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Preparation method of lvsong peptide rhzomorph C

A technology of peptidocin and actinomycetes, applied in the field of biopharmaceuticals, can solve the problems of high cost, complex structure, low yield and the like

Active Publication Date: 2021-02-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex structure of luzonin C, the two total synthetic routes require about 20 steps, high cost and low yield

Method used

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  • Preparation method of lvsong peptide rhzomorph C
  • Preparation method of lvsong peptide rhzomorph C
  • Preparation method of lvsong peptide rhzomorph C

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Experimental program
Comparison scheme
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Embodiment 1

[0044] 1. The construction of mutant strain DS-1, the construction steps are as follows:

[0045]1. The whole genome of Actinomadura sp. DSM43766 was sequenced to identify the biosynthetic gene cluster of luzonidura. The gene cluster is 48kb long, as shown in SEQ ID NO.1, including 30 genes, such as figure 1 shown.

[0046] Further functional analysis was carried out on 30 genes, and a gene encoding acetylase was determined. The nucleotide sequence of the gene is shown in SEQ ID NO.2, and it was named luz27. The amino acid sequence of the acetylase encoded by the gene is shown in SEQ ID NO.3.

[0047] 2. Based on the Escherichia coli-Streptomyces shuttle plasmid vector pSET152 (Genbank sequence number AJ414670.1), the luz27 gene was knocked out by homologous double crossover method.

[0048] Two homology arms (2.2 kb and 2.6 kb in length, respectively) upstream and downstream of the target knockout region were amplified by polymerase chain reaction using the genomic DNA of ...

Embodiment 2

[0058] Preparation of Luzonin C

[0059] Actinomadura sp. (Actinomadura sp.) strain DS-1 was cultured in MS medium (1L) for 12 days on a large scale, soaked in ethyl acetate for 3 times, the extracts were combined and filtered, and the ethyl acetate was evaporated to dryness The crude extract was obtained, which was dissolved in 4 ml of methanol and subjected to Sephadex LH20 gel chromatography (pure methanol mobile phase). Aliquot tubes of 5 ml each to receive the eluate. 20 microliters of components in each tube were analyzed with EC-C18 chromatographic column (mobile phase ratio: 0-25min, acetonitrile: water = 10:90-80:20; 25-26min, acetonitrile: water = 80:20~ 100:0; 26-28min, acetonitrile: water = 100:0; 28-29min, acetonitrile: water = 100:0-10:90; 29-30min, acetonitrile: water = 10:90. The mobile phase contains 0.05% Formic acid, the detector is a diode array detector, the flow rate is 1.2mL / min). By comparing with the standard substance of luzopeptide C, find the com...

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Abstract

The invention discloses a preparation method of lvsong peptide rhzomorph C, and belongs to the field of biological pharmacy. The preparation method comprises the following steps: taking actinomyces lschsoni DSM43766 as a starting strain, inactivating an acetylase gene luz27 by virtue of a gene inactivation technology, and enabling the nucleotide sequence of the acetylase gene luz27 to be shown asSEQ ID NO.2. The invention further discloses the application of the actinomyces lschsoni DSM43766 in preparation of the acetylase gene luz27. And then inoculating the constructed mutated strain of actinomyces lschsonii into a fermentation culture medium, carrying out fermentation culture, and separating and purifying a fermentation product to obtain the lschsonii peptide rhzomorph C. According tothe invention, the acetylase gene luz27 of actinomyces lschsonii is inactivated by a genetic engineering means to block the acetylation modification process of lschsonii bacteriocin C, so that the component lschsonii bacteriocin C with higher biological activity is specifically accumulated. The lvsong peptide bacteriocin C is specifically produced through a microbial fermentation method, and the method is green, economical and efficient and has industrial production prospects.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a green, efficient and specific method for preparing the antiviral active molecule luzonin C. Background technique [0002] Luzopeptin (luzopeptin) is a kind of double intercalation family non-ribosomal peptide compound produced by Actinomadura sp.DSM43766. The strain was isolated in 1980 by Japanese scientists from soil samples in Luzon, Philippines. Lusopeptide has good antitumor activity and antibacterial activity, and is also a highly effective HIV reverse transcriptase inhibitor. [0003] Actinomyces madura luzon can produce A, B, C and other components under different fermentation conditions. The general structural formula of luzonin A~C is as formula (I), [0004] [0005] Lusopeptide A:R 1 =R 2 =CH 3 CO; Lusopeptin B:R 1 =H,R 2 =CH 3 CO; Lusopeptin C:R 1 =R 2 =H. [0006] Lureotin C contains two 4-hydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid stru...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N1/21C12N15/74C12P21/02C07K7/06C07K1/14C07K1/16C07K1/20C12R1/03
CPCC12N9/1029C12N15/74C12P21/02C07K7/06
Inventor 杜艺岭施鑫杰黄立明
Owner ZHEJIANG UNIV
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