ABSTRACT The diversity of non-canonical amino acids (ncAAs) endows proteins with new features for a variety of biological studies and biotechnological applications. The genetic code

expansion strategy, which co-translationally incorporates ncAAs into specific sites of target proteins, has been applied in many organisms. However, there have been only few studies on

pathogens using genetic code expansion. Here, we introduce this technique into the human pathogen _Salmonella_ by incorporating _p_-azido-phenylalanine, benzoyl-phenylalanine, acetyl-lysine,

and phosphoserine into selected _Salmonella_ proteins including a microcompartment shell protein (PduA), a type III secretion effector protein (SteA), and a metabolic enzyme (malate

BIOLOGICAL INSIGHTS Article Open access 14 September 2021 A 68-CODON GENETIC CODE TO INCORPORATE FOUR DISTINCT NON-CANONICAL AMINO ACIDS ENABLED BY AUTOMATED ORTHOGONAL MRNA DESIGN Article

23 August 2021 ENGINEERING A GENOMICALLY RECODED ORGANISM WITH ONE STOP CODON Article Open access 05 February 2025 INTRODUCTION Non-canonical amino acids (ncAAs) are powerful tools for

protein studies. In the past few years, more than 150 different ncAAs have been incorporated into proteins in both prokaryotic and eukaryotic organisms using varied approaches. One of the

most powerful methods of ncAA incorporation is genetic code expansion1,2,3,4,5,6,7,8. This approach uses an orthogonal aminoacyl-tRNA synthetase (AARS)/tRNA pair, which does not cross-react

with host AARSs and tRNAs, to direct the incorporation of an ncAA at an assigned codon (usually the amber stop codon). This allows the introduction of ncAAs with novel functional groups at a

precise position in a protein of interest. Scientists have already used this strategy to insert photocrosslinkers for mapping weak, transient, or pH sensitive protein interactions, to

install post-translational modifications for regulating biological processes or identifying modifying enzymes, to incorporate photo-caged amino acids for controlling signaling and channeling

by light, and to introduce biophysical probes and labels for providing exquisite insights into protein dynamics. These approaches have been combined with imaging systems, single-molecule

studies, biophysical techniques, structural biology, and mass spectrometry to answer biological questions that are difficult or impossible to address by most classical

methods9,10,11,12,13,14. As the key components of genetic code expansion, a number of orthogonal AARS/tRNA pairs have been developed. In _Escherichia coli_, three pairs have been most

successful: (1) an evolved orthogonal pair based on the _Methanocaldococcus jannaschii_ tyrosyl-tRNA synthetase (_mj_TyrRS) and its cognate tRNA, which has been used to install a diverse

array of tyrosine and phenylalanine derivatives15; (2) a natural orthogonal pair of pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from _Methanosarcinaceae_ species, which

facilitates the incorporation of a variety of lysine and phenylalanine analogs16,17; and (3) the phosphoseryl-tRNA synthetase (SepRS) and its cognate tRNA from methanogenic archaea, which

were engineered for phosphoserine incorporation18,19. _Salmonella_ infects both human and animals, causing millions of illnesses every year. It is also an important model organism for

gastrointestinal and extraintestinal pathogenesis studies. Due to its significance in public health, we sought to expand the genetic code in _Salmonella_ with a variety of ncAAs, increasing

the diversity of approaches that can be used in _Salmonella_ studies. We utilized the _mj_TyrRS, PylRS, and SepRS systems to incorporate _p_-azido-phenylalanine, benzoyl-phenylalanine,

acetyl-lysine, and phosphoserine in _Salmonella_, individually. For validation, we incorporated these ncAAs into _Salmonella_ native proteins including a microcompartment shell protein, a

type III secretion effector protein, and a TCA cycle enzyme respectively to demonstrate practical applications of genetic code expansion in protein labeling, photocrosslinking, and

post-translational modification studies in _Salmonella_. RESULTS EXPRESSION OF TARGET PROTEINS AND EFFICIENCY OF ORTHOGONAL PAIRS IN _SALMONELLA_ First, we compared the expression of target

proteins from different vectors in _Salmonella_. We used superfolder green fluorescent protein (sfGFP) as a reporter, which was engineered to have improved tolerance of circular permutation,

greater resistance to chemical denaturants, and enhanced folding kinetics20, and put its gene in either a modified pBAD vector with the arabinose promoter or a modified pET vector with the

