Efficient rational modification of non-ribosomal peptides by adenylation domain substitution

Abstract

Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis

“…The suitable recombination sites between the Cs and A domains need to be further investigated in detail for the efficient engineering of lipopeptides. The recently reported research on the modification of NRPSs by A domain alone substitution also supported that C domain specificities were not as strict as we previously thought 36 . Our results suggested that the use of full-length Cs domain as a swapping unit is a feasible way to modify lipopeptides.…”

“…Thus, the reason for the failure could be donor specificity of the downstream C domain. The relatively broad specificities of RzmA-Cs and HolA-Cs to acceptor substrates explain why the aforementioned Cs domain swapping led to the successful production of acyl-changed rhizomide, holrhizin, and glidobactin derivatives, and support the very recent study that novel nonribosomal peptides can be generated by the substitution of A domains alone 36 . Subdomain (N-lobe) swapping of GlbF-Cs with that of GlpC-Cs was also conducted, but no expected compounds were detected.…”

“…This finding also supports the Cs domain swapping strategy to change the acyl chains (Figs. 2 c, d, 3a, b ), and the NRPS module swapping strategy at the C–A linker region for the combinatorial biosynthesis of nonribosomal peptides 36 , 38 .…”

The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity

“…Therefore, selection of recombination events that maintain the structural integrity of the C–A interface appears plausible, although recent engineering studies have proofed that the C–A linker can be exploited for the reengineering of domains ( Bozhüyük et al. 2018 ; Calcott et al. 2020 ; Kaniusaite et al.…”

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

“…Given the antibiotic resistance crisis, a detailed understanding of their underlying biosynthetic mechanisms becomes increasingly important to help in facilitating the exploitation of NRPS templates by combinatorial approaches for the discovery of new compounds. [1][2][3][4][5] The structural and functional organization of NRPSs is reminiscent of an assembly line. Arrays of single domains act together in a sequential manner to synthesize the peptide product from N to C terminus under the consumption of ATP.…”