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Saccharomyces cerevisiae engineering strain with high yield of ergosterol as well as construction method and application of saccharomyces cerevisiae engineering strain

A technology of Saccharomyces cerevisiae strains and Saccharomyces cerevisiae, which is applied in the field of Saccharomyces cerevisiae engineering strains with high production of ergosterol, can solve the problems of unsatisfactory annual ergosterol production, increased demand for ergosterol, and inability to effectively increase ergosterol content, etc., to achieve easy Achieve and control the effect of high output and low investment cost

Pending Publication Date: 2022-07-08
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If 24(28)-dehydroergosterol cannot be further converted into ergosterol, the ergosterol content in yeast cells will not be effectively increased
[0004] The annual output of ergosterol in my country cannot meet the needs of consumers and needs to be imported from abroad, but overall, the demand for ergosterol in my country is increasing year by year

Method used

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  • Saccharomyces cerevisiae engineering strain with high yield of ergosterol as well as construction method and application of saccharomyces cerevisiae engineering strain
  • Saccharomyces cerevisiae engineering strain with high yield of ergosterol as well as construction method and application of saccharomyces cerevisiae engineering strain
  • Saccharomyces cerevisiae engineering strain with high yield of ergosterol as well as construction method and application of saccharomyces cerevisiae engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1Construction of SEC14 gene expression vector

[0087] The following amplification primers were designed according to the nucleotide sequence of SEC14:

[0088] pRS426-P TPI1 -SEC14-3HA-F:

[0089] 5'-TCTATAACTACAAAAAAACACATACAATGGTTACAGTATGTTGTTTGC-3';

[0090] pRS426-P TPI1 -SEC14-3HA-R:

[0091] 5'-TTTCATCGAAAAGGCTTCCGGACATAGTCAGGAACATCGTATGGGTA-3';

[0092] PCR reaction system: FastPfu Buffer 10μL, 2.5mM dNTPs 4μL, template DNA 1μL, upstream and downstream primers 0.5μL each, FastPfu DNApolymerase 1μL, ddH 2 O fills up to a total volume of 50 μL;

[0093]The SEC14 fragment was obtained by PCR amplification with BY4741 genomic DNA as the template. PCR reaction conditions were as follows: 95°C for 5 min, 95°C for 30s, 56°C for 30s, 72°C for 1 min, cycle 35 times, 72°C for 5 min, and 4°C incubation. After the PCR product was analyzed by agarose gel electrophoresis, the target fragment was recovered by cutting the gel ( figure 1 ).

[0094] The fol...

Embodiment 2

[0099] Example 2 Construction of Saccharomyces cerevisiae engineering strain overexpressing SEC14 gene

[0100] Utilize the method of genetic engineering, use restriction endonuclease Xho I and Not I to SEC14-3HA fusion gene, P TPI1 The promoter and pRS426 vector were connected by double digestion, and the recombinant plasmid was obtained for double digestion verification ( image 3 ), sent to Goldwisdom for sequencing, and the correctly sequenced recombinant plasmid was named pRS426-P TPI1 -SEC14-3HA. The obtained recombinant plasmid was transformed into Saccharomyces cerevisiae BY4741 strain, and then the monoclonal recombinant strain was obtained by screening on uracil auxotrophic (-ura) solid yeast medium.

Embodiment 3

[0101] Example 3 Growth curve of Saccharomyces cerevisiae engineered strains overexpressing SEC14 gene

[0102] (1) The ergosterol-producing Saccharomyces cerevisiae engineering strain and the control strain were respectively inoculated into test tubes containing 5 mL of uracil auxotrophic (-ura) liquid medium, and cultured at 30°C and 220 r / min for 12-16 h ;

[0103] (2) Take 20 μL, 30 μL and 40 μL of bacterial liquid respectively and add them to a 1.5 mL EP tube containing 1 mL of uracil auxotrophic (-ura) liquid medium, mix well, add 300 μL to a 100-well plate, and simultaneously take 300 μL urine Pyrimidine auxotrophic (-ura) liquid medium was used as blank control. The 100-well plate was placed in an automatic growth curve analyzer, incubated at 30°C, and the absorbance value at a wavelength of 600 nm was measured every 1 h.

[0104] (3) Taking culture time (X) as abscissa, OD 600 The value (Y) is the ordinate, and the growth curve is drawn.

[0105] The result is as ...

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Abstract

The invention discloses a saccharomyces cerevisiae engineering strain capable of efficiently producing ergosterol, the saccharomyces cerevisiae engineering strain is constructed by overexpressing a lipid transporter gene SEC14 as shown in SEQ ID NO.1 in a saccharomyces cerevisiae host, the nucleotide sequence of the lipid transporter gene SEC14 is SEQ ID NO.1, and the amino acid sequence coded by the lipid transporter gene SEC14 is SEQ ID NO.2. The invention also discloses a saccharomyces cerevisiae engineering strain capable of efficiently producing ergosterol. The ergosterol contained in the constructed saccharomyces cerevisiae engineering strain is 0.95%-1.20% of the dry weight of saccharomycetes, and is 26%-31% higher than that of a wild type saccharomycetes strain. The engineering bacterium provided by the invention not only has the advantage of high yield, but also can be fermented through relatively simple method conditions, is easy to implement and control in industry, is low in investment cost, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an engineering strain of Saccharomyces cerevisiae with high ergosterol production, a construction method and application. Background technique [0002] Ergosterol, also known as ergosterol, is a 28-carbon steroid that is widely present in yeast and is an important component of fungal cell membranes. Integrity and transport of cellular material play an important role. Ergosterol is an important pharmaceutical chemical raw material used in the production of sterols such as "cortisone" and "hormone progesterone". Ergosterol is also vitamin D 2 The precursor of vitamin D can be obtained by ultraviolet irradiation 2 . In the pharmaceutical industry, vitamin D 2 It is an important drug for the prevention and treatment of rickets, dental caries and senile osteoporosis. Vitamin D 2 It can also be added to the feed as a feed additive to increase the egg production rate and hatching rat...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/31C12N15/62C12N15/81C12P33/00G01N21/31G01N30/02G01N30/74C12R1/865
CPCC12N1/18C07K14/395C12N15/81C12P33/00G01N30/02G01N30/74G01N21/31C07K2319/42
Inventor 王敏屠琳娜王雪薇孔繁萌杜娟申雁冰郑宇宋佳夏梦雷
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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