UC Berkeley

Natural products have played a critical role in various fields for decades. Studying natural product biosynthesis facilitates the discovery of novel bioactive compounds and innovates engineering strategies to create molecules with specific scaffolds or functional groups. In addition to producing tailored molecules, biosynthetic engineering can also be used to achieve mass production of useful compounds. Among all kinds of natural products, terminal alkene and alkyne containing compounds have garnered significant attention due to the reactivity of their terminal functionalities and broad applications in chemical biology research and pharmaceutical industry. Therefore, it is important to understand the biosynthesis of terminal alkene and alkyne containing natural products and engineer pathways to leverage the biosynthetic machinery.Salivabactin is a terminal alkene containing natural product isolated from Streptococcus salivarius K12, which exhibits potent antimicrobial activities towards gram-positive bacteria. Considering its bioactivity and unavailability though synthesis, it is necessary to understand the biosynthesis of salivabactin. Based on enzymatic assays in vitro, we elucidated the biosynthesis and uncovered the minimal set of enzymes required for salivabactin production. Notably, an unprecedented thioesterase involved in the terminal alkene formation was characterized by site-directed mutagenesis and structural elucidation. With the understanding of biosynthesis, we were able to engineer the pathway to increase compound titer. Through heterologous expression and growth optimization in E. coli, we achieved a remarkable seven-fold enhancement in titer compared to that in the native host. This work broadens our understanding of terminal alkene biosynthesis and facilitates the mass production of new antibiotics to combat emerging multi-drug resistance. In addition to terminal alkene containing natural products, there are also diverse natural products with the terminal alkyne moiety, formed through various biosynthetic mechanisms. Among them, we focused on the family of membrane-bound desaturases/acetylenases, represented by JamB and TtuB. These enzymes are capable of catalyzing the terminal alkyne formation on acyl carrier protein (ACP)-tethered substrates. Intriguing mechanistic questions surrounding JamB and TtuB prompted the biochemical and structural studies, necessitating solubilized and active forms of these membrane-bound enzymes. To achieve this, we employed different strategies, including residue swapping, cell-free protein synthesis and lipoprotein shielding. Our efforts yielded promising results in terms of solubilization and enzyme activity, paving the way for further mechanistic exploration into terminal alkyne biosynthesis. Meanwhile, with the successful engineering to generate terminal alkyne tagged polyketides, we extended the applicability of terminal alkyne tagging to lipopeptides, as demonstrated by two NRPS systems in vivo and in vitro. Notably, we employed an innovative engineering strategy involving a non-elongating ketosynthase domain, which facilitated the transfer of acyl chains between ACPs. This establishes a versatile method for incorporating non-cognate ACPs into assembly lines.