In Vivo Biocombinatorial Synthesis of Lipopeptides by COM Domain-Mediated Reprogramming of the Surfactin Biosynthetic Complex

Abstract

The intermolecular communication within NRPS complexes relies on the coordinated interplay of donor and acceptor communication-mediating (COM) domains. In this study, the potential of COM domains was exploited in vivo by establishing a system for the true biocombinatorial synthesis of lipopeptides via directed reprogramming of a natural NRP biosynthetic assembly line (i.e., surfactin). By means of COM domain swapping, these experiments verified the decisive role of COM domains for the control of protein-protein interactions between NRPSs, demonstrated the functionality of COM domain pairs even in the context of a heterologous host and NRPS system, and allowed for the intended skipping of a biosynthetic enzyme within a multienzymatic biosynthetic complex. Ultimately, abrogation of the selectivity barrier provided by COM domains afforded the successful simultaneous, biocombinatorial synthesis of distinct lipopeptide products.

Characterization of the Complete Zwittermicin A Biosynthesis Gene Cluster fromBacillus cereus

“…This strongly suggests the N terminus of ZmA initially contained a fatty acid, along with D-Asn. Finally, Chiocchini and colleagues and Hahn and Stachelhaus have shown that E domains and C domains that interact with each other contain communication domains that function as sites of specific protein-protein interactions between E and C domains on different NRPS polypeptides (7,15,16). Analysis of the C terminus of the E domain of ZmaO and the N terminus of ZmaK identified sequences consistent with communication domains (data not shown).…”

Surfactin and Other Lipopeptides from Bacillus spp.

“…Chiocchini et al (2006) show that a heterologous COM domain pair can replace the native COM domain pair. Indeed, the replacement of COM…”

“…Chemoenzymatic approaches were used to characterise the specificity of the thioesterase domain. Different heptapeptides were synthesised by chemical means, and a thioesterase domain was cloned and produced as an isolated enzyme (Chiocchini et al 2006).…”

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

“…The modular organization of protein domains in NRPSs has stimulated efforts to swap genes encoding heterologous domains into NRPSs with the goal of producing nonnatural variants of the original small-molecule product (3,4). Although limited success has been achieved with the production of nonribosomal peptide (NRP) variants (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), the resultant chimeric NRPSs are usually nonfunctional or heavily impaired, often yielding too little of the new product to make production feasible. It is not yet known why chimeric NRPSs suffer large reductions in activity, although it is likely that domain swapping disrupts quaternary interactions between protein domains in the assembly line (15).…”