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Carbonyl reductase expressed recombination engineering bacterium and application thereof

A technology of recombinant engineering bacteria and reductase, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of carbonyl reductase expression level and insufficient enzyme activity, and achieve the effect of optical purity and high conversion rate

Active Publication Date: 2013-04-03
NINGBO MENOVO PHARMA +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] The invention provides a recombinant engineering bacterium expressing carbonyl reductase to solve the problem of insufficient expression level and enzyme activity of wild strain carbonyl reductase

Method used

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  • Carbonyl reductase expressed recombination engineering bacterium and application thereof
  • Carbonyl reductase expressed recombination engineering bacterium and application thereof
  • Carbonyl reductase expressed recombination engineering bacterium and application thereof

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Embodiment Construction

[0033] (1) strain

[0034] Kluyveromyce marxianus CGMCC2.1977 can be purchased from China General Microorganism Culture Collection Center (CGMCC).

[0035] Escherichia coli DH5α is a commercial product, and Escherichia coli (E.coli) Rosetta (DE3) competent cells were purchased from Biotech Biotech.

[0036] (2) culture medium

[0037] PYD solid medium: glucose 10g, yeast extract 10g, peptone 20g, agar 15g, add water to 1000mL, pH natural.

[0038] PYD liquid medium: glucose 10g, yeast extract 10g, peptone 20g, add water to 1000mL, pH natural.

[0039] LB solid medium: tryptone 10g, yeast extract 5g, NaCl 10g, agar 15g, add water to 1000mL, pH7.0.

[0040] LB liquid medium: tryptone 10g, yeast extract 5g, NaCl 10g, add water to 1000mL, pH7.0.

[0041] 2. Cloning of the target gene

[0042]Pick K. marxianus (K.marxianus) CGMCC2.1977 preserved strain and activate it on agar slant medium at 28°C for 48h, then inoculate it in 30mL PYD liquid medium, put it in a 250mL conical f...

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Abstract

The invention discloses a carbonyl reductase expressed recombination engineering bacterium and application thereof. The engineering bacterium comprises a host cell and a recombinant vector transferred into the host cell, wherein the recombinant vector consists of an original vector and a target gene connected into the original vector, the host cell is Escherichiacoli Rosetta (DE3), the target gene is a carbonyl reductase gene, and the base sequence is shown in SEQ ID NO.1. According to the recombination engineering bacterium, the expression level of a carbonyl reductase is high, bacterial cells which are prepared through induced culture are used for converting N,N-dimethyl-3-oxy-3-(2-thienyl)-l-propylamine hydrochloride into (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-l-propylamine or converting 4-chloroethyl acetoacetate into (S)-4-chloro-3-hydroxyethyl butyrate, and the two products are higher in both optical purity and conversion rate.

Description

technical field [0001] The invention relates to a recombinant engineering bacterial strain, in particular to a recombinant engineering bacterial expressing carbonyl reductase and application thereof. Background technique [0002] Duloxetine (duloxetine), the chemical name is (+)-(S)-N-methyl-γ-yl-(1-naphthyloxy)-2-thienyl hydrochloride, which is the effective drug of Cymbalta Element. It is a dual serotonin (5-HT) and norepinephrine (NE) reuptake inhibitor that treats depression by increasing serotonin and norepinephrine levels in the synaptic cleft, and by blocking Disrupting pain signals to the brain to treat neuropathic pain. On August 3, 2004, the drug was approved by the U.S. Food and Drug Administration for marketing in the United States for the treatment of major depression, and on September 3 of the same year, it was approved for the treatment of diabetic peripheral neuropathic pain (DPN). At present, it has become the most outstanding representative of 3-aryloxy-...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/00C12P7/62C12R1/19
Inventor 金志华胡升杨岳微姚成志王爱珂金庆超杨郁梅乐和高然
Owner NINGBO MENOVO PHARMA
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