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Aldo-keto reductase gene recombinant coexpression vector, engineering bacteria and application thereof

A technology of co-expression vector and genetically engineered bacteria, applied in the fields of genetic engineering, oxidoreductase, application, etc., can solve the problems of complex chiral synthesis control, many cultivation batches, low chiral purity of products, etc. The effect of cell immobilization

Active Publication Date: 2017-07-07
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 of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate has many defects: poor safety; unfriendly environment; complex control of bichiral synthesis, low chiral purity of products; low yield of products , high production cost
The common aldehyde and ketone reductase and glucose dehydrogenase are expressed separately and then synergistically catalyzed to synthesize 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester. There are many batches of cell culture, and the coenzyme is carried out in different cells. Oxidation and reduction require multiple transmembrane transports, which increases mass transfer resistance

Method used

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  • Aldo-keto reductase gene recombinant coexpression vector, engineering bacteria and application thereof
  • Aldo-keto reductase gene recombinant coexpression vector, engineering bacteria and application thereof
  • Aldo-keto reductase gene recombinant coexpression vector, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning of aldehyde and ketone reductase gene (klakrm) and glucose dehydrogenase gene (esgdh) and co-expression recombinant system construction

[0031] Kluyveromyces lactis XP1461 (gene sequence number: XM_455342.1). The strain has been preserved in the China Typical Culture Collection Management Center, the preservation date is August 14, 2014, the preservation number is CCTCC NO: M 2014380, and the preservation address is Wuhan, China, Wuhan University, postcode 430072, and has been applied for a patent ( 201410641987.5). The aldehyde and ketone reductase mutant (nucleotide sequence is Shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2), the mutant enzyme activity is improved 14.2 times compared with the original, to tert-butyl 6-cyano-(5R)-hydroxyl-3-carbonyl hexanoate Exhibits strict poor selectivity (d.e p >99%), has been disclosed in the patent application (201610124451.5), and the mutant enzyme gene has been connected to the ex...

Embodiment 2

[0035] Example 2: Expression of aldehyde ketone reductase and glucose dehydrogenase in different co-expression systems

[0036] The different co-expression strains obtained in the above-mentioned Example 1 were inoculated in LB medium containing 50 μg / mL, cultured at 37° C. for 9 hours, and the obtained seed liquid was transferred to fresh Incubate in 50 μg / mL LB medium at 37°C for 2-2.5 hours. When OD 600 When it reaches 0.6-0.8, use the final concentration of 9g / L lactose as the inducer, induce the temperature at 28°C, and induce for 10 hours. The fermented liquid obtained from the induction is centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant is discarded to obtain wet bacteria. The cells were washed twice with physiological saline, centrifuged at 8000 rpm for 10 minutes at 4°C, and the cells were collected. Add pH 7.0, 200mM phosphate buffer to resuspend, and crush on ice (ultrasonic crushing conditions: power 400W, crush for 1s, pause for 1s) for 8 min...

Embodiment 3

[0037]Example 3: Comparison of asymmetric reduction efficiency and specific enzyme activity of different co-expression strains

[0038] Definition of enzyme activity: under standard conditions, the amount of enzyme required to generate 1 μmol of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester per minute is 1 unit of enzyme activity, and the apparent specific enzyme activity is The number of enzyme activity units per gram of dry bacteria. Sample detection method: tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate, tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, 6-cyano-(3S ,5R)-dihydroxyhexanoic acid tert-butyl ester is detected under high performance liquid chromatography, and wherein chromatographic column is Hypersil ODS C18 post (250 * 4.6mm, 5 μ m; Thermo, the U.S.), the ratio of mobile phase acetonitrile to water volume= 1:3(v / v), flow rate 1.0mL / min, injection volume 20μL, detection wavelength 210nm, column temperature 40℃. Under the above conditions, tert-buty...

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Abstract

The invention discloses an aldo-keto reductase gene recombinant coexpression vector, engineering bacteria and application thereof. The aldo-keto reductase gene recombinant coexpression vector is based on plasmid pET-28b, plasmid pETDuet-1 or plasmid pCDFDuet-1 and comprises an aldo-keto reductase mutant gene of which the nucleotide sequence is shown in SEQ ID NO:1, and a glucose dehydrogenase gene of which the nucleotide sequence is shown in SEQ ID NO:3. On the basis that the maximum substrate feeding amount of a method that two enzymes are combined and catalyzed after being separated and independently expressed is 50g / L (which is disclosed in the patent application (201610124451.5)), the maximum substrate feeding amount is further increased to 60g / L or greater, meanwhile a two-enzyme coexpression system is beneficial to whole-cell immobilization, and relatively good industrial application prospects can be achieved.

Description

[0001] (1) Technical field [0002] The invention relates to a recombinant bacterium co-expressing aldehyde and ketone reductase-glucose dehydrogenase, and using the aldehyde ketone reductase-glucose dehydrogenase to co-express the recombinant bacterium to asymmetrically reduce 6-cyano-(5R)-hydroxyl-3- Synthesis of atorvastatin calcium bichiral side chain 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester catalyzed by tert-butyl carbonylhexanoate. [0003] (2) Background technology [0004] Cardiovascular disease is one of the most threatening diseases to humans today, and its morbidity and mortality have surpassed tumor diseases and ranked first. The rise of blood low-density lipoprotein cholesterol (LDL-C) level is an important factor in inducing cardiovascular disease, and 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase is the key rate-limiting enzyme of cholesterol synthesis. Atorvastatin calcium can competitively inhibit the key rate-limiting enzyme of cholesterol ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/0006C12N9/0008C12P13/002C12Y101/9901
Inventor 郑裕国王亚军沈炜喻寒
Owner ZHEJIANG UNIV OF TECH
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