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Three-enzyme co-expression recombinant bacterium and application thereof in synthesis of (S)-citronellol

A technology of recombinant bacteria and co-expression, applied in applications, recombinant DNA technology, enzymes, etc., to achieve the effect of simple operation, high selectivity, and mild conditions

Pending Publication Date: 2021-11-30
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no co-expression of old yellow enzyme NemR-PS, alcohol dehydrogenase YsADH and glucose dehydrogenase BmGDH M6 However, there is no report on the construction of the old yellow enzyme NemR-PS, alcohol dehydrogenase YsADH and glucose dehydrogenase BmGDH M6 Two-step Reduction of (E / Z)-citral to (S)-citronellol by Recombinant Bacteria

Method used

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  • Three-enzyme co-expression recombinant bacterium and application thereof in synthesis of (S)-citronellol
  • Three-enzyme co-expression recombinant bacterium and application thereof in synthesis of (S)-citronellol
  • Three-enzyme co-expression recombinant bacterium and application thereof in synthesis of (S)-citronellol

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1, acquisition of old yellow enzyme NemR-PS coding gene

[0036] Utilize the published source from Providencia stuartii (Providencia stuartii; Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal) old yellow enzyme NemR-PS coding gene (GenBank accession number is KNZ86848), After codon optimization, artificially synthesized (Hangzhou Qingke Biotechnology Co., Ltd. provides gene synthesis service) the gene encoding the old yellow enzyme NemR-PS, the nucleotide sequence and amino acid sequence are respectively as SEQ ID NO.1 and SEQ ID NO. 2.

Embodiment 2

[0037] Example 2, Acquisition of Alcohol Dehydrogenase Encoding Gene

[0038] Utilizing the published gene encoding alcohol dehydrogenase YsADH (GenBank accession number KF887947) derived from Yorkella sp. CCTCC NO.M2013099 (disclosed in patent application 2013101888839), after codon optimization, the Synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd. provides gene synthesis service) the gene encoding alcohol dehydrogenase, the nucleotide sequence and amino acid sequence are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

Embodiment 3

[0039] Embodiment 3, the acquisition of the gene encoding glucose dehydrogenase

[0040] The GenBank accession number of glucose dehydrogenase BmGDH derived from Bacillus megaterium is AAA22475, and the amino acid sequence of glucose dehydrogenase BmGDH was replaced by Q252L / E170K / S100P / K166R / V72I / K137R to obtain mutant BmGDH M6 (Already disclosed in patent application 2020103075429). mutant BmGDH M6 The coding gene was artificially synthesized after codon optimization (Hangzhou Qingke Biotechnology Co., Ltd. provides gene synthesis service), and the nucleotide sequence and amino acid sequence are respectively shown in SEQ ID NO.5 and SEQ ID NO.6.

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Abstract

The invention discloses a three-enzyme co-expression recombinant bacterium and application thereof in synthesis of (S)-citronellal. The three-enzyme co-expression recombinant bacterium is constructed by jointly introducing the coding genes of old yellow enzyme NemR-PS, alcohol dehydrogenase YsADH and glucose dehydrogenase BmGDHM6 into a host cell. According to the three-enzyme co-expression recombinant bacterium, two-step reduction of 400mM (E / Z)-citral is catalyzed in a one-pot cascade manner by using the recombinant bacteria of co-expression old yellow enzyme NemR-PS, alcohol dehydrogenase YsADH and glucose dehydrogenase BmGDHM6, after reaction is performed for 24 hours, a wet cell of the recombinant bacteria for expressing the glucose dehydrogenase BmGDHM6 is supplemented, after the reaction is performed for 36 hours, a substrate is completely converted into the product (S)-citronellol, the e.e. value of the product is greater than 99%, and chemical selectivity and enantiomer selectivity are high. The three-enzyme co-expression recombinant bacterium has an environmentally-friendly and efficient method and mild conditions, is simple in operation and is suitable for large-scale industrial production.

Description

(1) Technical field [0001] The invention belongs to the field of biocatalysis, and relates to a recombinant bacterium co-expressing yellow enzyme, alcohol dehydrogenase and glucose dehydrogenation and its catalytic selective reduction of (E / Z)-citral to synthesize (S)-citronella in whole cells Alcohol application. (2) Background technology [0002] As an important spice raw material, citronellol is often used in the preparation of rose-flavored, citrus-flavored spices and high-end perfumes. Citronellol is also an important edible spice, used to make food such as flower cakes, baked goods, beverages and candies. Citronellol also has medicinal properties, including antibacterial and antifungal effects in vitro, and analgesic and anticonvulsant effects in vivo. (S)-citronellol is also an important intermediate in chiral synthesis, which can be used to synthesize all-cis rose ether, which has an elegant aroma. [0003] The synthesis methods of citronellol and (S)-citronellol ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/70C12P7/04C12R1/19
CPCC12N9/0006C12N9/001C12N15/70C12P7/04C12Y101/01C12Y103/01C12Y101/9901C12N2800/101Y02P20/584
Inventor 应向贤周雪婷王崎舟
Owner ZHEJIANG UNIV OF TECH
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