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Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof

A technology of yeast aldehydes, ketones, and reductases, which is applied in the direction of oxidoreductases, fermentation, etc., can solve the problems of low asymmetric reduction activity and low substrate feeding amount

Active Publication Date: 2019-12-17
ZHEJIANG UNIV OF TECH
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  • Claims
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Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that the existing aldehyde and ketone reductase has low asymmetric reduction activity to tert-butyl 6-substituted-(5R / S)-hydroxy-3-carbonylhexanoate and low substrate dosage, and provides a Stereoselective aldehyde and ketone reductase series mutants and recombinant bacteria using the aldehyde and ketone reductase mutants or their crude enzyme solution as catalysts for the asymmetric reduction synthesis of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tertiary For chiral alcohol compounds such as butyl ester and tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate, the catalyst activity is 4.1 times higher than that of KmAKR-W297H / Y296W / K29H / Y28A, and the substrate dosage is increased to 450g / L, which is the highest level in all literature reports

Method used

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  • Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof
  • Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof
  • Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction and screening of aldehyde and ketone reductase mutant library

[0036] The Kluyveromyces marx aldoketone reductase mutant library was prepared by one round of site-directed saturation mutagenesis. The primers were designed as shown in Table 1. E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A (see patent application CN201910072740.9 for construction) in the vector pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A as a template, using Thr63-F and Thr63-R in Table 1 as primers, through saturation mutation PCR, the The 63rd threonine of the aldehyde and ketone reductase KmAKR-W297H / Y296W / K29H / Y28A amino acid sequence shown in SEQ ID NO.2 was mutated into the remaining 19 kinds of amino acids, and transformed, plated, and screened by dominant strains (Table 2) , to obtain the aldehyde and ketone reductase mutant KmAKR-W297H / Y296W / K29H / Y28A / T63A (denoted as M5-A, that is, the 63rd threonine of the amino acid shown in SEQ ID NO.2 is mutated to alanine), KmAK...

Embodiment 2

[0040] Embodiment 2: Induced expression of control group aldehyde and ketone reductase, mutant and glucose dehydrogenase

[0041] Glucose dehydrogenase genetically engineered bacteria: insert the glucose dehydrogenase gene (GenBank NO.KM817194.1) from E.sibirium DSM 17290 into pET28b(+) to construct a recombinant expression vector, and transfer the expression vector into E.coli BL21 (DE3) E. coli BL21(DE3) / pET28b(+)-esgdh was produced.

[0042] The starting strain E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A from Example 1 and the aldehyde and ketone reductase mutant strain screened in Example 1 and E.coli BL21(DE3) / pET28b (+)-esgdh were respectively inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, and inoculated into fresh LB medium containing a final concentration of 1.5% (v / v) 50μg / mL kanamycin LB liquid medium, cultured at 37°C, 180rpm for 2h, then added 0.15mM IPTG to the culture solution, cultu...

Embodiment 3

[0043] Example 3: Mutant library screening

[0044]Mix the wet cells of the mutant strain induced and expressed in Example 2 and the wet cells of glucose dehydrogenase at a dry weight ratio of 3.5:1 (w / w) to form a mixed cell, add pH 7.0, 100mM PBS buffer to resuspend, Obtain the mixed bacterial liquid of mutant strains. Under the same conditions, the control strain E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A was used to replace the wet cells of the mutant strain to prepare a mixed bacterial solution of the control strain.

[0045] The mixed bacterial solution of the mutant strain and the mixed bacterial solution of the control strain were used as catalysts, tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate was used as the substrate, glucose was used as the auxiliary substrate, and no external derived NADPH or NADP + , using the endogenous NADPH of bacterial cells to establish a coenzyme circulation system. The reaction system is selected as 10mL, the amount of...

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Abstract

The invention discloses a kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and an application thereof. The aldehyde ketone reductase mutant is obtained through performing fixed-point saturation mutation on a 63rd site of an amino acid sequence as shown in SEQID NO.2. The specific enzyme activity of the constructed aldehyde ketone reductase mutant M5-A is increased by 1.1 times than that of aldehyde ketone reductase in a control group, the specific enzyme activity of the constructed aldehyde ketone reductase mutant M5-L is increased by 3.2 times than that of the aldehyde ketone reductase in the control group, the specific enzyme activity of the constructed aldehyde ketone reductase mutant M5-M is increased by 4.1 times than that of the aldehyde ketone reductase in the controlgroup, wherein the mutant KmAKR-Y296W / W297H / K29H / Y28A / T63M has remarkably-improved catalytic activity on 6-cyano-(5R)-hydroxy-3-carbonyl hexanoate tert butyl, 6-chloro-(5S)-hydroxy-3-carbonyl hexanoate tert butyl and the like. The feeding quantity of the largest substrate namely the 6-cyano-(5R)-hydroxy-3-carbonyl hexanoate tert butyl can reach 450g / L, the substrate conversion rate is higher than99%, the de value of products is always maintained to be 99.5% or above, and the time and space yield in the biocatalysis process is as high as 1224.3g / L d.

Description

[0001] (1) Technical field [0002] The present invention relates to the construction of a mutant of aldehyde and ketone reductase KmAKR derived from Kluyveromyces marx, and the development of aldehyde and ketone reductase recombinant bacteria and enzymes in atorvastatin, rosuvastatin, pitavastatin and other "super statins" Application of chiral biocatalytic synthesis of side-chain bichiral diol 6-substituted-(3R,5R / S)-tert-butyl dihydroxyhexanoate. [0003] (2) Background technology [0004] "Super statins" such as atorvastatin, rosuvastatin, and pitavastatin are important lipid-lowering drugs for the treatment of cardiovascular and cerebrovascular diseases. Disease morbidity and mortality. So far, the cumulative sales of atorvastatin calcium have exceeded 100 billion US dollars, and it is the most successful single drug variety in the history of the human pharmaceutical industry. [0005] Most statin drugs contain 6-substituted-(3R,5R / S)-tert-butyl dihydroxyhexanoate struct...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P7/02
CPCC12N9/0006C12P7/02
Inventor 王亚军邱帅李树芳程峰翁春跃郑裕国
Owner ZHEJIANG UNIV OF TECH
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