A method for promoting the synthesis of phloretin by recombinant Yarrowia lipolytica

A technology of yeast and phloretin, applied in the field of phloretin biosynthesis, can solve the problems of low extraction efficiency, chemical pollution, complicated separation and purification steps, etc., and achieve the effects of increasing yield and increasing content

Active Publication Date: 2022-06-17
SHAANXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] The object of the present invention is to provide a kind of Yarrowia lipolytica as host bacterium, the method for the heterologous synthesis of phloretin by microorganisms, from p-hydroxyphenylpropionyl-CoA and malonyl-CoA that satisfy the biosynthesis of phloretin Starting from a single precursor substance, by increasing the accumulation of these two precursor substances, the production of endorretin in the host fungus is finally increased, so as to solve the problems of low extraction efficiency, cumbersome separation and purification steps, and serious chemical pollution in existing extraction methods.

Method used

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  • A method for promoting the synthesis of phloretin by recombinant Yarrowia lipolytica

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Experimental program
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Effect test

Embodiment 1

[0021] 1. According to the amino acid sequence of the target gene and the codon preference of Yarrowia lipolytica, the base sequence of the gene was optimized and sent to a commercial gene synthesis company to synthesize the target gene: tyrosine ammonia lyase gene (TAL, Gene ID: 54319287 ), enoate reductase gene (2-ER, Gene ID: 44999865), 4-coumaroyl-CoA ligase gene (4CL, Gene ID: 110880015), chalcone synthase gene (CHS, Gene ID: 110908971 ), acetyl-CoA carboxylase gene (ACC1, Gene ID: 2909424). After synthesis, the codon-optimized tyrosine ammonia lyase gene (TAL) and the expression vector pJN44 (containing the promoter P TEF and terminator T xpr2 ) were treated with restriction endonuclease SmaI at 37 °C for 2 h, purified and gelled to recover the DNA fragments of the target gene and vector; the two recovered DNA fragments were ligated with NEB DNA ligase (NEB Company, product number: M0367S) The ligation reaction was carried out under the action to obtain the plasmid pJN...

Embodiment 2

[0029] In this example, the plasmid pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 was constructed according to the method of Example 1, and then the plasmid pURA-GUT2 L&R-TAL / 2 was modified with restriction enzymes BamHI and HindIII. - The original promoters of the ACC1 expression cassette and 4CL expression cassette on ER / 4CL / CHS / ACC1 were double-enzymatically digested, and BamHI was added to both ends of the POX2-1 and POX2-2 promoter mutant fragments screened in Example 1, respectively and HindIII restriction sites, and then the POX2-1 and POX2-2 promoter mutant fragments were ligated into the pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 vector, replacing the original ACC1 and 4CL expression cassettes, respectively If there is a promoter, follow the instructions of the yeast transformation kit (Zymo Research corporation, USA) to transform the recombinant plasmid into Yarrowia lipolytica, and use homologous recombination to integrate the expression cassette containing five target genes into Th...

Embodiment 3

[0031] In this example, the plasmid pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 was constructed according to the method of Example 1, and then the plasmid pURA-GUT2 L&R-TAL / 2 was modified with restriction enzymes BamHI and HindIII. - The original promoters of the ACC1 expression cassette and 4CL expression cassette on ER / 4CL / CHS / ACC1 were double-enzyme digested, and BamHI was added to both ends of the POX2-4 and POX2-3 promoter mutant fragments screened in Example 1, respectively and HindIII restriction sites, and then the POX2-4 and POX2-3 promoter mutant fragments were ligated into the pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 vector, replacing the original ACC1 and 4CL expression cassettes, respectively If there is a promoter, follow the instructions of the yeast transformation kit (Zymo Research corporation, USA) to transform the recombinant plasmid into Yarrowia lipolytica, and use homologous recombination to integrate the expression cassette containing five target genes into The chrom...

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Abstract

The invention discloses a method for promoting the synthesis of phloretin by recombinant Yarrowia lipolytica. The method uses tyrosine as a raw material, recombinant Yarrowia lipolytica as a host bacterium, and is catalyzed by several enzymes in the host bacterium. The reaction obtains phloretin. The present invention utilizes the high accumulation of acetyl-CoA in Y. lipolytica cells, and introduces a beneficial mutant fragment of the POX2 promoter to drive the conversion of acetyl-CoA into malonyl-CoA key genes ACC1 and p-hydroxyphenylpropanoid The key gene for acyl-CoA synthesis, 4CL, was efficiently overexpressed to increase the accumulation of malonyl-CoA and p-hydroxyphenylpropionyl-CoA, two important precursors for the synthesis of phloretin, so as to finally increase the production of phloretin in the host bacteria. The invention effectively avoids large-scale extraction and separation processes by constructing a phloretin synthesis and metabolism pathway in microorganisms, is environmentally friendly and pollution-free, and meets the new requirements of today's green production.

Description

technical field [0001] The invention belongs to the technical field of phloretin biosynthesis, in particular to a method for promoting the synthesis of phloretin by recombinant Yarrowia lipolytica. Background technique [0002] Phloretin is a new type of natural skin whitening agent recently researched and developed abroad. It is mainly distributed in the peel and root bark of juicy fruits such as apples and pears. Phloretin is pearl white crystalline powder, soluble in ethanol and acetone, and almost insoluble in water. Its moisturizing effect is very strong, and it can promote the absorption and utilization of functional factors in the formula, so that it can exert a good effect. It can be used in facial masks and skin care. In creams, lotions and serums. In addition, phloretin also has the function of improving memory and various important biological activities such as anti-cancer, anti-oxidation, and anti-tumor, and has broad application prospects in the development of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/26C12N15/81C12N15/60C12N15/53C12N15/54C12N15/52C12R1/645
CPCC12P7/26C12N15/815C12N15/52C12N9/88C12N9/001C12N9/93C12N9/1037C12Y403/01023C12Y103/01031C12Y602/01012C12Y604/01002C12Y203/01074
Inventor 孟永宏杨雪言郭玉蓉邓红李封辰
Owner SHAANXI NORMAL UNIV
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