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Application of oxidoreductase and mutant thereof in biosynthesis of noottanone

A biosynthesis and reductase technology, applied in the field of bioengineering, can solve the problems of unsatisfactory industrial production, low substrate concentration, poor salt tolerance, etc., and achieve excellent effect, good substrate tolerance, and good substrate tolerance. degree of effect

Active Publication Date: 2022-05-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of an oxidoreductase and its mutants in the biosynthesis of narone, aiming to solve the problem of dehydrogenation of existing alcohols in the process of biosynthesis of narone The enzyme can tolerate low substrate concentration, low conversion rate, poor salt tolerance and unsatisfactory problems in industrial production

Method used

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  • Application of oxidoreductase and mutant thereof in biosynthesis of noottanone
  • Application of oxidoreductase and mutant thereof in biosynthesis of noottanone
  • Application of oxidoreductase and mutant thereof in biosynthesis of noottanone

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Cloning of oxidoreductase NRRL and construction of expression vector

[0042] The marine killing yeast Wickerhamomyces anomalus M15 was inoculated in 5mL YPD (glucose 20g / L, yeast extract 10g / L, peptone 20g / L) liquid medium and cultured at 30°C until logarithmic growth phase. Genomic total DNA was extracted from the marine killing yeast using the Yeast DNAKit kit (purchased from Omega).

[0043] Aiming at the hypothetical protein [named WICANDRAFT_92107 (NCBI accession number is XP_019039214.1) derived from marine killer yeast (Wickerhamomyces anomalus NRRL Y-366-8), it contains a total of 264 amino acids; its coding gene (NCBI accession number is XM_019186339. 1) The nucleotide sequence contains a total of 795 nucleotides] A pair of specific primers are designed for the coding gene sequence, and each primer introduces a base sequence homologous to the vector at the 5' end for homologous recombination Cloning method To construct the YEp352 expression vector,...

Embodiment 2

[0062] Example 2: Construction of recombinant expression cells

[0063]Using the yeast transformation kit S.c.Easy Comp Transformation Kit (purchased from Invitrogen, USA), the recombinant expression plasmid YEp352-TDH3 constructed in Example 1 was p -NRRL-ADH1 t Transformed into S.cerevisiae CEN.PK2-1 Ca competent cells.

[0064] 1) Add 500ng of recombinant plasmid and 250μL of Solution III (Transformation solution) to every 25μL of competent cells;

[0065] 2) Oscillate evenly with a vortex shaker, and place in a constant temperature incubator at 30°C for 15 minutes;

[0066] 3) Shake and mix again after taking it out, place it at 30°C for 15 minutes, and repeat the operation twice;

[0067] 4) Then take 200 μL of recombinant yeast cells and spread them evenly on the auxotrophic plate SD / ΔUra (6.7g / L YNB, 2g / L amino acid mixture, 20g / L glucose, 20mg / L leucine, 20mg / L tryptophan) Spread the plate and incubate at 30°C for 2 to 4 days.

[0068] Until a single clone grows o...

Embodiment 3

[0069] Example 3: Catalytic preparation of naringone by whole cells in vitro

[0070] Pick the recombinant expression cell S.c.CEN.PK2-1 Ca (YEp352-TDH3 p -NRRL-ADH1 t ) into a test tube containing 5 mL of SD / ΔUra liquid medium, and cultured at 30° C. and 220 rpm for 24 hours. Then press initial OD 600 The inoculum size =0.05 was transferred to a 250 mL shake flask filled with 50 mL SD / ΔUra liquid medium, and cultured at 30° C. and 220 rpm for 24 hours. Subsequent collection of total OD 600 Centrifuge at 3000rpm, 4°C for 5min, and discard the supernatant. Then the recombinant expression cells were resuspended to 1mL with potassium phosphate buffer (50mM, pH 7.4) to obtain 50OD 600 / mL of reaction solution. Add 20 μL of 100 mM grapefruit alcohol solution (containing 1% (v / v) Triton-100, dissolved in dimethyl sulfoxide) to make the final substrate concentration 2 mM, and catalyze at 25° C. and 220 rpm for 24 hours.

[0071] Product detection method is as follows (the prod...

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Abstract

The invention discloses an application of oxidoreductase and a mutant thereof in biosynthesis of noottanone, and belongs to the technical field of bioengineering. According to the present invention, the oxidation-reduction enzyme having high nodulariol conversion ability is obtained from the marine killer yeast (Wickhamomyces anomalusM15) for the first time, and the oxidation-reduction enzyme mutant having high ability is obtained through site-specific mutagenesis; compared with the existing oxidoreductase capable of catalyzing nodulariol, the oxidoreductase and the mutant thereof have good substrate tolerance, high conversion rate and high salt tolerance. According to the oxidoreductase and the mutant thereof provided by the invention, conditions are provided for synthesizing noottanone in an in-vitro enzyme catalysis manner, and noottanone products are synthesized by catalyzing noottanol in a green and efficient manner. According to the invention, an important tool enzyme is provided for the synthesis of noottanone, and huge economic benefits are brought to the synthesis industry of noottanone.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the application of an oxidoreductase and a mutant thereof in the biosynthesis of naringone. Background technique [0002] Nootkatone (also known as Nootkatone), is a sesquiterpene compound with pomelo aroma and slightly bitter taste, which was first extracted from Alaska cedar and exists in plants such as grapefruit and citrus . Pure pomelo ketone is a white to slightly yellow crystal, approved by FDA and EPA for the deployment of pomelo, orange, tropical fruit and other food flavors and tobacco flavors. In spray form, narone is an effective insecticide against lone star ticks and deerticks, and also has a good killing effect on mosquitoes, bedbugs, lice, etc. Because narone has good volatility and is non-toxic to humans, it is considered to be an environmentally friendly insecticide. In 2014, the CDC officially approved two companies to produce narone-based...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N9/02C12N15/53C12N15/81C12N1/19C12R1/865
CPCC12P7/26C12N9/0004C12N15/81C12N2800/102C12N2800/22Y02A50/30
Inventor 李爽陈东莹李文朱晁谊
Owner SOUTH CHINA UNIV OF TECH
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