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Recombined aldehyde ketoreductase mutant, gene, carrier, engineering bacteria and application of recombined aldehyde ketoreductase mutant

A technology of genetically engineered bacteria and mutants, applied in genetic engineering, oxidoreductase, applications, etc., can solve the problems of borane compounds such as flammability, low optical purity of products, high energy consumption, etc., and achieve stereoselectivity and activity improvement Effect

Active Publication Date: 2017-08-18
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many defects in this technical method. First, the optical purity of the product is low, which can only reach 98%. Second, the borane compound is flammable. Third, the reaction requires cryogenic conditions and high energy consumption.

Method used

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  • Recombined aldehyde ketoreductase mutant, gene, carrier, engineering bacteria and application of recombined aldehyde ketoreductase mutant
  • Recombined aldehyde ketoreductase mutant, gene, carrier, engineering bacteria and application of recombined aldehyde ketoreductase mutant
  • Recombined aldehyde ketoreductase mutant, gene, carrier, engineering bacteria and application of recombined aldehyde ketoreductase mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the construction of wild-type aldehyde and ketone reductase KmAKR gene clone and its recombinant expression vector

[0033] (1) Cloning of the target gene

[0034] Using Kluyveromyces marxianus (CICC32920) genomic DNA as a template, through the upstream primer: 5'- CCATGG CCATGACAAAACCAAAAGTTCTTTACTT-3', downstream primer: 5'- GCGGCCGC TCACTTCTGGGATTCAGAAT-3' was amplified by PCR to obtain the amplified product. Take 10 μL of the PCR amplification product and detect it by 0.9% agarose gel electrophoresis. figure 1 As shown, a band appeared around 1000bp;

[0035] Composition of PCR reaction system (total volume 100 μL): 10 times Pfu DNA polymerase buffer (containing Mg 2+ ) 10 μL, 0.5 μL of 10 mM dNTP mixture (dATP, dCTP, dGTP, dTTP each 2.5 mM), 0.5 μL each of the clone upstream primer and downstream primer at a concentration of 50 μM, 1 μL of genomic DNA, 1 μL of Pfu DNA polymerase, and 86.5 μL of nucleic acid-free water. Using a Biorad PCR instru...

Embodiment 2

[0038] Example 2 Construction of aldehyde and ketone reductase mutant KmAKR-W297H recombinant expression vector

[0039] Based on the pET28a(+)-kmakr constructed in Example 1, in order to improve the stereoselectivity of the original type aldehyde and ketone reductase and the substrate 6-cyano-(5R)-hydroxyl-3-oxoylhexanoic acid tert-butyl Two rounds of point-saturation mutagenesis were performed on the ester activity. In the first round of mutation, the gene sequence of pET28a(+)-kmakr was used as a template, and W297-F and W297-R were used as upstream and downstream primers (Table 1) for PCR amplification (PCR reaction parameters: 95°C for 4min; 95°C for 15s , 55°C for 15s, 72°C for 7min, repeat 30 cycles; 72°C for 10min). The PCR product recovered after amplification was digested with DpnI for 3 h, purified by using a purification kit, and then transformed into E.coliJM109 recipient bacteria, spread on LB solid plates with a final concentration of 50 μg / mLKan, 37 After cul...

Embodiment 3

[0043] Example 3 Purification of wild type aldehyde and ketone reductase KmAKR and recombinant aldehyde and ketone reductase mutants KmAKR-W297H, KmAKR-Y296W / W297H

[0044] According to the method in embodiment 1 and 2, obtain E.coli BL21 (DE3) / pET28a (+ )-kmakr-W297H, and the E.coli BL21(DE3) / pET28a(+)-kmakr-Y296W / W297H of the double-site mutation gene expression vector were cultured.

[0045] (1) Induction culture: E.coli BL21(DE3) / pET28a(+)-kmakr, E.coli BL21(DE3) / pET28a(+)-kmakr-W297H and E.coli BL21(DE3) / pET28a(+) -kmakr-Y296W / W297H, respectively inoculated into Lysogeny-Broth (LB) liquid medium containing Kan at a final concentration of 50 μg / mL, cultured at 180 rpm for 10-12 hours at 37°C, and then inoculated with an inoculum volume concentration of 2% to contain The LB liquid medium with a final concentration of 50 μg / mL Kan was used for expansion culture, and cultured at 37°C and 180 rpm to the OD of the culture medium 600 Between 0.6 and 0.8, add a final concentrat...

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Abstract

The invention discloses a recombined aldehyde ketoreductase mutant, a gene, a carrier, an engineering bacteria and application of the recombined aldehyde ketoreductase mutant in preparing 6-cyan-(3R, 5R)-tert-butyl dihydroxycaproate; the mutant is obtained by mutating the 297th bit tryptophan of amino acid sequence shown in SEQ ID No.1 to histidine, mutating the 296th bit tyrosine to tryptophan and the 297th bit tryptophan to histidine. The aldehyde ketoreductase mutants W297H and Y296W / W297H have significantly improved three-dimensional selectivity and activity to 6-cyan-(3R, 5R)-tert-butyl dihydroxycaproate; the conversion rate is up to 99%; the product reaches the optical purity with the d.e. value greater than 99.5%.

Description

[0001] (1) Technical field [0002] The invention relates to a recombinant aldehyde and ketone reductase mutant, a carrier, an engineering strain and an application thereof, and also includes the synthesis of atorvastatin chiral side chain by recombinant Escherichia coli chiral biocatalysis highly expressing the aldehyde and ketone reductase mutant Method for tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate. [0003] (2) Background technology [0004] Cardiovascular and cerebrovascular diseases seriously endanger human health, and its fatality rate ranks first among all kinds of diseases. Atherosclerosis and its complications are the main causes of death from cardiovascular and cerebrovascular diseases, and the culprit behind it is abnormal blood lipids, especially abnormally elevated serum total cholesterol. Atorvastatin can competitively inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key rate-limiting enzyme in the synthesis of cholesterol in the liv...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/0008C12P13/002
Inventor 王亚军应彬彬郑裕国沈炜喻寒程英男
Owner ZHEJIANG UNIV OF TECH
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