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Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof

A technology of genetically engineered bacteria and reductases, applied in genetic engineering, oxidoreductases, applications, etc., can solve the problems of low product value, difficult boride waste disposal, and high energy consumption.

Inactive Publication Date: 2015-04-08
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional chemical synthesis process of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate starts from ketoacids (esters) and constructs chiral centers through asymmetric synthesis. The reaction route is complex and requires the use of flammable and explosive Borane, n-butyllithium, etc. and special conditions such as cryogenics lead to low product d.e. value, low yield, and high energy consumption
Moreover, the boride waste produced by the reaction is difficult to deal with, and needs to go through tedious repeated methanol quenching and vacuum distillation.

Method used

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  • Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof
  • Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof
  • Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 produces aldehyde and ketone reductase microbial screening

[0027] Enrichment medium (bean sprouts juice medium): fresh bean sprouts juice (fresh soybean sprouts, add water at 100g / L and boil for 30 minutes, filter the bean sprouts juice, replenish water to the initial volume) 100mL / L, glucose 50g / L, natural pH, Divide into 250mL shaker flasks, fill 50mL, and sterilize at 115°C for 30 minutes. Add sterile tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate 2 g / L. Solid medium: bean sprout juice medium, add 20g / L agar, natural pH.

[0028] Soil samples collected from all over the country were dispersed with normal saline in a certain proportion and then inoculated into the enrichment medium. Under the conditions of 30°C and 150 rpm, the shaker cultured for 24 hours. The enriched culture solution was diluted step by step, spread on a solid plate medium and cultured at 30°C until an observable single colony was formed. Pick a single colony and transfer it to a ...

Embodiment 2

[0038] Embodiment 2 strain identification

[0039] Physiological and biochemical identification and 18S rDNA sequence analysis were performed on the strains with aldehyde and ketone reductase activity screened in Example 1.

[0040] Physiological and biochemical identification: Utilize Vitek 2 microbial automatic identification system to measure bacterial strain XP1461 and bacterial strain XP1462 to the identification result of 46 kinds of identification tests ( as table 1 , Table 2 Shown), through the Vitek 2 reader analysis metabolic fingerprint analysis, it is determined that the bacterial strain XP1461 belongs to the lactis species of the genus Kluyveromyces (kluyveromyces), with a homology of 96%, and the bacterial strain XP1462 belongs to the bailii species of the genus Zygosaccharomyces (Zygosaccharomyces). Origin 98%.

[0041] 18S rDNA sequence analysis: Using the extracted total DNA of strain XP1461 and strain XP1462 as templates, the designed primers: pITS1:5'-TC...

Embodiment 3

[0043] Example 3: Cloning of aldehyde and ketone reductase gene and construction of recombinant plasmid

[0044] (1) Cloning of the target gene

[0045] Kluyveromyces lactis (Kluyveromyces lactis) XP1461 ( Collection No. CCTCC M 2014380) and Bayer yeast (Zygosaccharomyces bailii) XP1462 ( Collection No. CCTCC M 2014381) total genomic DNA of the bacteria.

[0046] Using K.lactis M 2014380 genomic DNA as a template, through the upstream primer (primer 1): 5'- CATATG ACTACTCAAAAGTTCTTTACTT-3', downstream primer (primer 2): 5'- GCGGCCGC TCACTTCTGGGATTCAGAAT-3' was amplified by PCR to obtain amplified product 1. Take 10 μL of PCR amplification product 1 and use 0.9% agarose gel electrophoresis to detect, agarose gel electrophoresis picture See picture As shown in (I) in 1, a band appeared at around 1000bp;

[0047] Using K.lactis CCTCC M 2014380 genomic DNA as a template, through the upstream primer (primer 3): 5'- AGATCT ATGACGTACTTAGCAGAAACAG-3', downstream primer ...

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Abstract

The invention discloses aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, a vector, engineering bacteria and an application thereof. A nucleotide sequence of aldehyde ketoreductase gene is one of a SEQ ID No. 1, a SEQ ID No. 3 and a SEQ ID No. 5, aldehyde ketoreductase gene shown by the SEQ ID No. 1 and the SEQ ID No. 3 comes from bacterial strain-kluyveromyces lactis XP1461, and aldehyde ketoreductase gene shown by the SEQ ID No. 5 comes from bacterial strain-baeyer zygosaccharomyces bailii XP1462. The disclosed aldehyde ketoreductase from K.lactis and Z. bailii can be firstly reported for biological catalyzed synthesis of 6-cyan-(3R, 5R)-dihydroxyhexanoate, optical voidness of the prepared sample 6-cyan-(3R, 5R)-dihydroxyhexanoate can be reached, existence of 6-cyan-(3R, 5R)-dihydroxyhexanoate can be hardly detected; high epimerase selectivity of aldehyde ketoreductase brings benefit, and the products extraction yield is high.

Description

(1) Technical field [0001] The invention relates to aldehyde and ketone reductase enzyme-producing microorganisms, enzyme genes, recombinant expression vectors for constructing these genes and enzyme aldehyde ketone reductase recombinant bacteria, and the development of aldehyde ketone reductase recombinant bacteria in atorvastatin chiral side chain 6-cyanide Application in biocatalytic synthesis of tert-butyl-(3R,5R)-dihydroxyhexanoate. (2) Background technology [0002] Cardiovascular and cerebrovascular diseases have become the number one killer of human health. It is estimated that by 2020, the number of deaths from cardiovascular diseases will account for 36% of the total number of human deaths. The incidence of cardiovascular and cerebrovascular diseases is closely related to high cholesterol levels in the human body. Atorvastatin can competitively inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key rate-limiting enzyme in the synthesis of c...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N1/21C12N1/19C12P13/00C12R1/645
Inventor 郑裕国王亚军罗希刘小青
Owner ZHEJIANG UNIV OF TECH
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