CZ2009278A3 - Gene cluster sequence encoding celesticetin biosynthesis - Google Patents

Abstract

The microorganism gene cluster of the Streptomyces caelestis actinomycetes encoding biosynthesis, biologically active substances, celesthetin. Use of this sequence to prepare lincosamide antibiotic hybrid substances.

Description

Gene cluster sequence encoding celesticetin biosynthesis

Technical field

The invention relates to the use of a cluster of genes for the biosynthesis of the celesticetin antibiotic for the production of hybrid substances based on lincosamide antibiotics.

BACKGROUND OF THE INVENTION

Linkomycin and celesticetin belong to the class of lincosamide antibiotics. Clinically significant lincomycin is produced by various types of actinomycetes incl. strain of Streptomyces lincolnensis and is used to treat infections caused by Gram-positive pathogens.

Celesticetin is the second natural lincosamide antibiotic and is produced by Streptomyces caelestis. Microbiological and semisynthetic preparation of several derivatives of celesticetin has been described. Celesticetine and its derivatives are much less potent than lincomycin and are therefore not used clinically.

In lincomycin biosynthesis, two propylproline precursors (PPL) and methylthiolincosamide (MTL) precursors are separately synthesized and coupled to Ndemethyllincomycin by condensation and further methylated to the resulting lincomycin. In the case of celesticetin, the sugar component of MTL is associated with proline and salicylate.

In 1995, a cluster sequence of genes encoding lincomycin biosynthesis proteins - the Imb cluster was published. This cluster contains the biosynthesis genes of both precursors, condensation reaction genes, regulatory genes and lincomycin resistance genes. The cluster for celesticetin biosynthesis has not been described yet.

SUMMARY OF THE INVENTION

The gene cluster sequence for celesticetin biosynthesis has been described and designated as ccb cluster. This sequence is useful for the preparation of new, biologically active substances.

For this purpose, a cosmid library was prepared from Streptomyces caelestis ATCC 15084 chromosomal DNA. Cosmides carrying parts of the celesticetin cluster were screened using homologous probes from the lincomycin cluster. Sequence analysis of cosmids revealed the presence of a whole cluster for celesticetin biosynthesis.

The celestetin cluster (ccb cluster) of 27350 bp is flanked by the genes ccr1 and ccb5 and contains a total of 24 genes. The cluster contains genes for sugar component (MTL) and salicylate biosynthesis, condensation reaction genes, regulatory genes and celesticetin resistance genes. The differences in lincomycin and celesticetin biosynthesis are reflected in the composition of both gene clusters. In celesticetin biosynthesis, the amino-sugar unit of IvITL is coupled with proteinogenic proline. Therefore, the ccb cluster lacks the homologues of lincomycin genes responsible for propylproline synthesis.

Another difference is the presence of ccbl-ccb5 genes encoding proteins for biosynthesis and attachment of a salicylate unit whose counterparts are missing in the lincomycin cluster. The ccb4 gene is found only in the celesticetin cluster and encodes the O-methyltransferase required for methylation of the amino sugar unit at the 7 'position.

The newly obtained gene cluster coding for celesticetin biosynthesis and its difference from the gene cluster coding for lincomycin biosynthesis can be advantageously used for the design and preparation of novel compounds based on lincosamide antibiotics. An interesting possibility seems to be the preparation of a producer carrying genes for biosynthesis of lincomycin and salicylate unit of celesticetin. The possibility of using celesticetin methyltransferase Ccb4 for methylation of the amino-sugar unit in the lincomycin molecule also appears to be promising.

Overview of pictures

Fig. 1: Structure of the lincomycin molecule

Giant. 2. Structure of celesticetin molecule

Fig. 3: Primers for PCR preparation of homologous probes, non-homologous sections are indicated by underlines

Fig. 4: Amino acid sequences of proteins coded by ccb cluster of genes

Fig. 5: Sequence of ccb cluster of genes encoding celesticetin biosynthesis and its surroundings

Chromosomal DNA S ¹ . caelestis ATCC 15084 was isolated from YEME grown culture

DETAILED DESCRIPTION OF THE INVENTION

Example 1 medium (yeast extract 3 g / l, malt extract 3 g / l, peptone 5 g / l, glucose 10 g / l, pH 7.2) (50 ml) with addition of glycine at a final concentration of 0 _? 5% and lysozyme at a final concentration of 1 mg / ml. Subsequently, the chromosomal DNA was partially digested with a restriction enzyme

BamHI and ligated into the SuperCos-1 cosmid vector (Stratagene) that had been pre-digested with XbaI and BamHI. The resulting ligation mixture was introduced into lambda phage, and the phage was b * transfected into E. coli strain XL1-Blue MR according to the Gigapack III XL Packaging protocol.

Kit (Stratagene). Cosmid DNA was isolated from the cells using the NucleoBond BAC kit

100 columns (Macherey-Nagel).

Example 2

The probes for the search for celesticetin genes in the Streptomyces caelestis cosmid library ATCC 15084 were designed based on putative conserved regions of the ImbJ, ImbM, ImbS and ImbZ genes from S. lincolnensis ATCC 25466. The primers used for PCR amplification probes are shown in Table 2. The probes were labeled with digoxigenin and used for hybridization with more than 1000 cosmid library clones.

DNA of the clones giving a positive signal with all probes was partially digested with the restriction enzyme Sau3AI. The restriction mixture was resolved by electrophoresis and 1-2 kpb fragments isolated and cloned into the pJAKO vector. The ligation mixture was

L was transformed into E. coli XL1-Blue MR cells.

Example 3

A library of DNA fragments of celesticetin cluster (Example 2) was sequenced using universal primers. The resulting sequences were compared with NCBI databases for the presence of homologues of lincomycin or other secondary metabolic genes. The sequences of twenty positive clones were used to design primers for sequencing the whole celestetin cluster by the primer walking method.

The sequences were analyzed using SeqMan (DnaStar software package) and Artemis (release 9, The Sanger Institute). DNA and protein homologues were retrieved using databases on the National Center for Biotechnology Information (Bethesda, MD.)

Industrial applicability

The gene cluster sequence encoding celesticetin biosynthesis can be utilized in the development of new biologically active substances, i.e. in pharmacy, medicine.

Claims (2)

PATENT CLAIMS PV 3.00H -25QJ 25 OU A gene cluster sequence encoding celesticetin biosynthesis designated ccb cluster. Use of a gene cluster sequence encoding celesticetin biosynthesis according to claim 1 for the preparation of hybrid substances of lincosamide antibiotics.