Accurate and precise component analysis of eurystatin analogs in fermentation broth was devised by HPLC methods with and without 2,4-dinitrophenylhydrazonation. Detailed optimization of fermentation conditions and strain improvement by HPLC analysis significantly increased the eurystatin productivity of Streptomyces eurythermus. Chemically defined fermentation media which produced eurystatins A and B at fermentation yields comparable to complex media were elaborated for radio-isotope fermentation studies and controlled biosynthesis. Radio-isotope incorporation study using 14C-labeled amino acids in chemically defined medium demonstrated that L-leucine and L-ornithine were the direct precursors for the L-leucine and L-ornithine moieties of eurystatins A and B, respectively. Based on this finding, L-valine and L-isoleucine were supplemented to the growing culture of S. eurythermus in chemically defined medium, which resulted in the controlled biosynthesis of new eurystatin analogs named eurystatins C, D, E and F.