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A kind of method of Saccharomyces cerevisiae fermenting and producing phloretin

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in the field of phloretin fermented by Saccharomyces cerevisiae, can solve the problems of serious pollution and chemical pollution, and achieve the effect of good purity, high content and easy production cost

Active Publication Date: 2021-11-05
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is discharge of chemical waste liquid in this method, and the chemical pollution is serious
In addition, apple peel is used as raw material, ethanol solution is used as extractant, concentrated, acid hydrolyzed and then extracted and dried to obtain phloretin. This method uses acid hydrolysis and serious pollution

Method used

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  • A kind of method of Saccharomyces cerevisiae fermenting and producing phloretin
  • A kind of method of Saccharomyces cerevisiae fermenting and producing phloretin
  • A kind of method of Saccharomyces cerevisiae fermenting and producing phloretin

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Plasmid construction.

[0043] According to the amino acid sequence provided in the present invention and Saccharomyces cerevisiae codon preference, design the nucleic acid sequence of the corresponding gene, send the gene Ha4CL and EbCHS to a commercial company for synthesis, design a homology arm with a size of about 20bp, and use a one-step cloning kit to clone the two The gene is constructed on the vector YCplac22 to obtain vector 1, as shown in Figure 2 (a), the expression vector YCplac22 has the ampicillin resistance of Escherichia coli and the selection marker trp gene of Saccharomyces cerevisiae;

[0044] Two genes, RlmatB and RlmatC, which convert malonate from rhizobia of leguminous plants into malonyl-CoA, were optimized according to the codon preference of Saccharomyces cerevisiae, and then the two genes were simultaneously constructed in Saccharomyces cerevisiae expression vector YCplac33 Obtain vector 2, as shown in Figure 2 (b), the expression v...

Embodiment 2

[0046] Embodiment 2 conversion process.

[0047] 1) Pick a single colony and shake it overnight for 12 hours in the corresponding medium;

[0048] 2) measure the OD600 value of the bacterium liquid that cultivates with spectrophotometer;

[0049] 3) Transfer to 50 mL of fresh YPAD medium with an initial OD600 value of 0.2 OD;

[0050] 4) Activate for 4-5 hours so that the OD600 value of the two-generation bacterial liquid of Saccharomyces cerevisiae is 0.8-0.9;

[0051] Centrifuge at 3600rpm for 5min to collect the bacteria, 25mL ddH 2 O washed twice (ddH used 2 O is preferably sterilized on the same day);

[0052]5) Resuspend 1mL of water into a sterile 1.5mL centrifuge tube; (for use in ultra-clean bench)

[0053] 6) Centrifuge at 13000rpm for 30s to collect the bacteria;

[0054] 7) Resuspend in 1 mL of water and aliquot 100 μL per tube for transformation, centrifuge the aliquoted bacterial solution in a hand-held centrifuge for 20 seconds, discard the supernatant and...

Embodiment 3

[0065] Embodiment 3 integration process

[0066] 1) Saccharomyces cerevisiae genome was extracted, and the upstream and downstream homology arms of the YPRCdelta15 integration site on chromosome 16 of Saccharomyces cerevisiae were extracted by PCR to extract fragments, upstream fragment 1 and downstream fragment 2.

[0067] 2) The upstream fragment and the screening tag leu gene, gene (including promoter and terminator) SeACS L641P , ScADH2, ScALD6, ScACCl S659A，S1157A The homologous arm is transferred into Saccharomyces cerevisiae W303. In Saccharomyces cerevisiae, using homologous recombination, Saccharomyces cerevisiae itself will integrate the introduced gene fragment into the genome of Saccharomyces cerevisiae.

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Abstract

The invention relates to a method for fermenting and producing phloretin by Saccharomyces cerevisiae, which uses p-hydroxyphenylpropionic acid as a raw material, Saccharomyces cerevisiae as a host bacterium, and is completed through the catalysis of several enzymes in the host bacterium. The present invention differs from the prior art in that: 1) low-cost compounds are used as raw materials, and the cost of process raw materials is low; 2) based on the technology of transforming microorganisms, the catalytic synthesis of biological enzymes is carried out in microorganisms, avoiding large-scale extraction, The separation and purification process is easy to control from the aspects of production cost and environmental friendliness; 3) the scale and quantity of the production equipment of this process are less than other methods, and it is easy for industrial transformation; 4) this process is only available in the final step of production. Separation and purification process, the product purification process is simple, the product quality is better than the general method, the purity is better, and the content is higher; 5) There is no waste liquid discharge during the synthesis process, which is environmentally friendly, pollution-free, and sustainable production.

Description

technical field [0001] The invention relates to a biosynthesis method of phloretin, in particular to a method for fermenting and producing phloretin by Saccharomyces cerevisiae. Background technique [0002] Phloretin molecular formula: C 15 h 14 o 5 ; English name: Phloretin; CAS: 60-82-2; Molecular weight: 274.28; Physical properties: easily soluble in methanol, ethanol and acetone, slightly soluble in chloroform, insoluble in water, petroleum ether and benzene, square prism or tablet crystals (ethanol or methanol). [0003] Phloretin is a new type of natural skin whitening agent newly researched and developed abroad. It is another natural beauty product after arbutin. It is mainly distributed in the peels and root bark branches and leaves of juicy fruits such as apples and pears. It is mainly used as a natural skin whitening agent. It can inhibit the activity of tyrosinase and melanin, and has a lightening effect on various skin spots. According to the latest researc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/26C12N15/81C12R1/865
Inventor 陈贤情刘晓楠江会锋王筱王文
Owner 嘉兴欣贝莱生物科技有限公司
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