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Lockwood, Colin W. J., Nash, Benjamin W., Newton-Payne, Simone E., van Wonderen, Jessica H., Whiting, Keir P. S., Connolly, Abigail, Sutton-Cook, Alexander L., Crook, Archie, Aithal, Advait R., Edwards, Marcus J., Clarke, Thomas A. 
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, 13 (9).
pp. 2833-2843.
ISSN 2161-5063
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Abstract
Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the 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 noncanonical 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 the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the 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 bioorthogonal functional groups could be used for efficient site-selective labeling 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 |
|---|---|
| Additional Information: | Data Availability Statement: The BocK-MtrC structures and the associated structure factors are deposited in the Protein Data Bank under the access codes 8QC9 for BocK at 239, 8QBZ for BocK at 344, and 8QBQ for BocK at 430. Data sets used to make figures are deposited at Figshare (DOI: 10.6084/m9.figshare.25491769). Funding information: The work was funded by a Leverhulme Trust Research Project Grant (RPG-2020-085) and the UKRI Biotechnology and Biological Sciences Research Council (Grant no. BB/S002499/1). B.W.N., A.L.S.-C., and A.R.A. were funded by the UKRI Biotechnology and Biological Sciences Research Council Norwich Research Park Biosciences Doctoral Training Partnership (Grant no. BB/T008717/1). A.C. was funded by a Summer Intern Scholarship from the School of Chemistry, University of East Anglia. Rights Retention Statement: For the purpose of open access, the authors have applied a CC BY public copyright license to any author-accepted manuscript version that arises. |
| Uncontrolled Keywords: | amber suppression,cytochrome,extracellular electron transfer,genetic code expansion,shewanella,biochemistry, genetics and molecular biology (miscellaneous),biomedical engineering,3* ,/dk/atira/pure/subjectarea/asjc/1300/1301 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology 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 |
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| Depositing User: | LivePure Connector |
| Date Deposited: | 07 Aug 2024 10:30 |
| Last Modified: | 13 Nov 2024 00:52 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96167 |
| DOI: | 10.1021/acssynbio.4c00248 |
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