A Framework for the In Vivo Production of Extensively Engineered Thiopeptides

Thiopeptides are a family of macrocycle-containing ribosomally synthesized and post-translationally modified peptides. Their elaborate scaffolds offer considerable potential for bioengineering toward thiopeptide-based pharmaceuticals and other practical applications. Among thiopeptides, lactazoles possess uniquely promiscuous biosynthetic machinery that enables the creation of diverse macrocyclic peptides. Previous bioengineering efforts have exploited this machinery to achieve the de novo design of bioactive lactazole-based thiopeptides in vitro. However, it remains unclear whether microbial systems can produce lactazole-based thiopeptides with dramatically engineered macrocycles, particularly those that are expanded and contain more than 50% amino acid divergence relative to native lactazole macrocycles. Here, we established a framework for the in vivo production of lactazole-based thiopeptides by tuning expression cassettes and selecting suitable heterologous hosts and culture conditions. Initially, we focused on transcriptional terminators in the 3′-untranslated region of the precursor gene and identified the lazA terminator as a critical determinant for lactazole production. We then evaluated several heterologous Streptomyces hosts and selected Streptomyces sp. TP-A0584 Δ godA for the production of lactazole-based thiopeptides. Under optimized conditions, including low-temperature cultivation, more than 90% of the tested lactazole-based thiopeptides were successfully produced, and a large part of them reached mg-scale production, with a maximum titer of 46.4 mg/L. Notably, although their macrocycles showed up to 73% amino acid divergence from those of native lactazoles, most of these lactazole-based thiopeptides were successfully produced in vivo. Our framework represents an initial step toward enabling the large-scale supply of lactazole-based thiopeptides and should facilitate the development of thiopeptide-based bioactive molecules.