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Method for promoting recombinant yarrowia lipolytica bacteria to synthesize phloretin

A yeast and phloretin technology, applied in the field of phloretin biosynthesis, can solve problems such as low extraction efficiency, cumbersome separation and purification steps, and chemical pollution, and achieve the effect of increasing production

Active Publication Date: 2021-06-04
SHAANXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Method for promoting recombinant yarrowia lipolytica bacteria to synthesize phloretin

Examples

Experimental program
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Effect test

Embodiment 1

[0022] 1. According to the amino acid sequence of the target gene and the codon preference of Yarrowia lipolytica, optimize the base sequence of the gene and send it 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 gel-recovered the DNA fragments of the target gene and the vector; the two recovered DNA fragments were treated with NEB DNA ligase (NEB Company, product number: M0367S) The ligation reaction was carried out to obtain the plasmid pJN44-TAL containing th...

Embodiment 2

[0030] In this example, the plasmid pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 was constructed according to the method in Example 1, and then the plasmid pURA-GUT2 L&R-TAL / 2 - The original promoters of the ACC1 expression cassette and the 4CL expression cassette on ER / 4CL / CHS / ACC1 were double digested, and BamHI was added to both ends of the POX2-1 and POX2-2 promoter mutant fragments screened in Example 1 and HindⅢ restriction sites, and then the POX2-1 and POX2-2 promoter mutant fragments were connected to the pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 vector to replace the original ACC1 and 4CL expression cassettes, respectively There is a promoter, and then according to the instructions of the yeast transformation kit (Zymo Research corporation, USA), the recombinant plasmid is transformed into Yarrowia lipolytica, and the expression cassette containing five target genes is integrated into the yeast by homologous recombination. lipolytica chromosome, and then obtain the recombinant Yarr...

Embodiment 3

[0032] In this example, the plasmid pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 was constructed according to the method in Example 1, and then the plasmid pURA-GUT2 L&R-TAL / 2 - The original promoters of the ACC1 expression cassette and the 4CL expression cassette on ER / 4CL / CHS / ACC1 were double digested, and BamHI was added to both ends of the POX2-4 and POX2-3 promoter mutant fragments screened in Example 1 and HindⅢ restriction sites, and then the POX2-4 and POX2-3 promoter mutant fragments were connected to the pURA-GUT2 L&R-TAL / 2-ER / 4CL / CHS / ACC1 vector to replace the original expression cassettes of ACC1 and 4CL There is a promoter, and then according to the instructions of the yeast transformation kit (Zymo Research corporation, USA), the recombinant plasmid is transformed into Yarrowia lipolytica, and the expression cassette containing five target genes is integrated into the yeast by homologous recombination. lipolytica chromosome, and then obtain the recombinant Yarrowia lipoly...

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Abstract

The invention discloses a method for promoting recombinant yarrowia lipolytica bacteria to synthesize phloretin. According to the method, tyrosine is used as a raw material, recombinant yarrowia lipolytica bacteria are used as host bacteria, and the phloretin is obtained through the catalytic reaction of a plurality of enzymes in the host bacteria. According to the invention, the high accumulation amount of acetyl coenzyme A in arrowia lipolytica bacterium cells is utilized, and by introducing a beneficial mutant fragment of a POX2 promoter, driving the acetyl coenzyme A to be converted into a key gene ACC1 of malonyl coenzyme A and carrying out high-efficiency overexpression on a synthetic key gene 4CL of p-hydroxybenzene propionyl coenzyme A, the accumulation amounts of two important precursor substances, i.e., the malonyl coenzyme A and the p-hydroxybenzene propionyl coenzyme A, for synthesizing the phloretin are increased, so that the yield of the phloretin in the host bacteria is finally increased. According to the method, a phloretin anabolic pathway is constructed in microorganisms, large-scale extraction and separation processes are effectively avoided, and the method is environment-friendly and pollution-free and meets the new requirements of current green production.

Description

technical field [0001] The invention belongs to the technical field of phloretin biosynthesis, and in particular relates 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 newly researched and developed abroad. It is mainly distributed in the peels and root barks of juicy fruits such as apples and pears. Phloretin is a pearl white crystalline powder, soluble in ethanol and acetone, but almost insoluble in water. It has a very strong moisturizing effect, can promote the absorption and utilization of functional factors in the formula, and make it play a good role. It can be used in facial masks, skin care In creams, lotions and serums. In addition, phloretin also has the function of improving memory and many important biological activities such as anti-cancer, anti-oxidation, anti-tumor, etc., and has broad application prospects in the development of new...

Claims

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Application Information

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Patent Type & Authority Applications(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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