DNA Sequencing and Transcriptional Analysis of the Kasugamycin Biosynthetic Gene Cluster from Streptomyces kasugaensis M338-M1

Abstract

Streptomyces kasugaensis M338-M1 produces the aminoglycoside antibiotic kasugamycin (KSM). We previously cloned, sequenced and characterized the KSM acetyltransferase, transporter, and some of the biosynthetic genes from this strain. To identify other potential genes in a chromosome walk experiment, a 6.8-kb EcoRI-PstI region immediately downstream from the KSM transporter genes was sequenced. Five open reading frames (designated as kasN, kasO, kasP, kasQ, kasR) and the 5Ј region of kasA were found in this region. The genes are apparently co-transcribed as bicistrons, all of which are co-directional except for the kasPQ transcript. Homology analysis of the deduced products of kasN, kasP, kasQ and kasR revealed similarities with known enzymes: KasN, D-amino acid oxidase from Pseudomonas aeruginosa (35% identity); KasP, F420-dependent H4MPT reductase from Streptomyces lavendulae (33% identity); KasQ, UDP-N-acetylglucosamine 2-epimerase from Streptomyces verticillus (45% identity); and KasR, NDP-hexose 3,4-dehydratase from Streptomyces cyanogenus (38% identity); respectively. A gel retardation assay showed that KasT, a putative pathway-specific regulator for this gene cluster, bound to the upstream region of kasN and to the intergenic region of kasQ-kasR, suggesting that the expression of these operons is under the control of the regulator protein.

Biosynthetic genes for aminoglycoside antibiotics

“…Kasugamycin lacks the amino functionality in the cyclitol moiety and therefore does not possess the aminotransferase gene in the gene cluster. 42 Several biosynthetic enzymes for bluensomycin 43 and spectinomycin 44 have also been characterized, but those studies have not been performed systematically yet. Biochemical characterization of many other enzymes is necessary to understand complex biosynthetic pathways for these aminoglycosides on the enzymatic reaction level.…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…1D) is involved in hydride transfer during reductive transformation of carbon dioxide and acetate into methane in methanogenic archaea (103,151,258,299,514). Coenzyme F 420 has also been found in certain streptomycetes, in which it serves as a cofactor in the biosynthesis of tetracycline and lincomycin (74,189,347,369). Mycobacterium and Nocardia spp.…”

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…However, comparative genome analysis indicates that there are numerous other F 420 -dependent LLHTs in actinomycetes, the majority probably serving as reductases (37). These have been implicated in a variety of roles, ranging from pyrrolobenzodiazepene antibiotic synthesis in streptomycetes (50,393,394,429) to cell wall metabolism in mycobacteria (37) and exogenous substrate mobilization by rhodococci (155). A bioinformatics analysis predicted that there are some 45 F 420 -binding LLHTs in M. smegmatis and 17 in M. tuberculosis, though this has yet to be validated experimentally (37).…”

“…16) (389, 391). The biosynthesis of other pyrrolobenzodiazepine antibiotics (392) are facilitated by equivalent F 420 H 2 -dependent imine reduction steps, namely, tomaymycin (Streptomyces achromogenes) (50), sibiromycin (Streptosporangium sibiricum) (393), kasugamycin (Streptomyces kasugaensis) (394), and anthramycin (Streptomyces rifuineus) (395). However, biochemical studies have yet to definitively identify which enzymes are responsible for these reactions.…”

“…However, biochemical studies have yet to definitively identify which enzymes are responsible for these reactions. The strongest candidates are the putative F 420 H 2 -dependent luciferase-like hydride transferases (LLHTs) encoded in the sequenced antibiotic synthesis gene clusters (50,391,(393)(394)(395) of each of these organisms. Because all research thus far has focused on the roles of F 420 in the secondary metabolism of streptomycetes, little is known about the roles of this cofactor in central metabolism of this genus; it is likely that streptomycetes use F 420 to support some important metabolic pathways, as they carry genes that encode homologs of mycobacterial enzymes such as the F 420 H 2 -dependent biliverdin reductase (30).…”