Biosynthetic Studies of the Antibiotic Uncialamycin

Microbial natural products (or secondary metabolites) have frequently been opted for use in medical and agricultural settings. Uncialamycin (UCM) produced by Streptomyces uncialis was identified as a potent bactericidal antibiotic that is active at nanomolar concentrations against Gram positive and negative bacteria, including Burkholderia cepacia that poses serious risk to patients with cystic fibrosis. Moreover, UCM is presently under development, as an antibody‐drug conjugate, for targeted cancer therapies. UCM has a unique structure where an anthraquinone moiety is conjugated to a 10‐membered ring enediyne moiety, thereby representing a fascinating molecule for biosynthetic studies. While we have cloned its biosynthetic gene cluster, we are still far from understanding its biosynthetic processes. Studies of UCM biosynthesis are currently limited by its production exclusively during growth on solid media. The objective of this study is to develop a platform that enables biosynthetic and bioengineering studies of UCM. To probe the activity of the UCM biosynthetic machinery, we have profiled the transcription of ucm genes during growth on ISP4 agar (a UCM‐producing condition) and submerged growth conditions. The ucm genes were inactive in most submerged growth conditions, suggesting the bottleneck is at transcriptional levels. We eventually discovered a growth condition that activated the transcriptions, although the estimated production titer was not more than 0.1 mg/L. To improve the production titer, rational engineering and random mutagenesis were conducted by inactivating the competing pathways, applying chemical mutagenesis as well as overexpressing the pathway‐specific transcription regulator‐encoding genes. While strain improvement is still ongoing, we have observed that the production titer has been improved by at least five folds. Using our newly developed platform for UCM production, we are now investigating the biosynthetic relationship between UCM and dynemicin (DYN). DYN has similar scaffold as UCM with an additional 6‐membered ring structure ( Fig. 1) and is interestingly produced by a non‐streptomycete strain. Comparative analyses of UCM and DYN gene clusters ( Fig. 2) have revealed several key genes that might have a role in diversifying the molecular structures. Supported by the development of genetic systems for S. uncialis , we are undertaking genetic and metabolomic approaches to investigate the function of the key genes. Knockout of the putative Rieske oxygenase‐encoding gene ( ucmM ) led to abolished production of UCM and accumulation of a new biosynthetic intermediate (UCM M1) that was not detected in the wild‐type extract. The ongoing structure elucidation of UCM M1 has suggested that the precursor molecule was perhaps larger than originally predicted. We further propose UCM M1 as a key intermediate for UCM and DYN ( Fig. 1). Further combinatorial biosynthesis strategy will ultimately decipher the biosynthetic relationship between UCM and DYN. Through this work, we have developed a submerged culture system for UCM production as well as rationally engineered S. uncialis mutants for titer improvement and biosynthetic studies of UCM family of natural products. Support or Funding Information US NIH grants CA78747 (to B.S.) and GM115575 (to B.S.) 1Structures of uncialamycin (UCM), dynemicin (DYN), and the new intermediate UCM M1. The anthraquinone and enediyne moieties are highlighted in blue and red, respectively. While biosynthetic relationship between UCM and DYN is currently unknown, UCM M1 is proposed to be a key intermediate for both UCM and DYN.2Genetic organization of ucm and dyn biosynthetic gene clusters. Homologous gene cassette or genes are indicated with the same color.