_lac_ promoter. For using the arabinose promoter, we removed the _araBAD_ gene cluster which encodes arabinose metabolic enzymes from the genome. Although the pET vector had higher

expression of sfGFP, the background of the pBAD vector was much lower (Fig. 1a). Furthermore, the expression of sfGFP in the pBAD vector can be modulated by the concentration of arabinose in

the media (Supplementary Fig. S1). So we chose the pBAD vector to express target proteins in later experiments. To allow varied applications, we chose four orthogonal pairs of AARS/tRNA

that are widely used in _E. coli_: (1) a recently evolved _mj_TyrRS variant which is highly specific for _p_-azido-phenylalanine (pAzF) incorporation21; (2) an optimized _mj_TyrRS variant

for incorporating a photocrosslinker, benzoyl-phenylalanine (Bpa); (3) an optimized PylRS system for the introduction of an important post-translational modification, acetyl-lysine (AcK)22;

and 4) an optimized SepRS system for the incorporation of phosphoserine (Sep), which is another major post-translational modification23. There are considerable genetic differences between

_E. coli_ and _Salmonella_ which may affect the incorporation efficiency of ncAAs in _Salmonella_ by these orthogonal pairs which were developed in _E. coli_. To modify these pairs for use

in _Salmonella_, we first optimized their expression. In _E. coli_, the genes of orthogonal AARSs and tRNAs are constructed with the _lpp_ promoter before AARS genes and the _ProK_ promoter

before tRNA genes22. Sequence alignments showed that the _lpp_ promoter in _Salmonella_ is highly similar to that in _E. coli,_ but the _ProK_ promoters are quite different between these two

organisms (Supplementary Fig. S2). Thus, we compared the effect of tRNA promoters on the incorporation of pAzF by using a sfGFP-based fluorescence assay24. By site-directed mutagenesis, a

stop codon (UAG) was introduced at position 151 of the sfGFP gene which was shown to be permissive at that site22,24,25. Higher pAzF incorporation results in better readthrough of the stop

codon thus generating higher fluorescence. The results showed that the _Salmonella ProK_ promoter (_Sal-ProK_) gave the highest ncAA incorporation (Fig. 1b). A similar approach was applied

to test the effect of AARS promoters, and the results showed the _Salmonella lpp_ promoter (_Sal-lpp_) had the best efficiency (Fig. 1c). To further optimize AARS and tRNA expression based

on varying promoter strength, we made a matrix of 16 combinations of four promoters for tRNA expression and four promoters for AARS expression. The results also showed that the combination

of _Sal-lpp_ for AARS expression and _Sal-ProK_ for tRNA expression was the best pair (Supplementary Table S1). Thus, we put the AARS genes after _Sal-lpp_ promoter and the tRNA genes after

the _Sal-ProK_ promoter. With the same fluorescence assay, we tested the incorporation of the other three ncAAs mentioned above. Promisingly, the incorporation efficiency of pAzF, Bpa, and

AcK was about 30% in average compared to the expression of the wild-type sfGFP (Fig. 1d). The incorporation of Sep was relatively low at ~15%, similar to that in _E. coli_, but it could

still produce ~50 mg protein in 1 L media which was sufficient for further studies. Finally, all the ncAA incorporation was confirmed with mass spectrometry (MS) analyses (Supplementary Fig.

S3). INCORPORATION OF _P_-AZIDO-PHENYLALANINE (PAZF) AS A HANDLE FOR PROTEIN LABELING BY CLICK CHEMISTRY Currently, the most popular tags to label proteins for imaging purposes are

fluorescent proteins (FPs)26. However, there are also disadvantages of this method. First, the relatively large size of FPs may perturb the structures and functions of target proteins.

Second, FP fusions are limited to the N- or C- termini of target proteins. Moreover, FPs need oxygen to produce fluorescence. Here, we applied a protein labeling method based on the

incorporation of pAzF, which is then reacted with an alkyne-fluorescent dye by the click reaction to produce fluorescence without oxygen. Moreover, due to its small size, pAzF can be

incorporated at essentially any site within a protein by using a UAG codon and the optimized pAzF incorporation system21. As for the target protein for validation, we chose a native

_Salmonella_ protein, PduA, one of the major shell proteins of 1,2-propanediol utilization (Pdu) microcompartments (MCPs) which are large multi-protein complexes, functioning as organelles

for 1,2-propanediol degradation27. Based on the crystal structure of PduA28, residue N67 which is at the outer surface of Pdu MCPs was selected for pAzF incorporation. First, we inserted an

amber stop codon (UAG) at the position 67 of PduA and overexpressed PduA from the pBAD plasmid. The purified pAzF-containing PduA was labeled by a fluorescent dye with an alkyne group

through click chemistry29. With a 5-minute reaction, the pAzF-containing PduA protein gave a clear and bright green band, while the wild-type PduA protein had a very low background (Fig.

