Shinta Ijichi

Abstract Nonribosomal peptide synthetases (NRPSs) are modular assembly-line enzymes that generate structurally diverse and biologically active natural products. Although adenylation (A) domain selectivity has been extensively characterized and engineered, the specificity of condensation (C) domains, which catalyze peptide bond formation and can constrain NRPS reprogramming, remains less well understood. Here, we report a systematic functional analysis of VibH, a stand-alone C domain VibH from the vibriobactin biosynthetic pathway. By integrating VibH with wild-type and engineered variants of the upstream aryl acid A domain EntE, we bypassed intrinsic A-domain constraints and independently interrogated donor- and acceptor-site substrate tolerance. The donor site of VibH exhibited stringent specificity, accepting only mono- and disubstituted benzoic acid derivatives closely resembling 2,3-dihydroxybenzoic acid (DHB), whereas bulkier aryl substrates were not processed. In contrast, the acceptor site displayed broad tolerance toward structurally diverse amines. Monoamines showed clear chain-length dependence, with productive turnover restricted to medium-chain substrates, whereas diamines were more broadly accepted. Systematic analysis further revealed distance-dependent steric tolerance, in which bulky substituents were accommodated when positioned distal to the reactive amine but were restricted beyond an upper steric threshold. This engineered reconstitution strategy enabled the synthesis of numerous non-native amide conjugates and uncovered an asymmetric substrate-recognition architecture characterized by a stringent donor site and a permissive yet spatially constrained acceptor site. Molecular docking analysis provided a structural rationale for these trends by suggesting distinct binding modes for native and non-native donor substrates and distance-dependent accommodation of bulky amine acceptors. These findings provide mechanistic insights into C-domain specificity and establish a framework for rational NRPS reprogramming.

Abstract 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 ΔgodA 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.