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Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae

A technology of derivatives and groups, used in the production and use of the following polypeptides, the identification of gene synthesis clusters

Inactive Publication Date: 2009-03-18
NOVACTA BIOSYSTEMS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it has been reported that macinamycin causes well-defined pores in flat bacterial membranes containing phosphatidylethanolamine

Method used

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  • Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae
  • Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae
  • Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae

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Experimental program
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Embodiment approach

[0114] The C-terminal formula of the derivative is -COR, where R represents the group -NR 1 R 2 , some examples, R 1 stands for hydrogen atom, R 2 Represents a general formula -(CH 2 ) n -NR 3 R 4 The group, where n represents an integer between 2 and 8, R 3 and R 4 independently represents a hydrogen atom or (C 1 -C 4 ) alkyl or R 3 with R 4 Together represent the following groups: -(CH 2 ) 3 -, -(CH 2 ) 4 -, (CH 2 ) 2 -O-(CH 2 ) 2 -, -(CH 2 ) 2 -S-(CH 2 ) 2 -or-(CH 2 ) 5 -. In these examples, R 3 and R 4 Preferably represents a hydrogen atom or (C 1 -C 4 )alkyl. More preferably represents (C 1 -C 2 ) alkyl, such as methyl. The integer n is preferably between 2-5, more preferably between 2-4, eg 3.

[0115] In another embodiment, R 1 and R 2 Together with the adjacent nitrogen atom, represents a piperazine. , preferably the 4-position N-substituent is selected from the following groups:

[0116] (a)(C 1 -C 4 )alkyl;

[0117] (b)(C 5 -...

Embodiment 1

[0233] Example 1 - Cloning of clustered genes

[0234] Identification and cloning of clustered genes for atagardine biosynthesis from Actinomyces garbardinogenes and Actinomyces liguria.

[0235] O / SBDIG-1 is a digoxigenin (DIG)-labeled degenerate oligonucleotide consisting of 48 bases. It is designed according to the known amino acid sequence of actigardine polypeptide and considering the codon preference of Actinomycetes genus. Southern hybridization analysis was performed on the genomic DNA extracted from Actinomyces garbartin-producing Actinomycetes and digested with the restriction endonuclease NcoI, and a DNA fragment of about 3 kb was identified that could bind to the probe OSBDIG-1 hybridize. After enriching the NcoI-digested genomic DNA, a DNA fragment of about 3 kb was isolated and cloned into the NcoI site of the vector pLITMUS28 (NEB). The cloned plasmid was transformed into Escherichia coli strain E.coli DH10B and analyzed by Southern hybridization using probe ...

Embodiment 2

[0396] Example 2 - Expression Cassette

[0397] Construction of expression cassettes

[0398] This example illustrates the method for constructing the expression cassette of the present invention. The plasmid pAGvarX in the expression cassette can efficiently construct the variant form of the LanA gene in the present invention, and then transfer it into a host cell, for example, in a strain of actinomycetes producing garbardine that does not contain wild-type actA (A .garbadinis Δ actA). This plasmid is derived from the vector pSET152 (Bierman et al. 1992), which can be integrated into the chromosome of the host cell through the attP attachment site. Variant forms of actA were produced by expressing the mutant form of the actA gene in combination with the wild-type gene of its own actagardine biosynthetic cluster gene by the host.

[0399] Construction of plasmid pAGvarX

[0400] All references to positions herein refer to the position on the sequence SEQ ID NO:100 unless ...

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Abstract

This invention relates to characterisation of the biosynthetic gene cluster for the lantibiotic actagardine, identification of a novel variant of actagardine and its biosynthetic cluster, and methods of production and use of actagardine, a novel actagardine variant, herein referred to as actagardine B, and variants of both of these produced according to this invention, utilizing genes from the characterised biosynthetic gene clusters.

Description

technical field [0001] The present invention relates to the characterization of biosynthetic cluster genes of lantibacterial antibacterial peptide actagardine, and the identification of a novel actagardine variant and its gene synthesis cluster. The invention also relates to methods of producing and using polypeptides comprising actagardine, a novel variant form of actagardine in the strain Actinomyces liguria, and the use of characterized Variant forms of these two polypeptides are produced by genes in the biosynthetic gene cluster of . Background technique [0002] Lantibiotics are peptides synthesized by Gram-positive bacteria with various activities including antibacterial ability. Among other types of residue modification, they cross-link the peptide chains to each other through the thioether amino acids lanthionine and methyllanthionine, forming a polycyclic structure. According to a classification standard that is not absolute, such antimicrobial peptides can be div...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/365
CPCC07K14/365C12R1/045A61P1/00A61P31/04C12R2001/045C12N1/205
Inventor 史蒂文·博克斯赫苏斯·科尔特斯巴尔加略迈克尔·约翰·道森
Owner NOVACTA BIOSYSTEMS LTD
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