Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments b bThe GenBank accession number for the sequence reported in this paper is AF235050.

Abstract

The coumarin antibiotic coumermycin A 1 contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A 1 biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A 1 production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A 1 , respectively. Cmethylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the nonmethylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A 1 in the wild-type.

Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mutasynthesis

“…In accordance with our previous studies on the generation of novobiocin and clorobiocin derivatives (5,20,41), the new compounds are called novclobiocins, with different numbers indicating different structures (Tables 1 and 2).…”

“…Heide and coworkers recently reported on the preparation of new aminocoumarins by combinatorial biosynthesis (5,20,41). The present investigation focused on the antimicrobial and DNA gyrase-inhibitory properties of novel aminocoumarin antibiotics obtained by mutasynthesis with a strain of the clorobiocin producer Streptomyces roseochromogenes (34).…”

Assembly and Heterologous Expression of the Coumermycin A1 Gene Cluster and Production of New Derivatives by Genetic Engineering

“…The level of coumermycin A 1 production of the S. coelicolorA C H T U N G T R E N N U N G (couMW16) integration mutants was on average 7 mg mL À1 and thereby superior to that found in the A C H T U N G T R E N N U N G natural producer Streptomyces rishiriensis DSM40489 (2-5 mg mL À1 ). [17] Inactivation of the C-methyltransferase gene couO…”

“…It has been shown previously that the methyltransferase CouO is responsible for C-methylation at position 8 in the aminocoumarin moiety in coumermycin A 1 . [17,18] Inactivation of couO was performed by the PCR targeting method. [12,13] The complete open reading frame of couO within cosmid couMW16 was replaced by an apramycin resistance cassette flanked by unique XbaI restriction sites, resulting in cosmid couMW17.…”

Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics The GenBank accession number for the sequence of cosmid K1F2 is AF329398.

“…The genes cloP, novP and couP, situated in the same relative position of their respective gene clusters, show homology to genes encoding O-methyltransferases and may be assigned to this reaction. CouP has recently been proven to be responsible for the methylation of 4-OH of the deoxysugar (Li et al, 2002).…”