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

Mark J. Calcott, Jeremy G. Owen and David F. Ackerley ()
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Mark J. Calcott: Victoria University of Wellington
Jeremy G. Owen: Victoria University of Wellington
David F. Ackerley: Victoria University of Wellington

Nature Communications, 2020, vol. 11, issue 1, 1-10

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-18365-0

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