Method for producing phloretin by fermentation of saccharomyces cerevisiae

A technology for Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in the field of Saccharomyces cerevisiae fermentation to produce phloretin, can solve problems such as serious pollution and serious chemical pollution, and achieve the effects of good purity, high content and simple operation.

Active Publication Date: 2018-01-16
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 soluti

Method used

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  • Method for producing phloretin by fermentation of saccharomyces cerevisiae
  • Method for producing phloretin by fermentation of saccharomyces cerevisiae
  • Method for producing phloretin by fermentation of saccharomyces cerevisiae

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Plasmid construction.

[0043] Design the nucleic acid sequence of the corresponding gene according to the amino acid sequence provided in the present invention and Saccharomyces cerevisiae codon preference, 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 S. Obtain vector 2, as shown in Figure 2 (b), the expression vector YCplac33 has the ampicillin resistance gene of Escherichia coli and the selection marker ura gene of Saccharomyces cerevisiae; ...

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 producing phloretin by fermentation of saccharomyces cerevisiae. The method is completed by performing catalysis on p-hydroxyphenylpropionic acid serving as a rawmaterial and the saccharomyces cerevisiae serving as host bacteria through a plurality of enzymes in the host bacteria. Compared with the prior art, the method disclosed by the invention is characterized in that (1) by taking a low-cost compound as the raw material, the technological raw material is low in cost; (2) on the technical basis of modification of microorganisms, catalytic synthesis ofbiological enzymes is carried out in the microorganisms, so that large-scale extraction, separation and purification processes are avoided, and the production cost and the environmental protection areeasy to control; (3) the scale and the quantity of production equipment in the process are less than the scale and the quantity in other methods, and industrial transformation is facilitated; (4) separation and purification procedures are only carried out in the last step of the production in the process, so that a product purification process is simple, the product quality is higher than that ina general method, and the content is higher; (5) no waste liquid is discharged in a synthesis process, so that the method is environmentally friendly and pollution-free and can realize 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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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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