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Aspergillus terreus-derived activity-enhanced acyltransferase mutant

An acyltransferase and activity-enhancing technology, applied in the field of biocatalysis to generate simvastatin, achieves high specific enzyme activity, mild reaction conditions, and enhanced activity

Inactive Publication Date: 2015-02-11
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the product simvastatin was found to compete with acyltransferases, significantly hindering the overall net rate of acylation
[0009]Acyltransferases are also highly prone to misfolding and aggregation when overexpressed in E. coli, making even whole-cell biocatalytic systems impractical for industrial scale production. too ideal
[0010] Zhu Liping expressed acyltransferase in Escherichia coli BL21(DE3) by optimizing fermentation conditions, fermentation medium, inoculum size, inducer concentration and induction time, etc. , the final expression level of acyltransferase was only 100mg / L, and the output of simvastatin was only 1.2g / L, far from meeting the requirements of industrial scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of wild-type and acyltransferase mutant expression vectors

[0028] After the polynucleotide (SEQ ID NO: 1) encoding the wild-type acyltransferase from Aspergillus terreus was sequence optimized according to the codon preference of strain DH1 (see Puigbò P, Guzmán E, Romeu A, Garcia- Vallvé S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res. 2007), through PCR-based gene synthesis method (PCR-based gene synthesis method) for gene synthesis. The optimized polynucleotide (SEQ ID NO: 1) encoding the acyltransferase was cloned under the control of the promoter of the expression vector (SEQ ID NO. 5) to obtain a plasmid capable of expressing the wild-type acyltransferase. The resulting plasmid was transformed into E. coli DH1 by standard methods. The cloning method used is homologous recombination, and the amplification primers used (which already contain homologous arms for homologous recombination) are:

...

Embodiment 2

[0032] Example 2 Preparation of Acyltransferase Mutants

[0033] Pick a single colony of Escherichia coli DH1 containing the target expression vector and inoculate it in 10ml of high-pressure sterilized medium: tryptone 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 3.55 g / L, diphosphate Potassium hydrogen 3.4 g / L, ammonium chloride 2.68 g / L, sodium sulfate 0.71 g / L, magnesium sulfate heptahydrate 0.493 g / L, ferric chloride hexahydrate 0.027 g / L, glycerin 5g / L, glucose 0.8g / L L, add kanamycin to 50mg / L after sterilization. Cultivate overnight at 30°C, 250rpm. The next day, take a 1L Erlenmeyer flask and inoculate it into 200ml of high-pressure sterilized medium at a ratio of 1:100: tryptone 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 3.55 g / L , potassium dihydrogen phosphate 3.4 g / L, ammonium chloride 2.68 g / L, sodium sulfate 0.71 g / L, magnesium sulfate heptahydrate 0.493 g / L, ferric chloride hexahydrate 0.027 g / L, glycerin 5g / L, glucose 0.3g / L. Add ...

Embodiment 3

[0035] Example 3: Catalytic synthesis of simvastatin sodium salt

[0036] Using the acyltransferase mutant prepared as described in Example 2, the steps to catalyze the formation of simvastatin sodium salt from monacolin J (Monacolin J) sodium salt are as follows. Add 10 g of Monacolin J (Monacolin J) to a 500 mL 3-neck round bottom flask, and then add 50 ml of deionized water. Stir the mixture vigorously until all solids are dissolved. Then 67ml of TE buffer solution (pH 8.5) was added, and the pH was adjusted to 9.0 with concentrated HCl. Add 0.2 g of mutant acyltransferase powder, and immediately stir at 25° C. for 3 minutes to mix the powder of mutant acylase with the liquid evenly. DMB-S-MMP (7.1 mL, 16.27 mmol ) was added, and the enzyme-catalyzed reaction was carried out at 25° C. and 350 rpm. After 5 μL of the reaction mixture was diluted 6 times with methanol, it was centrifuged at 12000g for 15 minutes to remove the precipitate and the supernatant was analyzed by ...

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Abstract

The invention relates to an aspergillus terreus-derived activity-enhanced acyltransferase mutant which has improved activity compared with aspergillus terreus-derived wild type acyltransferase. The invention further provides a polynucleotide coding the acyltransferase mutant, a host cell capable of expressing the mutant, and a production method of the acyltransferase mutant. The aspergillus terreus-derived activity-enhanced acyltransferase mutant can be used for synthesizing important statins compound simvastatin on a large scale.

Description

technical field [0001] The present disclosure relates to methods and materials for the biocatalytic production of simvastatin. More specifically, the present disclosure relates to non-naturally occurring mutant acyltransferases having improved activity compared to wild-type acyltransferases, polynucleotides encoding said mutant acyltransferases, compounds comprising such polynucleotides A host cell, a production method of the acyltransferase mutant, and a method and material for biocatalytically producing simvastatin using the acyltransferase mutant. Background of the invention [0002] Simvastatin is a hypolipidemic drug developed by Merck Company, trade name Zocor, simvastatin (simvastatin) is a semi-synthetic derivative of lovastatin, and lovastatin is a natural product isolated from the fermentation broth of Aspergillus terreus . The pharmacological effect of simvastatin is to inhibit the activity of HMG-CoA reductase (HMG-CoA reductase) in liver cells as a competitiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/06C12R1/66
CPCC12N9/1025C12P17/06
Inventor 丁雪峰
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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