Decoding and reprogramming fungal iterative nonribosomal peptide synthetases

Abstract

Nonribosomal peptide synthetases (NRPSs) assemble a large group of structurally and functionally diverse natural products. While the iterative catalytic mechanism of bacterial NRPSs is known, it remains unclear how fungal NRPSs create products of desired length. Here we show that fungal iterative NRPSs adopt an alternate incorporation strategy. Beauvericin and bassianolide synthetases have the same C1-A1-T1-C2-A2-MT-T2a-T2b-C3 domain organization. During catalysis, C3 and C2 take turns to incorporate the two biosynthetic precursors into the growing depsipeptide chain that swings between T1 and T2a/T2b with C3 cyclizing the chain when it reaches the full length. We reconstruct the total biosynthesis of beauvericin in vitro by reacting C2 and C3 with two SNAC-linked precursors and present a domain swapping approach to reprogramming these enzymes for peptides with altered lengths. These findings highlight the difference between bacterial and fungal NRPS mechanisms and provide a framework for the enzymatic synthesis of non-natural nonribosomal peptides.

Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering

“…Crude ethyl acetate extracts of the cell pellets were analyzed by MALDI-TOF-MS and LC-ESI-MS. Unlike previous approaches in Saccharomyces cerevisiae , 10 the GST-tagged C 1 deletion constructs (SYNΔC 1 ; ∼324 kDa) were successfully produced in E. coli co-expressing NpgA (ESI Fig. 8 † ).…”

“… 9 Recently, the linear mode has been strongly favored, stating that the growing chain is shuttling between the T 1 and T 2a/b domains which alternately switch their donor and acceptor roles. 10 For efficient macrocyclization of the full-length linear depsipeptide precursor, the N-terminal C 1 and C-terminal C 3 domain have previously been proposed to interact by forming a cavern. 9 In contrast, the C 3 domain alone was very recently found to mediate macrocyclization, 10 which is in agreement with the established function of so-called termination C (C term ) domains in fungal linear NRPSs.…”

“… 10 For efficient macrocyclization of the full-length linear depsipeptide precursor, the N-terminal C 1 and C-terminal C 3 domain have previously been proposed to interact by forming a cavern. 9 In contrast, the C 3 domain alone was very recently found to mediate macrocyclization, 10 which is in agreement with the established function of so-called termination C (C term ) domains in fungal linear NRPSs. 11 Still, various biosynthetic features of CDP synthetases are only partially understood, including mechanistic insights into how hexa- and octa-CDPs are assembled.…”

“…While we were preparing this manuscript, Yu et al proposed a linear CDP biosynthesis model, which complements our findings. 10 …”

Strategies for Engineering Natural Product Biosynthesis in Fungi

“…Chimeric enzymes have been assembled through swapping domains between multifunctional proteins to reprogram the enzymes in PKSs, iterative NRPSs, NRPS-like enzymes, and terpene synthases [58][59][60][61][62]. Entire NR-PKSs can even be deconstructed into mono-and multidomains and recombined in vitro with deconstructed domains from other systems to generate novel functional enzymes [63].…”