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Recombinant saccharomyces cerevisiae engineering strain for producing cherry glycoside and application of recombinant saccharomyces cerevisiae engineering strain

A technology of prunin and strains, which is applied in the field of genetic engineering and bioengineering, can solve problems such as difficult industrial production, and achieve the effect of increasing production and increasing supply

Pending Publication Date: 2021-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of chemical synthesis may offer an alternative, but is difficult to industrialize due to the complex chemical structure of the desired product

Method used

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  • Recombinant saccharomyces cerevisiae engineering strain for producing cherry glycoside and application of recombinant saccharomyces cerevisiae engineering strain
  • Recombinant saccharomyces cerevisiae engineering strain for producing cherry glycoside and application of recombinant saccharomyces cerevisiae engineering strain
  • Recombinant saccharomyces cerevisiae engineering strain for producing cherry glycoside and application of recombinant saccharomyces cerevisiae engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 Construction of recombinant Saccharomyces cerevisiae strain containing glycosyltransferase

[0065] The EXG1 site of Saccharomyces cerevisiae E32 was selected as the integration site of glucosyltransferase UGT73C6 (nucleotide sequence such as SEQ ID NO.1), and the endogenous gene SPR1 of Saccharomyces cerevisiae was knocked out. The specific steps are:

[0066] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the UGT73C6 gene shown in SEQ ID NO.1, and use the primers PCCW12-F / PCCW12-R in Table 3 to amplify the start from the genome of Saccharomyces cerevisiae CEN.PK2-1D Sub P CCW12 , using primers TADH1-F / TADH1-R to amplify the terminator T ADH1 , using primers EXG1-armup-F / EXG1-armup-R and EXG1-armdown-F / EXG1-armdown-R to amplify the sequence of 575bp upstream homology arm and 472bp sequence downstream homology arm of EXG1 site respectively. The above fragments were assembled using OE-PCR. PCR products were recovered by ethanol precipit...

Embodiment 2

[0073] Example 2 Optimization of endogenous UDP-glucose biosynthesis pathway

[0074] In order to promote the supply of UDP-glucose, overexpress the endogenous gene PGM1 of Saccharomyces cerevisiae (nucleotide sequence such as SEQID NO.2), and integrate the gene PGM1 into the GAL10 site. The specific steps are:

[0075] Use primer PGM1-F / PGM1-R to amplify gene PGM1 from Saccharomyces cerevisiae CEN.PK2-1D genome, use primer PTDH3-F / PTDH3-R in Table 3 to amplify from Saccharomyces cerevisiae CEN.PK2-1D genome promoter P TDH3 , using primers TTYS1-F / TTYS1-R to amplify the terminator T TYS1 , using primers GAL-armup-F / GAL-armup-R and GAL-armdown-F / GAL-armdown-R to amplify the 820bp sequence of the upstream homology arm of the GAL10 site from the genome of Saccharomyces cerevisiae CEN.PK2-1D And downstream homology arm 723bp sequence. Use primer UGP1-F / UGP1-R to amplify the gene UGP1 (nucleotide sequence such as SEQ ID NO.3) from the Saccharomyces cerevisiae genome, and use the...

Embodiment 3

[0077] Example 3 Expression of heterologous sucrose phosphorylase galU to strengthen the supply of UDP-glucose

[0078] With Escherichia coli Top10 genome as template, use primer galU-F / galU-R to amplify sucrose phosphorylase gene galU (nucleotide sequence as shown in sequence table SEQ ID NO.5), use primer PTDH3-galU-F / PTDH3 -galU-R amplifies promoter P from Saccharomyces cerevisiae CEN.PK2-1D genome TDH3 , using primers TGPM1-F / TGPM1-R to amplify the terminator T from the genome of Saccharomyces cerevisiae CEN.PK2-1D GPM1 , using primers 1014a-armup-F / 1014a-armup-R and 1014a-armdown-F / 1014a-armdown-R to amplify the 469bp sequence of the upstream homology arm of the 1014a site from the Saccharomyces cerevisiae CEN.PK2-1D genome respectively And downstream homology arm 419bp sequence. The above fragments were assembled using OE-PCR. PCR products were recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 500ng of sgRNA were transformed into Sac...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae engineering strain for producing cherry glycoside and application of the recombinant saccharomyces cerevisiae engineering strain, and belongs to the technical field of genetic engineering and bioengineering. The recombinant saccharomyces cerevisiae capable of synthesizing the cherry glycoside is obtained by expressing the recombinant saccharomyces cerevisiae of the glucosyltransferase UGT73C6, an endogenous UDP-glucose biosynthesis pathway is further strengthened, the supply of the UDP-glucose is improved, a heterologous sucrose phosphorylase pathway and a shikimic acid pathway are constructed and strengthened, the yield of the cherry glycoside is increased, the yield of the cherry glycoside of the saccharomyces cerevisiae fermented for 120 hours reaches 136.7 mg / L, and a foundation is laid for subsequent biosynthesis of flavonoid compounds.

Description

technical field [0001] The invention relates to a recombinant yeast engineering strain for producing prunin and its application, belonging to the technical fields of genetic engineering and bioengineering. Background technique [0002] Prunin is a kind of glycosylated flavonoid, which is an important component of citrus plants and has important beneficial effects on human health. Prunin has various biological activities such as antiviral, anti-inflammatory, hypoglycemic, and cholesterol-lowering, and has great application potential in the treatment of diabetes. Prunin is present in grapefruit and can be extracted from waste products such as the peel and seeds. Its hydrolyzed product naringenin has great application potential in the fields of medicine, cosmetics and food industry. [0003] At present, the main method of industrial production of flavonoids is plant extraction, but the accumulation level of high value-added flavonoid prunin in plant tissues is very low, and t...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/55C12N15/61C12N15/81C12P19/60C12N1/19C12R1/865
CPCC12N9/1051C12N9/16C12N9/90C12N15/52C12N15/81C12P19/60C12Y204/01007C12Y204/01024C12Y504/02002C12Y301/04046Y02E50/10
Inventor 周景文李宏彪徐沙曾伟主余世琴陈坚
Owner JIANGNAN UNIV