The route of incorporation of nitrogen atom into rifamycin during its biosynthesis

In this paper, it was first shown that under various conditions of nitrogen supply the rifamycin yield was positively correlated with the mycelial specific activity of glutamine synthetase (GS), then the enhancing effect of glutamine, the product of GS, on rifamycin biosynthesis was demonstrated with resting cell system. The stimulatory effect of glutamine was more pronounced than that of glutamate, and not reduced by a GS specific inhibitor, DON. However, the increase in yield brought about by glutamate, and by asparagine was strongly inhibited by this inhibitor. Glutamine-CO15NH2 amd glutamate-alpha-15NH2 were synthesized and compared for the incorporation of 15N into rifamycin. It was found that the amide nitrogen was incorporated to a much greater extent than the alpha-amino nitrogen, showing that glutamine was a direct precursor of the nitrogen atom in rifamycin. In addition, synthesis of A-32 (C7N), an intermediate secreted by an inactive mutant rif 1, was also greatly stimulated by glutamine, and the synthetic C7N was found to be able to stimulate the biosynthesis of rifamycin. Based upon the above results, the route of incorporation of nitrogen atom into rifamycin is summarized as follows: (Formula: see text).