Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome

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.


Tables
Table S1: Key proteins in the maturation and secretion of MtrC with their respective stop codon.
Table S2: Strains and plasmids used in this work.
Table S3: Primers used to introduce the Amber stop codon into mtrC.
Table S4: Data collection and refinement statistics for crystallographic analysis of MtrC BocK proteins.Table S1.Key proteins in the maturation and secretion of MtrC with their respective stop codon.Amber stop codons (UAG) are highlighted in bold.

Figure S4 :
Figure S4: Deconvoluted mass spectra for proteins purified by affinity chromatography from culture of MR-1.Mj.CxxxUAG strains with arabinose and no ncAA.

Figure S6 :
Figure S6: SDS-PAGE gel images for samples of ncAA-containing MtrC proteins incubated with functionalized Cy5 dyes as indicated.

Figure
Figure S2.SDS-PAGE gel images for spent media from culture of MR-1.C, MR-1.Pyl.C and MR-1.Mj.C with arabinose (Arb), BocK and AzF as indicated.Proteins visualized by Coomassie stain.Samples correspond to those of Figure 3 in the main text.Arrow indicates the expected migration of wtMtrC.

Figure S3 .
FigureS3.Optical density (OD) at 600 nm for the indicated cultures.At OD600 nm approximately 0.4, arabinose was added to a final concentration of 5 mM and the indicated ncAA was added to a final concentration of 4 mM.

Figure S4 .
Figure S4.SDS-PAGE gel images for spent media from culture of MR-1.C, MR-1.Pyl.C and MR-1.Mj.C with arabinose (Arb), BocK and AzF as indicated.Proteins visualized with heme stain (upper) and Coomassie stain (lower).Samples correspond to those presented in Figure 4 of the main text.Arrows indicate the migration of wtMtrC.

Figure S5 .
Figure S5.Deconvoluted mass spectra for MtrC proteins.A) Proteins purified by affinity chromatography from culture of MR-1.Mj.CxxxUAG strains with arabinose and no ncAA.Intact mass values are consistent with insertion of phenylalanine at the site encoded by the amber stop codon: calculated (calc.)and observed (obs.).Thus, spectra are labelled for the corresponding protein MtrCxxxF where xxx is the residue encoded by the amber stop codon.The deconvoluted mass spectrum of wtMtrC is included for reference.B) MtrC344AlkK protein purified by affinity chromatography from a culture of MR-1.pyl.344xUAG with arabinose and alkyne lysine.The deconvoluted mass spectrum of wtMtrC is included for reference.

Figure S6 .
Figure S6.Crystallographic analysis of BocK containing MtrC proteins.(A) Pairwise alignment of the heme cofactors in MtrC293BocK (Blue), MtrC344BocK (coral) and Mtr430BocK (green) and wtMtrC (red) overlaid on the secondary structure of wtMtrC (gold).H5 indicates Heme 5 and H10 indicates Heme 10 where Hemes are numbered in order of their sites of attachment to the MtrC peptide, protein domains are labelled I-IV.(B)The 2Fo-Fc (blue) and Fo-Fc (green/red) electron density map (contoured to 1.2 and 3.5 sigma respectively) for the indicated proteins, resulting from (left) refinement of the BocK MtrC structure model against the BocK MtrC data and (right) refinement of the wtMtrC structure (PDB ID: 4LM8) against the BocK MtrC data.

Figure S7 .
Figure S7.Alpha-fold model of S. oneidensis MR-1 MtrCAB.Outer membrane spanning MtrA (purple) and MtrB (blue) with extracellular MtrC (gold).Domains to IV of MtrC are indicated, hemes are red and the C carbons of residues 293, 344 and 430 are shown as blue, salmon and green spheres, respectively.

Figure S8 .
Figure S8.Introduction of fluorescent probes to AzF-containing MtrC proteins using bioorthogonal chemistry.SDS-PAGE gel images for the reaction products from incubation of dibenzocyclooctyne sulfo-cyanine 5 (DBCO-Cy5) with (A) MtrC293AzF (B) MtrC344AzF and MtrC344Phe.Gels imaged by protein stain, heme stain or fluorescence emission (excitation at 635 nm) as indicated.Masked lanes carry samples not relevant to this study.Reaction time = 18 hours.

Figure S9 .
Figure S9.SDS-PAGE gel images for of ncAA-containing MtrC proteins incubated with functionalized Cy5 dyes as indicated.Top: Cy5 dye visualized by fluorescence emission (excitation at 635 nm).Bottom: proteins visualized by Coomassie stain.Arrows indicate the expected migration of MtrC proteins.

Figure S10 .
Figure S10.Spectroscopic analysis of ncAA containing MtrC proteins.The electronic absorbance spectrum of oxidized (air equilibrated) wtMtrC compared to that of the BocK, AzF and Alk containing proteins as indicated.Samples MtrC (0.5 to 0.8 µM) in 100 mM Tris-HCl, 150 mM NaCl, pH 8.5.

Table S2 .
Table S1 continued.Strains and plasmids used in this work.

Table S4 .
Data collection and refinement statistics for crystallographic analysis of MtrC BocK Proteins (with the corresponding PDB accession code).
*Values in parentheses are for highest-resolution shell.