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Biosynthetic method and gene cluster of a kind of compound balanol

A technology for biosynthesis and chemical compounds, applied in the fields of biochemical equipment and methods, plant genetic improvement, chemical instruments and methods, etc., which can solve the problem of undetected products, etc.

Active Publication Date: 2020-04-24
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Through genome sequencing, a PKS-NRPS hybrid gene cluster containing an AflR regulatory factor was found in the genome sequence of Cordyceps macrocystis, but no product corresponding to the gene cluster was detected in the fermentation product, and it was speculated that it was a recessive gene cluster

Method used

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  • Biosynthetic method and gene cluster of a kind of compound balanol
  • Biosynthetic method and gene cluster of a kind of compound balanol

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Embodiment 1

[0042] Taking this method to activate balanol biosynthesis in Cordyceps macrocystis as an example, the specific implementation steps are as follows:

[0043] 1) Analyze and obtain the PKS-NRPS hybrid gene cluster containing the Zn2Cys2 family transcription factor AflR analog gene blnR in the genome sequence of the Cordyceps magnum strain, and obtain the gene by PCR cloning. The cloned blnR gene DNA fragment (its base sequence is as follows: Shown in SEQ ID NO: 1) was connected into the pFG vector containing pTEF strong promoter and kanamycin resistance gene (neo) and chlorimuron-methyl resistance gene (SUR), to obtain plasmid pTblnR;

[0044] 2) Transform the plasmid into Agrobacterium tumefaciens EHA105 by electrotransformation, select for streptomycin (Str) and kanamycin resistance, culture at 28°C for 2 days, and obtain positive recombinant Agrobacterium ApTblnR by colony PCR identification ;

[0045] 3) Inoculate the positive recombinant Agrobacterium ApTblnR monoclonal i...

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Abstract

The invention discloses a biosynthesis method of a compound balanol and a gene cluster thereof. The biosynthesis method is effective and convenient and comprises the following steps: connecting a blnRgene segment into a carrier, thus obtaining an expression plasmid; utilizing electroporation to convert the expression plasmid into agrobacterium tumefaciens EHA105, screening and culturing, thus obtaining the agrobacterium with the expression plasmid; inoculating into a liquid culture medium, culturing by shaking under a shading condition, taking the culture solution and then culturing by shaking under the shading condition, thus obtaining a recombined agrobacterium EHA105 bacteria solution; collecting large cysticercus spores, thus obtaining a large cysticercus spore suspension; mixing therecombined agrobacterium EHA105 bacteria solution with the large cysticercus spore suspension and co-culturing in a black place, thus obtaining co-culture mixed bacteria; obtaining an OE::blnR large cysticercus spore converter, performing fermentation culturing and separating the fermenting solution, thus obtaining the compound balanol.

Description

technical field [0001] The invention relates to the technical field of biosynthesis of the compound balanol, in particular to a biosynthesis method and a gene cluster of the compound balanol. Background technique [0002] Fungal natural products are a treasure trove of natural drug sources for humans, but many gene clusters are recessive under natural conditions. In recent years, many new technological advances, including sequencing and detection methods, have made it easier to activate new products. Cordyceps Datuan is a traditional Chinese medicine used to treat ailments such as metrorrhagia and irregular menstruation. It is distributed in Asia and Europe. [0003] Through genome sequencing, a PKS-NRPS hybrid gene cluster containing an AflR regulatory factor was found in the genome sequence of Cordyceps macrocystis, but no product corresponding to the gene cluster was detected in the fermentation product, and it was speculated that it was a Stealth gene cluster. By const...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12P17/10C12N15/31C07K14/37C12N15/54C12N15/61
CPCC07K14/37C12N9/1029C12N9/90C12N15/52C12N15/80C12P17/10
Inventor 李永泉陈新爱何弦
Owner ZHEJIANG UNIV
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