Cypemycin precursor peptide mutant and application thereof and prepared cypemycin analogues

A technology of sepramycin and precursor peptide, applied in the field of biotechnology engineering

Pending Publication Date: 2020-07-28
南京凯沫比尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, some important amino acids play a key role in its biosynthesis, so mutating the ...

Method used

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  • Cypemycin precursor peptide mutant and application thereof and prepared cypemycin analogues
  • Cypemycin precursor peptide mutant and application thereof and prepared cypemycin analogues
  • Cypemycin precursor peptide mutant and application thereof and prepared cypemycin analogues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction of recombinant plasmids containing mutant precursor peptides

[0063] Firstly, use the PCR method, using forward CypA-F and reverse CypA-R as primers, Streptomyces sp.OH4156 (references: Claesen J, Bibb M. Genome mining and genetic analysis of cypemycin biosynthesis reveal an unusual class of posttranslationally modified peptides. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(37): 16297-16302.) total DNA is template amplification target fragment cypA (nucleic acid sequence such as SEQ ID NO.27 in the sequence listing shown); the PCR reaction conditions were: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s; annealing at 58°C for 15 s; extension at 72°C for 20 s; extension at 72°C for 7 min; cycle 30 times.

[0064]Two strategies are mainly used to design point mutation primers: if the target amino acid to be mutated is located at an important amino acid at the C-terminal of the cor...

Embodiment 2

[0069] Example 2 Conjugative Transfer Obtaining a Bacterial Strain Containing a Mutant Propeptide

[0070] The vector pIJ10257 was digested simultaneously with restriction endonucleases HindIII and NdeI (see: Claesen J, Bibb M. Genome mining and genetic analysis of cypemycin biosynthesis reveal anunusual class of posttranslationally modified peptides. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010,107(37):16297-16302.) and DNA amplified fragment cypA, construct plasmid pHB-CypA-wt, clone plasmid into competent cell E.coli ET12567 (commercially available obtained) and combined with the recipient bacterial cell S.coelicolor M1414(ΔcypA) (construction method reference: Claesen J, Bibb M. Genome mining and genetic analysis of cypemycin biosynthesis reveal an unusual class of posttranslationally modified peptides.[J].Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(37): 16297-16302.) The wild-type...

Embodiment 3

[0072] Example 3 Obtaining Sepromycin and Sepromycin Structural Analogues by Fermentation

[0073] (1) Optimization of wild-type and mutant fermentation conditions: analysis of liquid phase mass spectrometry (LC-MS) experiment results found that the yield of septomycin was very low, which was not conducive to the analysis of septomycin and its structural analogues. To improve the yield of septomycin, it is urgent to further optimize the fermentation conditions of the replenished wild-type strain S.coelicolor M1422, set the volume of fermentation broth (100mL), the volume of extraction solvent (100mL), and the volume of solvent for extracting mycelium (100mL) must, on the basis of the septomycin fermentation medium MarM (for details, see: Komiyama, K., Otoguro, K., Segawa, T., Shiomi, K., Yang, H., Takahashi, Y., Hayashi, M., Oxani, T., and Omura, S. (1993) A new antibiotic, cypemycin. Taxonomy, fermentation, isolation and biological characteristics. J. Antibiot. 46, 1666-1671)...

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Abstract

The present invention discloses a cypemycin precursor peptide mutant and an application thereof and prepared cypemycin analogues. The mutant is prepared by mutating one or more threonines in a core peptide to serine, lysine, alanine or leucine; and/or, mutating one or two glutamines in the core peptide to glutamic acid; and/or, mutating serine in the core peptide to alanine or tyrosine; and/or, mutating valine at a 12nd and/or 21st position of a sequence shown in SEQ ID NO.1 to lysine, alanine, isoleucine, or cysteine; and/or, mutating cysteine at a 19th position of the sequence shown in the SEQ ID NO.1 to serine or tyrosine; and/or, mutating cysteine at a 33rd position and/or proline at a 36th position of a sequence shown in SEQ ID NO.2 to alanine. A series of cypemycin analogues can be prepared by using the mutant and provide a basis for screening compounds with higher activity.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and specifically relates to a mutant of a septomycin precursor peptide, an application thereof and a prepared septomycin analogue. Background technique [0002] Natural products are the material basis for drug discovery and development, and have received great attention from microbiologists and pharmacologists in the past few decades, among which penicillin, streptomycin, and abamectin have been favored by Nobel Prizes one after another. At present, 60% of clinical anticancer drugs and 70% of antibiotics are derived from them. In recent years, with the emergence of various drug-resistant bacteria and super-resistant bacteria, many natural products have good development prospects in the field of new drug research and development in the future because of their good biological activity. [0003] Cypemycin (Septomycin, also known as Cypemycin) is a "star" compound of the linaridin (linear de...

Claims

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Application Information

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IPC IPC(8): C07K14/36C12N15/31C12N15/76C12N1/21A61K38/16A61P35/02A61P31/04C12R1/465
CPCC07K14/36C12N15/76C12N1/20A61P35/02A61P31/04A61K38/00
Inventor 张琪莫天录丁伟
Owner 南京凯沫比尔生物科技有限公司
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