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Mutants of fatty acid adenosine monophosphate (AMP) ligase with improved substrate affinities and application thereof

A mutant, ligase technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of long cycle and high randomness

Inactive Publication Date: 2016-07-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the relatively complete research on fermentation technology, domestic breeding of high-daptomycin-producing strains is mainly to find high-daptomycin-producing strains through mutagenesis and resistance screening or to improve the ability of Streptomyces roseospora to decanoic acid. Tolerance, increase the production of daptomycin by increasing the amount of capric acid. Due to the high randomness and long cycle of mutagenesis and resistance screening, it is necessary to carry out directional modification at the gene level to improve the production of daptomycin. Nutrient production has become a research hotspot in recent years

Method used

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  • Mutants of fatty acid adenosine monophosphate (AMP) ligase with improved substrate affinities and application thereof
  • Mutants of fatty acid adenosine monophosphate (AMP) ligase with improved substrate affinities and application thereof
  • Mutants of fatty acid adenosine monophosphate (AMP) ligase with improved substrate affinities and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Design of mutant genes

[0024] Using computer simulation method, using MOE software for homology modeling, molecular docking of fatty acid AMP ligase and decanoic acid, simulated determination of its affinity, and replacement of some active centers or nearby amino acids that may affect the affinity. For the size of affinity, select sites 296 and 302 with improved affinity.

Embodiment 2

[0025] Embodiment 2: the acquisition of mutant strain

[0026] Adopt the method of PCR amplification and OVERLAPPCR, obtain the amino acid sequence such as the fatty acid AMP ligase gene shown in SEQNO.4 and SEQNO.5, then the gene is connected to pET-22b (+) and then transformed into Escherichia coli E.coliDH5α and carried out Amplify, screen and transform into Escherichia coli E.coliBL21 with correct sequencing, and name the transformants with correct screening as EC296 and EC302.

Embodiment 3

[0027] Embodiment 3: the verification of mutant strain

[0028] Seed culture: Inoculate an appropriate amount of recombinant bacteria EC296 and EC302 from a glycerol tube into LB medium (100ug / ml ampicillin) with a liquid volume of 10ml / 50ml, culture overnight at 37°C on a shaker at 220rpm.

[0029] Shake flask fermentation: Inoculate the overnight cultured seed solution with 2% inoculum into LB medium, and when the OD value is 0.6-0.8, add IPTG with a final concentration of 0.2mM for induction, and induce at 18°C ​​for 18-24h.

[0030] Purification of fatty acid AMP ligase: centrifuge the recombinant bacterial fermentation broth at 8000r / min for 20min, take the precipitate, stir with hypertonic sucrose solution for 20min at low temperature, centrifuge at 12000r / min for 30min, collect the supernatant, and stir the precipitate with hypotonic solution for 20min at 12000rr / min centrifuged for 30min, and the supernatant was collected. The supernatants collected twice were combin...

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Abstract

The invention relates to mutants of a fatty acid adenosine monophosphate (AMP) ligase and application of the mutants, and belongs to the field of bioengineering. The fatty acid AMP ligase comes from streptomyces roseosporus. The 296th glycine of the fatty acid AMP ligase is changed into alanine, and the 302th isoleucine of the fatty acid AMP ligase is changed into valine to respectively obtain the mutants pAL296 and pAL302. The mutants of the fatty acid AMP ligase can improve the binding level with exogenous precursor decanoic acid to enhance the yield of daptomycin. According to the invention, an encoding gene dptE of the fatty acid AMP ligase is subjected to site-directed mutagenesis in vitro by using computer simulation, overlap extension PCR and site-directed mutagenesis technologies to screen out the mutants pAL296 and pAL302 of which the affinities are obviously improved, recombinant shuttle plasmids pAK296 and pAK302 are constructed and sequentially led to the streptomyces roseosporus, and zygotes are screened to obtain streptomyces roseosporus DCE296 and DCE302 of the fatty acid AMP ligase with improved substrate affinities to lay a foundation for improving the production level of daptomycin.

Description

technical field [0001] The invention relates to two fatty acid AMP ligase mutants with improved affinity, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Daptomycin is a cyclic lipopeptide compound produced by Streptomyces roseosporus. The biosynthesis of this class of antibiotics is accomplished through the ribosome-independent non-ribosomal peptide synthetase (Non-ribosomal peptide synthetase, NRPS) pathway. Yes, it has potent antibacterial activity against Gram-positive microorganisms in vitro. [0003] Daptomycin is isolated from the fermentation broth of Streptomyces roseospora and consists of 13 amino acid residues. Under normal circumstances, Streptomyces roseospora fermentation produces a group of cyclolipopeptide antibiotics: A21978C1-3, Daptomyces Daptomycin is a derivative of A21978C1-3. During the fermentation process of Streptomyces roseospora, adding exogenous precursor capric acid can increase the proportio...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/76C12P21/02C12R1/465
CPCC12N9/93C07K7/08
Inventor 王旻吴旻王露璇王世静
Owner CHINA PHARM UNIV
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