Nybo, E. | Search bioRxiv

bioRxiv2021

A BioBricks toolbox for multiplexed metabolic engineering of central carbon metabolism in the tetracenomycin pathway

The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. We developed a BioBricks(R) genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to {beta}-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Altogether, this system is compatible with the BioBricks(R) [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE.

synthetic biology↗

bioRxiv2019

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

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.

microbiology↗