Lipopeptide compounds Totopotensamides and preparation method and application thereof

The technology of a compound, ppww50-totr1, is applied in the field of new antibiotic compounds, which can solve the problems such as the difficulty of making natural products into drugs

Active Publication Date: 2020-07-07
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As we all know, due to the influence of various factors, it is very diff

Method used

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  • Lipopeptide compounds Totopotensamides and preparation method and application thereof
  • Lipopeptide compounds Totopotensamides and preparation method and application thereof
  • Lipopeptide compounds Totopotensamides and preparation method and application thereof

Examples

Experimental program
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Embodiment example 1

[0228] Implementation Case 1: Construction of recombinant strain S.lividans TK64 / pCSG5549

[0229] According to the tot biosynthetic gene cluster ( figure 1 ) of the upstream gene orf (-1) and downstream gene totR5 design primers orf (-1) testF / R and totR5testF / R (primer sequences are shown in Table 1), screened out in the BAC library of bacterial strain S.pactum SCSIO 02999 Clone pCSG5549, through terminal sequencing ( figure 2 ), it was confirmed that pCSG5549 contained all the genes of the totopotensamides biosynthetic gene cluster. The pCSG5549 was introduced into the heterologous host S. lividans TK64 by conjugative transfer of three parents. The process of conjugative transfer is described as follows: S.lividans TK64 was cultured by streaking on the SFM medium plate for 5-7 days, and the grown spores were collected in the TSB medium with a sterile cotton swab, and vortexed to disperse the spores; the hyphae were separated by filtration The spores and spores were susp...

Embodiment example 2

[0230] Example 2: Construction of recombinant strain S.lividans TK64 / pCSG5549 / ΔtotR5i / ΔtotR3i

[0231] By PCR-targeting method, the neo gene in cosmid pCSG4809 (totR5 gene was deleted in the same frame) was replaced with oriT / aadA fragment ( image 3 ). The specific instructions are as follows: (1) The plasmid pCSG4809 (see literature Chen, R.D., Zhang, Q.B., Tan, B., Zheng, L.J., Li, H.X., Zhu, Y.G., and Zhang, C.S. (2017) Genomemining and activation of a silent PKS / NRPS gene cluster direct the production of totopotensamides, Org. Lett.19, 5697-5700.) into Escherichia coli E.coli BW25113 / pIJ790 to obtain E.coli BW25113 / pIJ790 / pCSG4809, with 10mmol L -1 The L-arabinose induces the expression of the λ / red recombinant system, and prepares it as an electroporation-competent cell for use; (2) Digest the plasmid pIJ778 with endonucleases EcoR I and Hind III, and recover about 1.4kb of which contains the transfer origin and the DNA fragment of the spectinomycin resistance gene, a...

Embodiment example 3

[0233] Example 3: Construction of recombinant strain S.lividans TK64 / pCSG5549 / ΔtotR5i / ΔtotR3i / ΔtotP2i

[0234] Using the PCR targeting method, the gene knockout primer totP2-tarF / R (Table 1) was designed for cosmidpCSG4801 (see literature Chen, R.D., Zhang, Q.B., Tan, B., Zheng, L.J., Li, H.X., Zhu, Y.G., and Zhang, C.S. (2017) Genome mining and activation of a silent PKS / NRPS genecluster direct the production of totopotensamides, Org.Lett.19, 5697-5700.) totP2 was knocked out ( Figure 5), its construction method is consistent with the replacement of neo gene in cosmid pCSG4809 by oriT / aadA, and the plasmid pCSG5580 is obtained. The plasmid pCSG5580 is introduced into E.coli DH5α / BT340 and grown on the Kan plate at 28°C. Point-to-point screening on LB solid plates of Kan and Spc, for single clones that grow on LB solid plates containing Kan but not on LB solid plates containing Spc, use the verification primer totP2-TestF / R (Table 1) After verification, a single clone with s...

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Abstract

The invention discloses lipopeptide compounds Totopotensamides and a preparation method and application thereof. The invention discloses antibiotic compounds Totopotensamides (TPMs) R1-1 to R1-6(1-6),R2-1(7), P3-1(8), P3-2(9), P2-1 to P2-7(10-16), U5-1(17), U5-2(18), H1 to H8(19-26) and HM1 to HM3(27-29). The lipopeptide compounds Totopotensamides with antiviral activity are obtained through separation and purification from mutant strains, especially the lipopeptide compounds Totopotensamides have inhibitory activity on 3CL hydrolase of a novel coronavirus and are expected to be used for preparing anti-novel-coronavirus drugs.

Description

Technical field: [0001] The invention belongs to the field of industrial microorganisms, in particular to new antibiotic compounds Totopotensamides (TPMs) R1-1 to R1-6 (1-6), R2-1 (7), P3-1 (8), P3-2 (9) , P2-1 to P2-7(10-16), U5-1(17), U5-2(18), H1 to H8(19-26) and HM1 to HM3(27-29) and their preparation methods and application. Background technique: [0002] TPMs are a class of lipopeptide compounds. The compounds in this family have diverse structures and have various biological activities such as antibacterial, antitumor, anti-inflammatory and antiviral. As we all know, due to the influence of various factors, it is very difficult to make natural products without structural modification into drugs. With the advancement of molecular biology and gene editing technology, it has become possible to optimize the structure of natural products from the perspective of natural product biosynthesis genetic modification and in vitro enzymatic catalysis, and has the advantages of s...

Claims

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Application Information

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IPC IPC(8): C07K5/12C12N1/21A61K38/07A61P31/12C12R1/465
CPCC07K5/126C07K14/36A61P31/12A61K38/00
Inventor 张长生张庆波谭彬朱义广金红波张丽萍陈思强
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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