Genetic Code Expansion\nin Shewanella\noneidensis MR‑1 Allows Site-Specific Incorporation\nof Bioorthogonal Functional Groups into a <i>c</i>‑Type\nCytochrome

Genetic code expansion has enabled cellular synthesis\nof proteins\ncontaining unique chemical functional groups to allow the understanding\nand modulation of biological systems and engineer new biotechnology.\nHere, we report the development of efficient methods for site-specific\nincorporation of structurally diverse noncanonical amino acids (ncAAs)\ninto proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the\nbiosynthetic machinery for ncAA incorporation is compatible and orthogonal\nto the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of <i>c</i>-type\ncytochromes, and protein secretion. This allowed the efficient synthesis\nof a <i>c</i>-type cytochrome, MtrC, containing site-specifically\nincorporated ncAA in S. oneidensis MR-1\ncells. We demonstrate that site-specific replacement of surface residues\nin MtrC with ncAAs does not influence its three-dimensional structure\nand redox properties. We also demonstrate that site-specifically incorporated\nbioorthogonal functional groups could be used for efficient site-selective\nlabeling of MtrC with fluorophores. These synthetic biology developments\npave the way to expand the chemical repertoire of designer proteins\nexpressed in S. oneidensis MR-1.