Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii HC34, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. We have been interested in the biosynthesis of vicenistatin, because the aglycon is distinct from regular polyketides solely consisting of lower fatty acid units. Isotope tracer experiments of ^<13>C-labeled acetate and propionate showed that C-1 to C-16 of the aglycon was derived from acetate and propionate in a standard polyketide biosynthetic pathway, and incorporation of intact acetate into C-17 and C-18 strongly suggested that a possible starter unit was derived from glutamate via 3-methylaspartate. Feeding experiments of deuterated glutamate, [^<15>N]-glutamate and deuterated (2S,3S)-3-methylaspartate showed that glutamate mutase, which is known to convert glutamate to 3-methylaspartate, was actually involved in the biosynthesis of the aglycon. However, (2S,3R)-3-methylaspartate and DL-3-amino-2-methylpropionate were not incorporated into vicenistatin. Thus, epimerization of the methylated site is involved in the starter biosynthesis, but its timing is yet to be clarified. The vicenistatin gene cluster (vin) spanning ca. 64kbp was successfully cloned and sequenced by using consensus sequences of dTDP-glucose 4,6-dehydratase and dTDP-4-keto-6-deoxyglucose 2,3-dehydratase which were usually involved in the early steps of the 2,6-deoxysugar biosynthesis. The vin cluster contains ORFs encoding proteins homologous to polyketide synthases (vinP1-4), glutamate mutase (vinH, I), acyl CoA ligase (vinN) and decarboxylase (vinO). VinN and VinO appear to be involved in the formation of the starter unit of aglycon from the earlier intermediate 3-methylaspartate generated by VinH and VinI. VinP1-4, consisting of eight extending modules and a terminal thioesterase domain, are responsible to produce the whole aglycon polyketide. Also contained in the cluster are ORFs homologous to 4,6-dehydratase (vinB), 2,3-dehydratase (vinD), aminotransferase (vinF), N-methyltransferase (vinG) and glycosyltransferase (vinC), for the vicenisamine biosynthesis. Furthermore, VinC was heterologously expressed in Escherichia coil and purified. The glycosyl transfer reaction from dTDP-vicenisamine to the aglycon was confirmed with the recombinant VinC. These results proved that the abovementioned gene cluster encodes the vicenistatin biosynthetic enzymes.
