Exploring the Biosynthetic Potential of <i>Tistrella</i> Species for Producing Didemnin Antitumor Agents

Didemnins are a class of cyclic depsipeptides derived from sea tunicates that exhibit potent anticancer, antiviral, and immunosuppressive properties. Although certain <i>Tistrella</i> species can produce didemnins, their complete biosynthetic potential remains largely unexplored. In this study, we utilize feature-based molecular networking to analyze the metabolomics of <i>Tistrella mobilis</i> and <i>Tistrella bauzanensis</i>, focusing on the production of didemnin natural products. In addition to didemnin B, we identify nordidemnin B and [hysp²]didemnin B, as well as several minor didemnin analogs. Heterologous expression of the didemnin biosynthetic gene cluster in a <i>Streptomyces</i> host results in the production of only didemnin B and nordidemnin B in limited quantities. Isotope-labeling studies reveal that the substrate promiscuity of the adenylation domains during biosynthesis leads to the accumulation of nordidemnin B and [hysp²]didemnin B. Additionally, precursor-directed biosynthesis is applied to generate eight novel didemnin derivatives by supplementing the culture with structurally related amino acids. Furthermore, we increased the titers of nordidemnin B and [hysp²]didemnin B by supplementing the fermentation medium with l-valine and l-isoleucine, respectively. Finally, both compounds undergo side-chain oxidation to enhance their biological activity, with their anticancer properties found to be as potent as plitidepsin.