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Whole-cell catalyst containing phenylpyruvic acid decarboxylase mutant and application to production of phenethyl alcohol

A technology of phenylpyruvate decarboxylase and whole-cell catalyst, which is applied in the field of bioengineering, can solve problems such as low phenylethanol tolerance, complicated operation process, and food safety issues, and achieve low catalytic efficiency, improved catalytic efficiency, The effect of increasing the conversion rate

Active Publication Date: 2019-11-12
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Previously, there were studies devoted to the co-expression of three enzymes (phenylalanine aminotransferase, ketoacid decarboxylase, and alcohol dehydrogenase) in yeast and Escherichia coli for whole-cell transformation to produce phenylethyl alcohol, but the method had the following drawbacks: (1) Escherichia coli is not a food-grade microorganism, and the endotoxin produced by Escherichia coli during the fermentation process will bring new food safety problems; (2) The operation process of using Escherichia coli as a whole-cell catalyst is complicated and requires Add inducer and control strict induction conditions; (3) yeast and Escherichia coli have low tolerance to phenylethanol, which is unfavorable for high-yield phenylethanol; (3) the time for cultivating yeast whole cell catalyst is too long, and the whole process is as long as 100h

Method used

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  • Whole-cell catalyst containing phenylpyruvic acid decarboxylase mutant and application to production of phenethyl alcohol
  • Whole-cell catalyst containing phenylpyruvic acid decarboxylase mutant and application to production of phenethyl alcohol
  • Whole-cell catalyst containing phenylpyruvic acid decarboxylase mutant and application to production of phenethyl alcohol

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Experimental program
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Embodiment 1

[0025] 1) Construction of recombinant plasmid pHY-kivD:

[0026] An α-ketoacid decarboxylase kivD gene was cloned from Lactococcus lactis subsp.lactis (CICC6246) by using kivD-F and kivD-R. Using the Bacillus subtilis 168 genome as a template, the P43 promoter was amplified with P43-F and P43-R primers; the amylase terminator TamyL was amplified with TamyL-F and TamyL-R primers. P43-F and TamyL-R were used as primers to perform SOE-PCR on the three fragments of P43, kivD and TamyL to obtain the fusion fragment P43-kivD-TamyL. Using the plasmid pHY300PLK as a template and pHY-T5-F and pHY-T5-R as primers, the whole plasmid was amplified by PCR to obtain a linearized pHY300PLK vector. After the above amplified products were checked by electrophoresis, the PCR products were purified and recovered using a gel recovery kit. Using the ClonExpress II one-step cloning kit, the fusion fragment was fused with the linearized vector pHY300PLK to obtain the recombinant plasmid pHY-kivD. ...

Embodiment 2

[0048] 1) Cloning of Bacillus licheniformis DW2 alcohol dehydrogenase coding gene yqdH

[0049] According to the amino acid sequence of Bacillus licheniformis alcohol dehydrogenase YqdH in the NCBI database, it is found that it has 38.65% homology with the alcohol dehydrogenase YqhD of Escherichia coli K12. YqhD is reported to have a broad substrate spectrum. In order to test the catalytic ability of Bacillus licheniformis YqdH to phenylacetaldehyde, primers yqdH-F1 and yqdH-R1 were designed according to the gene sequence of yqdH, and the genomic DNA of Bacillus licheniformis was used as a template to amplify the yqdH sequence. The alcohol dehydrogenase gene yqdH fragment was digested with BamHI and XhoI, and ligated with the expression vector pET28a(+) after the same digestion with T4DNA ligase, and the ligated product was transformed into E.coliDH5α competent cells, and the positive The clones were transferred to liquid LB medium for overnight culture, and the recombinant e...

Embodiment 3

[0058] Construction of co-expression recombinant plasmid pHY-F542W-yqdH:

[0059] Using yqdH-F2 and yqdH-R2 to amplify the yqdH gene from the Bacillus licheniformis DW2 genome, using the Bacillus subtilis 168 genome as a template and P43-F1 and P43-R as primers to amplify the P43 promoter. P43-F1 and yqdH-R2 were used as primers to perform SOE-PCR on the two fragments of P43 and yqdH to obtain the fusion fragment P43-yqdH. Using plasmid pHY-F542W or pHY-kivD as a template and amp-T5-F and amp-T5-R as primers, the whole plasmid was amplified by PCR to obtain linearized pHY-F542W and pHY-kivD. After the above amplified products were checked by electrophoresis, the PCR products were purified and recovered using a gel recovery kit. The yqdH fragments were inserted into the expression vectors pHY-F542W and pHY-kivD by the ClonExpress II one-step cloning kit, and the recombinant co-expression plasmids pHY-F542W-yqdH and pHY-kivD-yqdH were successfully constructed by colony PCR and ...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a whole-cell catalyst containing a phenylpyruvic acid decarboxylase mutant and application to production ofphenethyl alcohol. According to the hole-cell catalyst containing the phenylpyruvic acid decarboxylase mutant, phenylethanol resistant bacillus licheniformis serve as an expression host, heterologousexpression of the phenylpyruvic acid decarboxylase mutant from lactococcus lactis and alcohol dehydrogenase from the bacillus licheniformis is achieved through free plasmid pHY300PLK, and the recombinant bacillus licheniformis with co-expression of the phenylpyruvic acid decarboxylase and the alcohol dehydrogenase are obtained. L-phenylalanine serves as a substrate, and the phenethyl alcohol is produced through whole-cell catalysis of recombinant bacteria. The advantages that the production cost is low, the production condition is mild, impurities in a transformation system is less, the technology steps are simple, and production operation is safe are achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a whole-cell catalyst containing a phenylpyruvate decarboxylase mutant and its application in the production of phenylethyl alcohol. Background technique [0002] Phenylethyl alcohol is an aromatic alcohol with a light, delicate and long-lasting rose aroma. Because of its special odor, phenylethyl alcohol is widely used as a fragrance substance in perfume, cosmetics and food industries. Phenylethyl alcohol can also be used as a pesticide and a new type of biofuel. Therefore, phenethyl alcohol has broad application prospects. [0003] At present, there are two main methods of synthesizing phenylethanol: chemical method and biological method. The biological method is divided into extraction method and fermentation method. In the biosynthesis process of phenylethanol, phenylalanine generates phenylpyruvate through transamination, and phenylpyruvate is further d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/54C12N15/53C12P7/22C12R1/10
CPCC12N9/88C12Y401/01043C12N9/0006C12N15/75C12P7/22
Inventor 陈守文占杨杨王欢石姣周梦林马昕
Owner HUBEI UNIV
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