Genetic code expansion in Shewanella oneidensis MR-1 allows site-specific incorporation of bioorthogonal functional groups into a c-type Cytochrome

Lockwood, Colin, Nash, Benjamin, Newton-Payne, Simone, van Wonderen, Jessica H., Whiting, Keir, Connolly, Abigail, Sutton-Cook, Alexander, Crook, Archie, Aithal, Advait, Edwards, Marcus J., Clarke, Tom ORCID: https://orcid.org/0000-0002-6234-1914, Sachdeva, Amit and Butt, Julea N. ORCID: https://orcid.org/0000-0002-9624-5226 (2024) Genetic code expansion in Shewanella oneidensis MR-1 allows site-specific incorporation of bioorthogonal functional groups into a c-type Cytochrome. ACS Synthetic Biology. ISSN 2161-5063 (In Press)

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Abstract

Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow understanding and modulation of biological systems and engineer new biotechnology. Here we report the development of efficient methods for site-specific incorporation of structurally diverse non-canonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated biorthogonal functional groups could be used for efficient site-selective labelling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.

Item Type:	Article
Uncontrolled Keywords:	3* ,/dk/atira/pure/researchoutput/REFrank/3_
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024) Faculty of Science Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Groups > Centre for Photonics and Quantum Science Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Energy Materials Laboratory Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Depositing User:	LivePure Connector
Date Deposited:	07 Aug 2024 10:30
Last Modified:	07 Aug 2024 10:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/96167
DOI:	issn:2161-5063

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