Recombinant saccharomyces cerevisiae expressing CBDAS and construction method and application thereof

A technology for recombining Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in the field of synthetic biology, can solve problems such as high cost, long culture time, and low efficiency, and achieve the effect of increasing production and improving expression activity

Pending Publication Date: 2022-02-01
SENRIS BIOTECHNOLOGY (SHENZHEN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of obtaining CBDA from cannabis plants has disadvantages such as long cultivation time, low efficiency, high cost, waste of biomass, low product purity and the risk of hallucinogenic THC

Method used

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  • Recombinant saccharomyces cerevisiae expressing CBDAS and construction method and application thereof
  • Recombinant saccharomyces cerevisiae expressing CBDAS and construction method and application thereof
  • Recombinant saccharomyces cerevisiae expressing CBDAS and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Transformation Example 1 Insert Fragment Combination Mode A

[0046] The starting strain ySC-31 was inoculated in 10 mL of liquid YPD medium and cultured overnight at 30°C and 200 rpm. The next day, the OD value of the bacterial solution was measured, and an appropriate amount of the bacterial solution was inoculated into 50 mL of YPD medium and diluted to an OD value of 0.2, and the culture was continued for 4.5 hours. For each construction, 5OD bacterial solution was taken, centrifuged at 3000 rcf at room temperature for 5 min, the supernatant was discarded, and washed twice with ddH2O to obtain yeast cells. Finally, 50 μL of DNA mixture (2 μg of 416d-Up, Gal1-CBDAS-CEN1-tADH1, and 416d-Down inserts, 250 ng of plasmid pCUT-416d, and ddH2O was added until the DNA mixture reached 50 μL) to suspend the cells. Then add the above-mentioned lithium acetate conversion mixture, and mix well. 40min in water bath at 42°C. 25°C, 5000rpm, centrifuge for 1min, discard the super...

Embodiment 2

[0047] Transformation Example 2 Insertion Fragment Combination Mode B

[0048] The starting strain ySC-31 was inoculated in 10 mL of liquid YPD medium and cultured overnight at 30°C and 200 rpm. The next day, the OD value of the bacterial solution was measured, and an appropriate amount of the bacterial solution was inoculated into 50 mL of YPD medium and diluted to an OD value of 0.2, and the culture was continued for 4.5 hours. For each construction, 5OD bacterial solution was taken, centrifuged at 3000 rcf at room temperature for 5 min, the supernatant was discarded, and washed twice with ddH2O to obtain yeast cells. Finally, 50 μL of DNA mixture (2 μg of 416d-Up, Gal1-CBDAS-CYB5-tADH1, and 416d-Down inserts, 250 ng of plasmid pCUT-416d, added ddH2O until the DNA mixture reached 50 μL) was used to suspend the cells. Then add the above lithium acetate conversion mixture and mix well. 40min in water bath at 42°C. 25°C, 5000rpm, centrifuge for 1min, discard the supernatant,...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae expressing CBDAS and a construction method and an application thereof. The CBDAS is positioned on a net membrane of endogenous endoplasmic reticulum positioning peptide of the saccharomyces cerevisiae to be expressed, the recombinant saccharomyces cerevisiae expressing the CBDAS is obtained, firstly, a genome of the saccharomyces cerevisiae serves as a template, and an upstream homologous arm 416d-Up fragment and a downstream homologous arm 416d-Down fragment are obtained through PCR amplification; then, by taking a plasmid pZF048 or pZF049 as the template, PCR amplification is carried out, so as to obtain a Gal1-CBDAS-CEN1-tADH1 fragment or a Gal1-CBDAS-CYB5-tADH1 fragment; the recombinant saccharomyces cerevisiae for expressing the CBDAS is obtained by transforming the fragments into the saccharomyces cerevisiae, and the heterologous protein CBDAS is expressed by using endoplasmic reticulum positioning peptide of the saccharomyces cerevisiae, so that the expression activity of the enzyme is improved, and the yield of the product CBDA is increased.

Description

technical field [0001] The present invention relates to the genetic engineering transformation technology in the technical field of synthetic biology, and more specifically, the present invention relates to a high-activity expression strain of CBDAS constructed by using an endogenous endoplasmic reticulum targeting peptide in Saccharomyces cerevisiae and its construction method and application. Background technique [0002] Cannabinoids are secondary metabolites of cannabis plants. With the industrial development and utilization of cannabinoids, the output value in 2020 is expected to reach 100 billion US dollars. At present, more than 100 types of cannabinoids have been discovered, mainly including cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBG) and cannabichromene (CBC), etc. Among them, CBD and THC, which are isomers of each other, have the highest content. Compared with the hallucinogenic THC, the non-psychoactive CBD has a better application prospect. CB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P7/42C12N9/10C12R1/865
CPCC12N15/815C12P7/42C12N9/1085
Inventor 张云丰李宗瑾许薷芳罗小舟
Owner SENRIS BIOTECHNOLOGY (SHENZHEN) CO LTD
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