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Construction of a dptc1 mutant for daptomycin biosynthesis

A technique for dptc1 and mutant proteins, applied in the field of construction of dptC1 mutants

Active Publication Date: 2022-03-11
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on the analysis of the relationship between structure and function at the molecular level, and the study of molecular design and transformation.

Method used

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  • Construction of a dptc1 mutant for daptomycin biosynthesis
  • Construction of a dptc1 mutant for daptomycin biosynthesis
  • Construction of a dptc1 mutant for daptomycin biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Establishment of dptC1 in vitro enzyme activity identification and screening method

[0136] In order to construct a screening method for in vitro enzyme activity identification of dptC1, we carried out a series of work such as strain construction, multi-enzyme expression and purification, and in vitro multi-enzyme reaction system construction. The specific operation is as follows:

[0137] 1.1 Construction of Escherichia coli strains for enzyme expression and in vitro purification of enzymes

[0138] The dptC1, dptPCP1, dptE, and dptF genes of Streptomyces roseospora daptomycin fatty acid-loaded related NRPS were constructed into the pET-28a(+) vector by molecular cloning technology. The N-terminus of the protein has a his tag and is purified by Ni-NTA gravity column. The purified buffer is 50mM Tris-HCl (pH 7.6), 0.5M NaCl, 10mM MgCl 2 . The nickel column was decontaminated with washing buffer containing 20mM and 50mM imidazole successively, and final...

Embodiment 2

[0149] Example 2: Design of dptC1 small intelligent mutation library

[0150] 2.1 Homology modeling and molecular docking

[0151] Because the crystal structure of dptC1 has not yet been resolved, the crystal structure of the homologous protein CDA-C1 (41.05% sequence homology) (PDB: 4JN3) was used as a template, and the SWISSMODEL online software was used for homology modeling to obtain dptC1 simulation structure. Centering on His139, the second His catalytic site in the conserved motif "HHxxxDG" in the C domain, the model substrate channel was positioned using Schrodinger molecular simulation software. Analysis localizes residues and regions important for catalysis and substrate binding.

[0152] 2.2 Construction of dptC1 substrate pocket region mutants

[0153] According to the results of protein-substrate molecular docking, select the vicinity of the fatty acyl chain that docks into the substrate pocket The residues within are used as key residues, and mutations a...

Embodiment 3

[0157] Example 3: Cloning, expression and purification of dptC1 mutants

[0158] The dptC1 mutant was point-mutated using the wild-type pET-28a(+) vector as a template, expressed in Escherichia coli BL21(DE3), and purified in vitro using a Ni-NTA gravity column.

[0159] The whole plasmid amplification system constructed by the mutants is shown in Table 2.

[0160] Table 2 Total plasmid amplification reaction system (50 μL)

[0161]

[0162]

[0163] The PCR reaction program was: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 95 s, a total of 30 cycles; finally, extension at 72°C for 5 min. After amplification, the whole plasmid PCR product was detected by 1% agarose gel electrophoresis. After the PCR product was purified, the template was digested with DpnI enzyme, and then the plasmid was transformed into Escherichia coli BL21(DE3), and a single clone was picked for sequencing to verify the suc...

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Abstract

The invention provides the construction of a dptC1 mutant used for the biosynthesis of daptomycin. Specifically, the present invention provides a dptC1 mutein, which is based on the first C domain of the wild-type non-ribosomal peptide synthetase NRPS (dptC1 protein) shown in SEQ ID NO: 2, selected One or more amino acid positions are mutated from the following group: glutamine at position 15 (Q), tyrosine at position 145 (Y), arginine at position 247 (R), threonine at position 314 (T) and the 355th valine (V); and, the dptC1 mutant protein has the activity of catalyzing the reaction of the decanoyl substrate into N-decanoyl compounds. The inventive dptC1 mutant protein of the present invention can improve the catalytic efficiency of decanoyl substrate, and realize high and high yield of daptomycin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a dptC1 mutant used for the biosynthesis of daptomycin. Background technique [0002] Daptomycin is an N-decanoylcyclic peptide secondary metabolite produced by Streptomyces roseospora, which has inhibitory effects on most Gram-positive bacteria in vitro, and is effective against many drug-resistant bacteria, such as vancomycin-resistant Enterococci with antibiotics, methicillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci still have strong activity and are clinically important cyclolipopeptide antibacterial drugs, which are considered to be "after vancomycin" Antibacterial last line of defense", has good application value and market prospect. [0003] Daptomycin is an antibiotic with important clinical significance and market value, but it is a trace component in the multi-component N-acyl product of its natural bacteria Stre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12P21/02C12Q1/25
CPCC12N9/93C12P21/02C07K7/08C12Q1/25C12Y603/02G01N2333/9015
Inventor 刘倩刘盼盼冯雁樊文杰
Owner SHANGHAI JIAOTONG UNIV
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