The macrolactam antibiotic vicenistatin, produced in Streptomyces halstedii HC34, is biosynthesized by the polyketide pathway, using a unique 3-methylaspartate-derived molecule as starter unit. The vinI gene in the vicenistatin biosynthetic gene cluster encoding glutamate mutase, which rearranges glutamate to 3-methylaspartate, was disrupted. The vinI disruption completely abolished the production of vicenistatin, while the disruptant recovered the production of vicenistatin when 3-methylaspartate was added to the culture. These results indicate that vinI is essential for the 3-methylaspartate formation in the vicenistatin biosynthesis. Furthermore, the mutant accumulated new vicenistatin derivatives (desmethylvicenistatins), which lacked a methyl group in the starter unit. The desmethylvicenistatins were shown by feeding experiments to be derived from aspartate instead of 3-methylaspartate as the starter unit. These results indicate that the vicenistatin polyketide synthase can accept alternative starter units toward the production of novel polyketides.