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A High-throughput Approach to NRPS Domain Substitution for Generation of Novel Peptides

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posted on 2023-05-08, 02:41 authored by Sarah Messenger

Many small, medically significant peptides are produced by non-ribosomal peptide synthetases (NRPSs). NRPSs are large modular enzymes that function as an assembly-line with each module adding one amino acid subunit to the final product. Each module is comprised of domains which  perform the functions of substrate specification, peptide bond  formation; and substrate loading and transfer. Novel peptides can be produced by modifying the NRPS template to enable a different amino acid to be incorporated into the peptide product. However, previous efforts at engineering these enzymes have been inefficient and not scalable, frequently resulting in non-functional proteins.


This study focussed on development of a powerful high-throughput method for the targeted mass modification of NRPS templates. A pyoverdine synthesising NRPS from Pseudomonas aeruginosa PAO1 was used as a model  system. Pyoverdine is a fluorescent green siderophore essential for growth on iron-restricted media thus it is easy to detect and quantify. Engineering efforts were targeted to the second module of PvdD, the final NRPS in the pyoverdine biosynthetic assembly-line.


To generate diversity, domains were amplified with degenerate primers from uncharacterised, highly diverse metagenomic DNA libraries and used for mass cloning into pvdD to create libraries of random variants. These libraries were then screened for the production of pyoverdine analogues and characterised using mass spectrometry. Two libraries were created in this manner and screened, leading to the discovery of four novel pyoverdines.


This is the first attempt to engineer NRPS enzymes in a high-throughput manner and shows promise for future engineering of NRPS systems. This approach could pave the way for creation of new pharmaceuticals and revolutionise current methods of NRPS engineering. 

History

Copyright Date

2019-01-01

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains All Rights

Degree Discipline

Biotechnology

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Drug Discovery and Development

ANZSRC Socio-Economic Outcome code

970106 Expanding Knowledge in the Biological Sciences

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Ackerley, David