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Saccharomyces cerevisiae engineering bacteria for producing campesterol and construction method

A technology for Saccharomyces cerevisiae and campesterol, which is applied in the fields of genetic engineering and bioengineering, can solve the problems of low activity of reductase DHCR7 and restricting efficient production of campesterol, etc.

Active Publication Date: 2021-05-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the prior art, although there are reports of adopting yeast cells to produce campesterol, due to the way (such as image 3 Shown) The activity of the key enzyme 7 dehydrocholesterol reductase DHCR7 is not high, which limits the efficient production of campesterol

Method used

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  • Saccharomyces cerevisiae engineering bacteria for producing campesterol and construction method
  • Saccharomyces cerevisiae engineering bacteria for producing campesterol and construction method
  • Saccharomyces cerevisiae engineering bacteria for producing campesterol and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Acquisition of related genes in the campesterol synthesis pathway

[0050] (1) Acquisition of 7-dehydrocholesterol reductase gene

[0051] According to the amino acid sequence of 7-dehydrocholesterol reductase DHCR7 derived from Pangasianodon hypophthalmus (Genbank registration sequence number is XP_026786162), the codons of the 7-dehydrocholesterol reductase gene have yeast preference through Saccharomyces cerevisiae codon optimization, producing The optimized gene sequence, that is, the nucleotide sequence of the gene dhcr7 encoding 7-dehydrocholesterol reductase is shown in SEQ ID NO 2.

[0052] (2) Acquisition of upstream and downstream homology arms of the erg5 gene locus

[0053] According to the upstream homology arm and downstream homology arm sequence of the erg5 gene locus in the Saccharomyces cerevisiae genome, primers 5'-TGGGAATACTGTACCAGATAATCAAACAT-3' and 5'-CAAAGTTCTGTTTTTCCCCCATTTGTTAAAAGGTATTTATTGTCTATTGGAATAGC-3' were designed to amplify th...

Embodiment 2

[0057] Embodiment 2: the acquisition of the Saccharomyces cerevisiae engineered strain that produces campesterol

[0058] Specific steps are as follows:

[0059] (1) Construction of the expression cassette: the upstream homology arm of the integration site prepared in Example 1, the yeast promoter, the 7-dehydrocholesterol reductase gene, the yeast terminator, and the downstream homology arm of the integration site were constructed to obtain 7 -Dehydrocholesterol reductase expression cassette element; the obtained 7-dehydrocholesterol reductase expression cassette element and the knockout plasmid containing Cas9 protein were introduced into Saccharomyces cerevisiae BY4742 cells through yeast transformation, and the homologous Recombination completes the assembly of elements to obtain a complete expression cassette;

[0060] The genetic manipulation methods involved are as follows:

[0061] The donor DNA fragment was amplified by PCR using primers generated by CASdesigner to ...

Embodiment 3

[0067] Embodiment 3: the fermentation of the Saccharomyces cerevisiae engineered strain that produces campesterol

[0068] Specific steps are as follows:

[0069] (1) Shake flask stage fermentation:

[0070] Saccharomyces cerevisiae engineered bacteria BY4742-Δerg5-GAL1p-dhcr7-ADH1t, BY4742-Δerg5-PGK1p-dhcr7-ADH1t, BY4742-Δerg5-GPM1p-dhcr7-ADH1t, BY4742-Δerg5-TDH3p-dhcr7 prepared in Example 2 -ADH1t, BY4742-Δerg5-TEF1p-dhcr7-ADH1t, BY4742-Δerg5-TPI1p-dhcr7-ADH1t, BY4742-Δerg5-GPD1p-dhcr7-ADH1t, BY4742-Δerg5-TEF2p-dhcr7-ADH1t, BY4742Tcr7p-dhAC -ADH1t, BY4742-Δerg5-TDH2p-dhcr7-ADH1t were activated by streaking on YPD solid medium, and after growing for 48 hours, a single colony was obtained;

[0071]Pick a single colony and put it into a 10mL / 50mL vial of liquid YPD medium, and culture it at 30°C and 200rpm for 24h to obtain a seed solution. The prepared seed solution was transferred to the liquid YPD medium of 50mL / 250mL with an inoculum size of 2% (v / v), and fermented at 30...

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Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria for producing campesterol and a construction method, and belongs to the field of genetic engineering and bioengineering. An expression cassette element of 7-dehydrocholesterol reductase is introduced into a yeast cell body, and is integrated into a yeast genome by utilizing a CRISPR / Cas9 gene operating system, C-22 sterol desaturase is knocked out, competition of an ergosterol branch path is relieved, and yeast synthesis from glucose to campesterol is realized. The method is simple in process, green and environment-friendly, and can be used for producing campesterol through fermentation. The yield of campesterol prepared by adopting the saccharomyces cerevisiae engineering strain BY4742-delta erg5-TEF1p-dhcr7-ADH1t provided BY the invention can reach 253.35mg / L in a shake flask stage, and the horizontal yield of a fermentation tank can reach 916.88mg / L, which is increased by 2.6 times compared with the yield of campesterol in the shake flask stage of the strain.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae engineering bacterium for producing campesterol and a construction method thereof, belonging to the fields of genetic engineering and bioengineering. Background technique [0002] In recent years, steroid drugs have been highly valued by the biopharmaceutical industry due to their good anti-inflammatory, anti-allergic and contraceptive effects. big drug. As people's demand for steroid hormone drugs continues to increase, the production process of steroid hormone drugs worldwide has undergone several changes, including chemical total synthesis, plant-extracted saponin plus chemical semi-synthesis, and new microbial synthesis. Up to now, the synthesis process of steroid hormone drugs is still being explored and innovated continuously. Campesterol is an important synthetic precursor of steroidal drugs (progesterone, androstenedione, hydrocortisone, etc.), and is also one of the main sterols derived fro...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/53C12N15/55C12P33/00C12R1/865
CPCC12N9/001C12N9/0071C12N15/81C12N15/905C12N9/22C12P33/00C12Y103/01021C12Y114/19
Inventor 饶志明周武林邵明龙杨套伟张显徐美娟
Owner JIANGNAN UNIV
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