Recombinant saccharymyces cerevisiae for producing ginsengenins as well as construction method and application of same

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied to recombinant Saccharomyces cerevisiae producing ginsenosides and its construction and application fields, and can solve the development and application of ginsenoside pharmaceutical raw material intermediates affecting the clinical application of ginseng, continuous cropping obstacles, ginsenosides The production of quasi-compounds cannot meet the needs of society, etc.

CN103484389AActive Publication Date: 2014-01-01TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2014-01-01

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Abstract

The invention discloses a recombinant saccharymyces cerevisiae for producing ginsengenins as well as a construction method and an application of the same. The recombinant saccharymyces cerevisiae capable of producing three ginsengenins of oleanolic acid, protopanoxadiol and protopanaxatriol simultaneously is Saccharymyces cerevisiae GY-1, with a collection number of CGMCC No. 7725 in the China General Microbiological Culture Collection Centre. The saccharymyces cerevisiae GY-1 is capable of producing three ginsengenins of oleanolic acid, protopanoxadiol and protopanaxatriol simultaneously, wherein the yields achieve 21.4 mg / L in fermentation broth, 17.2 mg / L in fermentation broth and 15.9 mg / L in fermentation broth respectively, the proportions of which in the total ginsengenin content are 39.3%, 31.5% and 29.2% respectively.
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Description

technical field

[0001] The invention relates to a recombinant saccharomyces cerevisiae producing ginsenogenin and its construction method and application. Background technique

[0002] Ginsenoside is the main active ingredient of Araliaceae plants Panax ginseng and Panax quinquefolium. There are more than 40 kinds of ginsenosides discovered so far, all of which have similar basic structures, including sterane steroid cores arranged in four rings with 30 carbon atoms. Depending on the structure of the glycoside group, ginsenosides are divided into three types: panaxadiol type, panaxatriol type and oleanolic acid type, as follows:

[0003] Panaxadiol type, which contains the most types of ginsenosides, such as ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2 and glycoside PD;

[0004] Panaxatriol type, including ginsenosides Re, Rg1, Rg2, Rh1 and glycoside PT;

[0005] Oleanane type, containing ginsenoside R0.

[0006] 1) Dammarenediol-II is a common precursor for the biosynth...

Examples

Embodiment 1

[0069] Example 1: Plasmid Construction

[0070] 1. The cloning of genetic elements is divided into the following three steps:

[0071] (1) Yeast genomic DNA extraction

[0072] Genomic DNA of Saccharomyces cerevisiae BY4742 (Saccharomyces cerevisiae BY4742, described in Carrie baker brachmann et al., 1998, YEAST, 14:115-132, available to the public from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.) was extracted.

[0073] (2) Acquisition of Arabidopsis cDNA

[0074] Total RNA was extracted from Arabidopsis thaliana (col-0 ecotype, recorded in Athanasios Theologis et al., 2000, Nature 408: 816-820, and the public can obtain it from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences), and was reverse-transcribed to obtain pseudo A. thaliana cDNA.

[0075] (3) PCR amplification and cloning of gene elements

[0076] Using the genomic DNA of Saccharomyces cerevisiae BY4742 (Saccharomyces cerevisiae BY4742, recorded in Carri...

Embodiment 2

[0153] Embodiment 2, construction of engineering bacteria Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY-βA

[0154] 1. Construction of BY4742-TRP engineering bacteria

[0155] Using the p-ΔTrp1 plasmid in Example 1 as a template and using the primers in Table 11, Trp-Loxp-HIS3 of about 2056 bp was amplified.

[0156] Culture Saccharomyces cerevisiae BY4742 overnight in YPD, take 1ml (OD about 0.6-1.0) and put it into 1.5ml EP tube, centrifuge at 10000g for 1min at 4°C, discard the supernatant, wash the precipitate with sterile water (4°C), do the same Centrifuge under conditions and discard the supernatant. Add 1ml of treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5), add DTT only when the treatment solution is used) to the bacteria, and place it at 25°C for 20min. Centrifuge, discard the supernatant, add 1ml of 1M sorbitol (0.22um aqueous membrane membrane sterilization) to resuspend the bacteria, centrifuge, discard the supernatant (res...

Embodiment 3

[0182] Embodiment 3, the construction of engineering bacteria recombinant Saccharomyces cerevisiae BY-OA

[0183] 1) Trp-HIS3-up, P PGK1 -GgbAS-T ADH1 , P TDH3 -AtCPR1-T TPI1 , P TEF1 -MtOAS-T CYC1 and Trp-down preparation

[0184] A 1861bp DNA fragment M1 (Trp-HIS3-up) shown in SEQ ID No. 7 was prepared, and M1 contained a homologous fragment upstream of the Trp1 site shown in positions 1429-1861 of SEQ ID No. 7. A 648bp DNA fragment M5 (Trp-down) shown in SEQ ID No. 12 was prepared, and M5 contained a homologous fragment downstream of the Trp1 site shown in positions 1-455 of SEQ ID No. 12. Use the PCR templates and primers described in Table 15 to perform PCR to obtain functional modules (DNA fragments M2-M4): about 3206bp of M2 (P PGK1 -GgbAS-T ADH1 ), about 3281bp of M3 (P TDH3 -AtCPR1-T TPI1 ) and about 2177bp of M4 (P TEF1 -MtOAS-T CYC1 ). M2 (P PGK1 -GgbAS-T ADH1 ) containing the expression cassette P of the GgbAS gene PGK1 -GgbAS-T ADH1 , the expressi...