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Metarhizium anisopliae o-methyltransferase and application thereof

An oxymethyltransferase and oxymethyl technology, applied in transferase, application, genetic engineering and other directions, can solve the problems of strong corrosiveness of reagents, serious environmental pollution, and many types of products, and achieve low cost, environmental safety, Simple to use effects

Active Publication Date: 2015-08-26
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many disadvantages in the modification by chemical reaction, such as long production cycle, many types of products, high separation cost, most of the reagents used are highly corrosive, and serious environmental pollution
Even the currently researched advanced molecular sieve catalysts cannot combine the advantages of high stability, good catalytic activity, and specific selectivity.
However, there is no report on the oxymethyltransferase with a wide range of substrates

Method used

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  • Metarhizium anisopliae o-methyltransferase and application thereof
  • Metarhizium anisopliae o-methyltransferase and application thereof
  • Metarhizium anisopliae o-methyltransferase and application thereof

Examples

Experimental program
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Effect test

Embodiment 114

[0055] Embodiment 114, The methylation modification of 15-Dehydrozearalenol

[0056] 1. MaOMT gene cloning:

[0057] 1) Metarhizium robertsii (Metarhizium robertsii) genomic DNA was extracted by phenol-chloroform extraction

[0058] 2) Using the genome of Metarhizium anisopliae as a template and MaOMT F and MaOMT R as primers (see Appendix 1), the MaOMT gene was amplified. Add two restriction sites upstream and downstream of the MaOMT gene by primers: add CAT upstream of the start codon ATG to form an Nde I restriction site, and add GTTTAAAC downstream of the stop codon TAA to form a Pme I restriction site

[0059] 3) Digest MaOMT PCR product and PRS425M vector with Nde I and Pme I

[0060] 4) Connect the two fragments of restriction enzymes to form the PRS425M-MaOMT plasmid (see the attached map figure 1 ), transformed into competent Escherichia coli, and applied to solid LB medium containing Amp (50μg / ml), 38°C, 16h

[0061] 5) Pick positive clones, transfer them to liqu...

Embodiment 2D

[0100] The methylation modification of embodiment 2 Desmethyl-Lasiodiplodin

[0101] The method is the same as in Example 1, except that the yeast transformation step is that pBDL6066, pBDL6067 and PRS425M-MaOMT are co-transformed into competent yeast cells.

[0102] HPLC analysis found that a peak (peak 4) appeared in front of Desmethyl-Lasiodiplodin (peak 3). Further structural analysis (NMR) proved that compound 3 was Desmethyl-Lasiodiplodin and compound 4 was Lasiodiplodin. See Table 3 and Table 4 for NMR data.

[0103] Table 3 compound 3NMR spectrogram analysis result

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[0105] Table 4 compound 4NMR spectrogram analysis result

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Abstract

The invention provides an o-methyltransferase with a wide range of substrates, and can be applied to oxygen methyl transfer in a biosynthesis process of an organic compound. An experiment proves the catalytic activity of the o-methyltransferase, and shows that the o-methyltransferase can be applied to a plurality of substrates. The invention further provides a synthesis method for a hydroquinone lactone compound. The hydroquinone lactone compound is obtained by transforming an o-methyltransferase gene and a hydroquinone lactone synthetic gene into yeast cells, carrying out fermentation cultivation and extracting a product.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an oxygen methyltransferase obtained from Metarhizium anisopliae. The invention also relates to the application of the oxygen methyltransferase in the biosynthesis of diphenol lactone compounds. Background technique [0002] Polyketide compounds have biological activities such as antibacterial and insecticidal, anticancer, blood lipid lowering, and immune suppression. Diphenol lactones are a large category of them, and they have great application potential in clinical and agricultural medicine. Two genes are needed for the synthesis of quinolactones: a reduced polyketide synthase gene and a non-reduced polyketide synthase gene, and these two genes are collectively referred to as quinolactone synthesis genes. [0003] The modification of natural diphenol lactone polyketide compounds can provide a way for the renewal and transformation of drugs. For example, after the formation of the ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12P7/22C12P17/08
Inventor 徐玉泉刘航张维张礼文
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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