Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

Abstract

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunAtunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.

Quinovosamycins: new tunicamycin-type antibiotics in which the α, β-1″,11′-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine

“…Bioinformatic analysis using tunB and tunD gene sequences to probe an actinomycetes genomic library The TUN biosynthetic operon of 12 essential tun genes (tunA-tunL) has been characterized in several diverse actinomycetes, 13,14,18 and TUN production is known to occur in various other actinobacteria. 8,19 Two genes, encoding for a radical SAM protein (TunB) and an unusual α-β-anomeric-to-anomeric glycosyltransferase (TunD) are highly selective to the TUN pathway.…”

“…10,11 A biosynthetic pathway has been proposed for TUN in which the 11-carbon dialdose sugar, tunicamine, is derived from uridine and N-acetylglucosamine. [12][13][14] The pseudoribosyl ring of tunicamine is N-glycosidically linked to uracil to form a structure highly analogous to the nucleoside uridine, and is presumed to mimic the UMP leaving group in the transition state for the translocase-catalyzed reactions. This inhibits the formation of N-acetylmuramyl-pentapeptide-undecaprenyl pyrophosphate in bacteria or N-acetylglucosamine-dolichol pyrophosphate in eukaryotes, which are essential intermediates in these organisms.…”

Determinants of Murein Hydrolase Targeting to Cross-wall of Staphylococcus aureus Peptidoglycan

“…At higher concentrations (i.e. the minimal inhibitory concentration of 10 -40 g), tunicamycin also blocks translocase I (MraY), a key enzyme for peptidoglycan synthesis (70). S. aureus was grown to logarithmic phase and treated for 30 min with 1 g/ml tunicamycin, a concentration that is known to specifically block TagO (52).…”

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

“…The biosynthesis gene clusters for a number of pyrimidine nucleoside antibiotics have been elucidated, including nikkomycin (4,46), polyoxin (8), blasticidin S (13), A-500359s and A-503803s (25), caprazamycin (38), lipisidomycin (39) and its analogue A-90289 (24), pacidamycins (50,60), muraymycin (12), and tunicamycin (9). However, biosynthesis studies for the amicetin group of disaccharide nucleoside antibiotics are not yet available.…”