Eurystatins A and B, new prolyl endopeptidase inhibitors. III. Fermentation and controlled biosynthesis of eurystatin analogs by Streptomyces eurythermus.

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.