Biosynthetic gene cluster of cyclic lipopeptide compounds, method for activating biosynthetic gene cluster and application thereof

A biosynthesis and compound technology, applied in the field of microbial genetic engineering, can solve problems such as products that have not been discovered

Active Publication Date: 2018-03-13
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

[0002] In recent years, with the development and wide application of genome sequencing technology, it has been found that almost every strain of actinomycetes h

Method used

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  • Biosynthetic gene cluster of cyclic lipopeptide compounds, method for activating biosynthetic gene cluster and application thereof
  • Biosynthetic gene cluster of cyclic lipopeptide compounds, method for activating biosynthetic gene cluster and application thereof
  • Biosynthetic gene cluster of cyclic lipopeptide compounds, method for activating biosynthetic gene cluster and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Including in situ knockout of the negative regulatory gene totR5 in the Totopotensamides biosynthesis gene cluster to construct a mutant ΔtotR5i; in situ knockout of the strain (Streptomyces sp.SCSIO 02999, deposit number: CCTCC NO: M 2012145), the key gene xiaP in the biosynthesis of the main product xiamycins, was used to construct a double mutant strain ΔtotR5i / ΔxiaPi; in the double mutant strain ΔtotR5i / ΔxiaPi, the negative regulatory gene totR3 was knocked out by insertion mutation, and a triple mutant strain ΔtotR5i / ΔxiaPi / ΔtotR3 was constructed; The positive regulatory genes totR1, totR2 and totR4 were inserted and knocked out in the double mutant strain ΔtotR5i / ΔxiaPi respectively, and the triple mutant strains ΔtotR5i / ΔxiaPi / ΔtotR1, ΔtotR5i / ΔxiaPi / ΔtotR2 and ΔtotR5i / ΔxiaPi / ΔtotR4 were constructed; in the double mutant strain ΔtotR5i / ΔxiaPi The positive regulatory gene totR1 was overexpressed in the medium, and the mutant strain ΔtotR5i / ΔxiaPi / totR1 was construct...

Embodiment 2

[0098] Example 2: The heterologous expression and activation method of the cyclolipopeptide compound Totopotensamides biosynthetic gene cluster.

[0099] Including the heterologous expression of the BAC plasmid 3-20G containing the complete gene cluster of Totopotensamides biosynthesis in S. lividans, the construction of the strain T3-20G, the in-frame deletion of the negative regulatory gene totR5 in the strain T3-20G, and the construction of the mutant strain ΔtotR5i / T3-20G.

[0100] The specific steps are as follows:

[0101] 1. Heterologous expression process of BAC library

[0102] Specific steps are as follows:

[0103] (1) First construct the BAC library of Streptomyces sp.SCSIO 02999, and then screen the BAC library containing the Totopotensamides biosynthesis gene cluster: use the upstream and downstream primers totR5F / R and Tot-orf(-1)F / R screened the BAC library of Streptomyces sp.SCSIO 02999, and screened three BAC plasmids, which were named BAC plasmid 3-20G,...

Embodiment 3

[0118] Embodiment 3: the preparation of ring lipopeptide compound Totopotensamides

[0119] (a) Preparation of the fermentation culture of the mutant strain ΔtotR5i / ΔxiaPi / totR1: insert the activated mutant strain ΔtotR5i / ΔxiaPi / totR1 into the seed medium, culture at 28°C and 200 rpm for 48 hours to obtain the seed liquid, and the seed liquid was A fraction of 10% of the inoculum was inserted into the fermentation medium, 28° C., 200 rpm, and vibrated for 7 days to obtain a fermentation culture. The formulations of the seed medium and fermentation medium were as follows, based on the mass fraction of 100%. , including soluble starch 2%, glycerin 1%, glycine 0.01%, alanine 0.01%, threonine 0.01%, isoleucine 0.01%, CaCO 3 0.5%, sea salt 3%, the balance is water, pH 7.0, mix the above ingredients evenly, sterilize and set aside.

[0120] (b) The fermentation culture is divided into fermentation broth and mycelium by centrifugation at 4000rmp and 10min. The secondary metabolites...

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Abstract

The invention discloses a biosynthetic gene cluster of cyclic lipopeptide compounds, a method for activating the biosynthetic gene cluster and application thereof. Nucleotide sequences of the biosynthetic gene cluster of the cyclic lipopeptide compounds Totopotensamides are shown as 2972nd-101763rd bit nucleotide sequences in SEQ ID NO.1. The biosynthetic gene cluster, the method and the application have the advantages that all gene and protein information related to the biosynthetic gene cluster of the cyclic lipopeptide compounds Totopotensamides can assist people to understand biosynthesismechanisms of the cyclic lipopeptide compounds Totopotensamides, and materials and knowledge can be increased for further genetic modification; bases can be provided for activating expression of silent gene clusters by means of controlling regulatory genes, natural product biosynthetic gene cluster banks can be enriched, and entity can be provided for discovering lead compounds of medicines.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a biosynthetic gene cluster of cyclolipopeptide compound Totopotensamides and its activation method and application. Background technique: [0002] In recent years, with the development and wide application of genome sequencing technology, it has been found that almost every strain of actinomycetes has a large number of secondary metabolite biosynthesis gene clusters, but most of them are in a silent state, and their encoded products have not been found yet. By changing fermentation conditions, ribosome engineering, promoter engineering, manipulating regulatory genes, and combinatorial biosynthesis, the expression of silent gene clusters can be activated to produce new secondary metabolites. Invention content: [0003] The object of the present invention is to provide a biosynthetic gene cluster of cyclolipopeptide compound Totopotensamides and its acti...

Claims

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Application Information

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IPC IPC(8): C12P21/04C12N15/76C12N15/31C12N15/52C12N1/21C12R1/465
CPCC07K14/36C12N9/00C12P21/02
Inventor 张长生陈瑞东张庆波谭彬郑六眷李慧贤朱义广
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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