Combined use of design of experiments and metabolic engineering for optimization of nogalamycin production

Abstract

Nogalamycin is an anthracycline antibiotic produced from Streptomyces nogalater that exerts its mechanism of action via inhibition of topoisomerase I. Nogalamycin has a unique tetracyclic structure composed of a 7-O-glycosidically linked L-nogalose sugar and a C2-C5\"-linked L-nogalamine residue that forms an epoxyoxacin ring. Nogalamycin was originally developed as an anticancer agent in the 1970s, however, it exhibited dose-limiting cardiotoxicity. Shortly after the discovery of nogalamycin, the semi-synthetic analogue 7-O-methylnogarol, or menogaril, was synthesized and exhibited a mild improvement in cardiotoxicity and anticancer efficacy. Menogaril lacks the 7-O-L-nogalose moiety and functions as a topoisomerase II inhibitor, like most anthracyclines, however this agent still proved too toxic for clinical use. Our laboratory is developing a production platform for microbial synthesis of novel nogalamycin analogs useful for treatment of human cancers or as antibiotics. Our initial hypothesis is that overexpression of structural genes responsible for synthesis of TDP-deoxysugar and polyketide precursor substrates, respectively, will increase carbon flux towards nogalamycin production. In this study, we have employed metabolic engineering to enhance nogalamycin production in Streptomyces nogalater. In this work, we used an optimized soytone glucose production medium to produce nogalamycin. We also overexpressed copies of structural biosynthetic genes to bolster substrate precursor building blocks for nogalamycin production. First, overexpression of the TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase enzymes (mtmDE) resulted in a 50% increase in nogalamycin production (160 mg/L) as compared to wildtype S. nogalater (100 mg/L). Secondly, overexpression of the minimal polyketide synthase genes (snoa123) resulted in a fourfold production increase in nogalamycin (400 mg/L). This production platform will serve as the fundament for production of nogalamycin analogues for future drug development efforts.