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Saccharomyces cerevisiae engineering strain for synthesizing ectoine through fermentation

A technology of tetrahydropyrimidine and Saccharomyces cerevisiae, which is applied in the directions of fermentation, genetic engineering, bacterial peptides, etc., can solve the problems of high environmental pressure, affecting the production of tetrahydropyrimidine, and corrosion of production equipment, and achieves the effect of wide application value.

Pending Publication Date: 2021-07-23
RAYTING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the shortcomings of high-salt fermentation waste liquid, which severely corrodes the production equipment and puts a lot of pressure on the environment.
In addition, the prior art also discloses recombinant E. coli that produces ectoine, but the construction of this genetically engineered bacteria relies on E. coli with a specific genotype, and at the same time, this kind of bacteria is an amino acid-deficient bacteria, which will inhibit the growth of the bacteria to a certain extent. Growth, causing a certain pressure on the bacteria, and then affecting the production of ectoine

Method used

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  • Saccharomyces cerevisiae engineering strain for synthesizing ectoine through fermentation
  • Saccharomyces cerevisiae engineering strain for synthesizing ectoine through fermentation
  • Saccharomyces cerevisiae engineering strain for synthesizing ectoine through fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 constructs the recombinant vector that separately optimizes ectA, ectB, ectC gene or its RBS sequence

[0038] Construction of the expression system: using the Halomonas elongata genome as a template, the primers s-ect-F / s-ect-R were used to amplify the DNA fragment of the ectABC gene cluster (as shown in SEQ ID NO.1) by PCR, and the resulting DNA fragment and use primer pEBS-F / pEBS-R to amplify pEBS (the construction method of described pEBS is disclosed in paper Yang S, Liu Q, Zhang Y, et al.Construction and Characterization of Broad-spectrum Promoters forSynthetic Biology.[J ]. Acs Synthetic Biology, 2017, 7(1): 287-291. The pEBS backbone of pEBS was assembled to generate pEBS-ectABC. Using pEBS-ectABC as a template, use primer R 1A -F / R 1A -R, R 2A -F / R 2A -R, R 3A -F / R 3A -R,P 1A -F / P 1A -R,P 2A -F / P2A -R,P 3A -F / P 3A -R, R 1B -F / R 1B -R, R 2B -F / R 2B -R,R 3B -F / R 3B -R,P 1B -F / P 1B -R,P 2B -F / P 2B -R,P 3B -F / P 3B -R, R 1C -F / R ...

Embodiment 2

[0039] Example 2 Combined optimization of ectA, ectB, ectC gene RBS, recombinant plasmid of promoter sequence

[0040] pEBS-P constructed in Example 1 1 - ectABC as template, use primer R respectively 1A -F / R 1 P 1 -R,R 2A -F / R 2 P 1 -R,R 3A -F / R 3 P 1 -R for circular PCR amplification, and then digested with DpnI to obtain the recombinant vector pEBS-P after promoter optimization 1 R 1 -ectABC,pEBS-P 1 R 2 -ectABC,pEBS-P 1 R 3 -ectABC.

[0041] pEBS-P constructed in Example 1 2 - ectABC as template, use primer R respectively 1A -F / R 1 P 2 -R, R 2A -F / R 2 P 2 -R, R 3A -F / R 3 P 2 -R for circular PCR amplification, and then digested with DpnI to obtain the recombinant vector pEBS-P after promoter optimization 2 R 1 -ectABC,pEBS-P 2 R 2 -ectABC,pEBS-P 2 R 3 -ectABC.

[0042] pEBS-P constructed in Example 1 3 - ectABC as template, use primer R respectively 1A -F / R 1 P 3 -R,R 2A -F / R 2 P 3 -R,R 3A -F / R 3 P 3 -R for circular PCR amplification...

Embodiment 3

[0049] Embodiment 3 separately optimizes ectA, ectB, the construction of the recombinant bacteria of ectC gene RBS or promoter sequence

[0050]Construction of recombinant bacteria: respectively transfer the linearized recombinant plasmid pEBS-R constructed in Example 1 into S.cerevisiae CEN.PK2-1C haploid competent cells 1 -ectABC,pEBS-R 2 -ectABC,pEBS-R 3 -ectABC,pEBS-P 1 -ectABC,pEBS-P 2 -ectABC,pEBS-P 3 -ectABC,pEBS-ectA-R 1 BC, pEBS-ectA-R 2 BC, pEBS-ectA-R 3 BC, pEBS-ectA-P 1 BC, pEBS-ectA-P 2 BC, pEBS-ectA-P 3 BC, pEBS-ectAB-R 1 C, pEBS-ectAB-R 2 C, pEBS-ectAB-R 3 C, pEBS-ectAB-P 1 C, pEBS-ectAB-P 2 C, pEBS-ectAB-P 3 c.

[0051] The above-mentioned 18 recombinant strains of Saccharomyces cerevisiae, positive control bacteria (genome integration linearized pEBS-ectABC) and negative control bacteria (genome integration linearization pEBS empty Plasmid) single clone was inoculated in 5mL YPD medium, and kanamycin with a final concentration of 50 μg / mL was ...

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Abstract

The invention discloses a saccharomyces cerevisiae engineering strain for synthesizing ectoine through fermentation, and belongs to the technical field of biological engineering. According to the invention, saccharomyces cerevisiae is taken as a host, a triple shuttle plasmid pEBS of bacillus subtilis, escherichia coli and saccharomyces cerevisiae is taken as an expression vector, an ectABC gene cluster derived from halomonas elongata is introduced into the saccharomyces cerevisiae, then strategies such as ectA, ectB and ectC single-gene RBS optimization, promoter optimization, single-gene RBS and promoter double optimization and the like are carried out, and efficient biosynthesis of ectoine is realized. The ectoine accumulation amount is up to 2.3 g / L when the recombinant saccharomyces cerevisiae strain constructed by the invention is cultured for 96 hours on a shake flask, and the recombinant saccharomyces cerevisiae strain has potential and wide application value in industry.

Description

technical field [0001] The invention relates to an engineering strain of Saccharomyces cerevisiae for fermenting and synthesizing ectoine, belonging to the technical field of bioengineering. Background technique [0002] Ectoine is a polar, soluble, uncharged small molecule organic compound within the physiological pH range. Studies have shown that ectoine is the most common regulator of osmotic pressure in aerobic moderately halophilic bacteria; it has positive effects on the conformation and activity of proteins, which can improve their functionality; Freezing, high salinity, high temperature, radiation, etc.) to protect nucleic acids, proteins, enzymes, etc.; widely used in medicine, cosmetics, enzyme industry and other fields. [0003] There are two main methods for the synthesis of ectoine: chemical synthesis and biosynthesis. Among them, the biosynthesis method mainly adopts enzyme catalysis method and fermentation method. There are many problems in the chemical syn...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/31C12P17/12C12R1/865
CPCC12N15/81C07K14/195C12P17/12
Inventor 康振陈坚堵国成杜养标魏伟伟
Owner RAYTING BIOTECHNOLOGY CO LTD
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