Recombinant yeast engineering bacterium with high yield of friedelin

A technology of recombining yeast and suberone, applied in genetic engineering, recombinant DNA technology, fermentation, etc., can solve the problems of loss of multiple plasmids and low production of suberone

Pending Publication Date: 2022-04-29
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the production of suberone synthesized by microorganisms that have been reported is low, and multiple plasmid forms are easy to cause loss

Method used

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  • Recombinant yeast engineering bacterium with high yield of friedelin
  • Recombinant yeast engineering bacterium with high yield of friedelin
  • Recombinant yeast engineering bacterium with high yield of friedelin

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Integration of multiple endogenous genes into the yeast genome

[0035] In this study, a modular two-step integration (M2S) technique was used to integrate multiple transcriptional units on yeast chromosomes. 1. Integration of overexpression modules at the chromosome level in yeast

[0036] The truncated gene tHMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase, the ERG20 gene encoding FPP synthase, The ERG9 gene encoding squalene synthase, the ERG1 gene encoding squalene monooxygenase, and the POS5 gene encoding NADH kinase. The gene sequence of UPC2.1 was obtained from literature (Dai [4] et al., 2012, Hu [5] et al., 2020). According to the instructions of the yeast genome extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), the genome of Saccharomyces cerevisiae strain BY4741 was extracted as a template to amplify the open reading frame (ORF) of the overexpressed gene. Then, according to the sequence information of genes pTDH3, pADH1...

example 2

[0039] Example 2 CRISPR / Cas9 technology to knock out genes on competitive pathways

[0040] gRNA expression vector p426-SNR52p-gRNA.CAN1.Y-SUP4t (ref.no.43803), Cas9 expression vector p414-TEF1p-Cas9-CYC1t (43802), universal vector for gene knockout pTY-U01, pTY- U02 is preserved in the laboratory (construction method reference Hu [5] et al., 2020). Specific gRNAs targeting BTS1, ROX1, YPL062w, and YJL064w genes were designed using open source tools (http: / / yeasttricion.tnw.tudelft.nl), and effective target sequences were screened. All gRNA target sequences used in this study are listed in Table 1. To obtain single gRNAs, equimolar solutions of 24nt oligonucleotides F and R are mixed and annealed to generate a double-stranded insert with overhangs at both ends. The double-stranded oligonucleotide fragment was inserted into the AarI recognition site of pTY-U01 using the Golden Gate assembly method to construct a plasmid expressing a single gRNA (Engler [10] et al., 2014; Le...

example 3

[0047] Example 3 Construction of Suberone Synthase Expression Vector

[0048] MiFRS (GenBank: KX147270.1) from Maytenusilicifolia and PdFRS (Genbank: KY931453.1) from Populus davidiana were synthesized by Ruibiotecch and cloned into the cloning site (BamHI / SalI) of pESC-Leu plasmid. FRSs sequences were downloaded from the National Center for Biological Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) database. Codon-optimized plasmid pYES2-TwOSC1 from T. wilfordii T502E Stored in the laboratory (see Zhou for the construction method [9] et al., 2019). The three recombinant vectors were transformed into the modified strains using the Frozen-EZYeast Transformation II Kit (Zymo Research, Irvine, CA, USA), and the empty pYES2 vector and pESC-Leu vector were transformed into yeast as controls. Positive clones were screened on corresponding solid culture plates and confirmed by colony PCR and DNA sequencing.

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Abstract

The invention relates to a recombinant yeast engineering bacterium capable of producing friedelin at high yield. The recombinant yeast engineering bacterium is used for over-expressing tHMG1, ERG1, ERG9 and ERG20; or tHMG1, ERG1, ERG9, and POS5; moreover, the BTS1, ROX1, YPL064w and YJL062w genes are knocked out, and after the tripterygium wilfordii friedelin encoding gene is introduced, friedelin can be efficiently expressed.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant yeast engineering bacterium used for producing suberone. Background technique [0002] Friedelin (friedelin) is a kind of triterpenoid-type pentacyclic triterpene component, which exists in the cork of various plants or the outer bark of stems. Pharmacological studies in recent years have shown that suberone has antioxidant, anti-inflammatory, and blood-lipid-lowering activities, and can inhibit the growth and metastasis of leukemia cells (Chang [2] et al., 2020), exhibited antidiabetic effects in rats (Sunil [8] etal.,2021), inhibit the growth of breast cancer MCF-7 cells (Subash-Babu [7] P, 2017). In addition, suberone plays a good anti-insect effect in plant protection (Baskar [1] et al., 2014). [0003] A series of triterpenoid derivatives based on suberone as the skeleton have been further isolated and identified, which have great potent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/61C12N15/53C12N15/54C12N15/31C12N15/81C12P33/00C12R1/865
CPCC12N9/90C12Y504/9905C12N15/81C12Y101/01034C12N9/0006C12Y205/0101C12N9/1085C12Y205/01021C12N9/0073C12Y114/13132C12Y207/01086C12N9/1205C07K14/395C12P33/00
Inventor 高伟高海云赵欢
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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