Pending Publication Date: 2022-03-22
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical method is synthesized by the hydrocarboxylation reaction of olefins, but the energy consumption is large, and the precursor is not easy to obtain

Method used

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  • Genetically engineered bacterium for producing free fatty acid under assistance of formate
  • Genetically engineered bacterium for producing free fatty acid under assistance of formate
  • Genetically engineered bacterium for producing free fatty acid under assistance of formate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0049] Existing microorganisms as hosts utilize formic acid and CO 2 Among the technologies, most of them use gene editing methods to modify the chassis of microorganisms so that they have this function, and to further improve the use of formic acid and CO by microorganisms 2 method and utilization of formic acid and CO 2 Proceeding to the production of microbial chemicals remains to be explored. In view of this, the present inventors use formic acid and CO 2 A certain amount of research has been carried out on the method of improving microbial fermentation to produce FFAs, a derivative product of acetyl-CoA. The specific research process is as follows:

[0050] The present invention selects Saccharomyces cerevisiae with mature gene manipulation technology and high robustness to perform formic acid and CO 2 utilization to produce FFAs. Formate can be used as an auxiliary carbon source, which can be converted into carbon dioxide under the action of formate dehydrogenase, ac...

Embodiment 1

[0131] Embodiment 1: Construction of knockout plasmid

[0132] The primers used in this example are shown in Table 1 above.

[0133] First, design the gRNA required for gene knockout through the website (https: / / www.atum.bio / eCommerce / cas9 / input), design the gRNA on the primer, and use the Pstg.Ura plasmid as a template to amplify the gene with uracil ( Ura) screened the marked fragments gRNA-hfd1, gRNA-pox1, gRNA-faa1, gRNA-faa4, and connected these four fragments with the plasmid pLacZ-SalI in turn, and sequenced to obtain the correct plasmids pCas9-hfd1, pCas9-pox1, pCas9 -faa1, pCas9-faa4. Subsequently, each gene was knocked out according to the traditional Cas9 knockout method of Saccharomyces cerevisiae gene.

Embodiment 2

[0134] Embodiment 2: Construction of recombinant plasmid

[0135] The primers used in this example are shown in Table 2 above.

[0136] The exogenous genes FDH1, FDH2, FDH3, FDH4, FDH4-TB1, FDH4-TB2, FDH4-TB3, FDH4-TB4, FDH4-TB5, FDH4-TB6 were synthesized by the company and connected to the plasmid Psp-GM1 respectively, After the experimental screening, the high-efficiency FDH was amplified using the respective plasmids as templates, and then integrated into the Psp-GM1 plasmid by restriction enzyme digestion, and the correct plasmids Psp-GM1-FDH3-FDH4 and Psp-GM1-FDH3 were obtained by sequencing - FDH4-TB1, Psp-GM1-FDH4-FDH4-TB1, Psp-GM1-FDH3-FDH4-FDH4-TB1.

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Abstract

The invention relates to a genetically engineered bacterium for producing free fatty acid under the assistance of formate, the genetically engineered bacterium is recombinant saccharomyces cerevisiae containing a formate dehydrogenase gene FDH, a 1, 5-diphosphate carboxylase/oxygenase gene RuBisCO and a ribulose phosphate kinase gene PRK, and the genetically engineered bacterium is subjected to chassis microbial modification of knockout of related genes of an FFAs catabolism pathway. The genetically engineered bacterium is safe and non-toxic, can enable microorganisms to produce FFAs by utilizing carbon dioxide and a derivative formic acid thereof through fermentation, and is environment-friendly, relatively low in production cost and wide in application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a genetically engineered bacterium that utilizes formate to assist the production of free fatty acids and its application. Background technique [0002] In recent years, massive fossil fuel combustion and excessive greenhouse gas emissions have seriously affected the environment and climate. Atmospheric CO 2 Concentrations have been stable at 200-280ppm for the past 40,000 years, but have risen sharply to nearly 400ppm in the last 50 years This could lead to an increase in global average temperature of 2°C compared to annual levels. Such a severe global temperature change will inevitably increase the risk of glacier calving and cause negative knock-on effects. With the continuous development of synthetic biology technology, scientists have gradually opened up a path to provide green alternatives for human activities (using microorganisms to produce biofuels and chemicals), in order t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/60C12N15/54C12P7/6409C12R1/865
CPCC12N15/81C12N9/0008C12N9/1205C12N9/88C12Y102/01002C12Y207/01019C12Y401/01039C12P7/6409
Inventor 谭天伟王凯王爽解荣陈必强王萌刘子鹤
Owner BEIJING UNIV OF CHEM TECH
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