Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin

Abstract

SummaryStreptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Streptomyces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identification of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decarboxylase gene ( vlmD ), a valanimycin resistance gene ( vlmF ), at least two regulatory genes ( vlmE , vlmI ), two genes encoding a flavin monooxygenase ( vlmH , vlmR ), a seryl tRNA synthetase gene ( vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of L -valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5 ¢ ¢ ¢ ¢ end.

Biochemical Characterization of VlmL, a Seryl-tRNA Synthetase Encoded by the Valanimycin Biosynthetic Gene Cluster

“…This finding suggests that an additional SerRS gene might be present in S. viridifaciens. The presence of a second SerRS gene (svsR) in S. viridifaciens has been subsequently confirmed by PCR experiments (21). As expected, the translation product of svsR exhibited a high degree of identity (91%) to the SerRS of S. coelicolor but a relatively low level of identity to VlmL (39%).…”

“…Previous investigations have shown that the valanimycin producer S. viridifaciens contains two genes that appear to code for SerRSs (21). Sequence analysis suggests that one of these genes (svsR) is likely to play a primary metabolic role, whereas the second gene (vlmL), which is embedded in the valanimycin gene cluster, appears to be involved in valanimycin biosynthesis (21).…”

“…work identified a housekeeping seryl-tRNA synthetase gene in S. viridifaciens in addition to vlmL (21). In the current studies, the S. viridifaciens housekeeping SerRS (svsR) and vlmL were amplified using primers that contained restrictions sites to facilitate the cloning of each gene into the protein expression vector pFLAG-CTC.…”

“…Furthermore, the azoxy nitrogen atoms in valanimycin have been shown to originate from the nitrogen atoms of isobutylamine and serine (17). Enzymatic and genetic investigations (18 -20) led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes (21). The functions of five proteins encoded by these genes have been elucidated.…”

“…A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that appears to encode a class II seryl-tRNA synthetase (SerRS) (21). The translation product of vlmL exhibits a relatively low identity (38%) to the single SerRS that is present in Streptomyces coelicolor.…”