2a). Then, we used the same approach to label Pdu MCPs in living cells of _Salmonella_. We mutated the position 67 of PduA gene to an amber stop codon in the genome, expressed the entire

_pdu_ operon to produce pAzF-containing Pdu MCPs, which were then purified by previous protocols30, and labeled with fluorescent dyes. After washing away excess fluorescent dyes, only

pAzF-containing MCPs had fluorescence (Fig. 2b). The SDS-PAGE gel also indicated the specific labeling of PduA in the whole Pdu MCP protein profile (Supplementary Fig. S4). The pAzF

incorporation in PduA was confirmed by LC-MS/MS analysis (Supplementary Fig. S5). INCORPORATION OF BENZOYL-PHENYLALANINE (BPA) FOR PHOTOCROSSLINKING Understanding how proteins interact is

one of the most common questions to be solved for any biological processes. However, it can be difficult when the interactions are weak, transient, pH-dependent, or when the interactions are

at particular subcellular locations such as membranes. The covalent nature of photocrosslinking enables detection of low-affinity interactions. Moreover, it can be used in living cells to

identify specific, direct protein-protein interactions31. Benzoyl-l-phenylalanine (Bpa) is a widely used photocrosslinker excited by UV at the wavelength of 365 nm. Here, we utilized an

optimized _mj_TyrRS variant32,33 to incorporate Bpa into proteins in _Salmonella_. As for the target protein for validation, we chose a native _Salmonella_ protein, SteA, a type III

secretion effector which contributes to the control of membrane dynamics of _Salmonella_-containing vacuoles34, globally affecting host cell proliferation, morphology, adhesion and

migration35. For minimal perturbation to the protein structure, we selected two tyrosine residues at positions 40 and 155 for Bpa incorporation, individually. First, we mutated these two

positions to the amber stop codon separately, and overexpressed SteA from the pBAD plasmid. Then, we exposed the purified Bpa-containing SteA proteins to the long wavelength 365 nm UV light

and analyzed the products by SDS-PAGE. Interestingly, the SteA (155-Bpa) formed a covalent dimer, while the SteA (40-Bpa) showed no crosslinking (Fig. 3). This indicated that residue Tyr155

may be at the dimer interface, so the SteA (155-Bpa) variant may affect further experiments to identify SteA-interacting proteins _in vivo_, while SteA (40-Bpa) variant should be ideal for

this purpose. The Bpa incorporation in SteA was confirmed by LC-MS/MS analyses (Supplementary Fig. S5). INCORPORATION OF ACETYL-LYSINE (ACK) AND PHOSPHOSERINE (SEP) FOR STUDYING PROTEIN

POST-TRANSLATIONAL MODIFICATIONS Acetylation and phosphorylation are two of the most common post-translational modifications in natue36,37. Previous studies have shown that lysine

acetylation regulates the functions of many proteins in _Salmonella_38,39,40. Although proteomic analyses revealed that more than 20% of proteins in bacteria are modified by acetylation or

phosphorylation41,42, limited validation has been done to confirm these modifications. One of the challenges is that it is difficult to synthesize proteins that are fully modified at desired

positions by most classical methods. Therefore, we applied the genetic code expansion strategy to directly incorporate AcK and Sep site-specifically into proteins in _Salmonella_ to

overcome such challenge. As for the target protein for validation, we chose a native _Salmonella_ protein, malate dehydrogenase (MDH), an important metabolic enzyme in the TCA cycle43.

Previous studies have shown that the acetylation of lysine residues in mammalian MDHs increases their enzyme activities, and is involved in the cross-talk between adipogenesis and the

intracellular energy level44,45. It has also been shown that bacterial MDHs are subject to both lysine acetylation and serine phosphorylation38,46. We selected residue Lys140 for AcK

incorporation based on the proteomic studies38, and residue Ser280 for Sep incorporation, as its corresponding position in _E. coli_ MDH (95% sequence identity) was shown to be

phosphorylated46. We mutated these two positions to amber stop codons separately, and overexpressed the MDH from pBAD plasmids (Fig. 4a). Enzyme assays of the purified MDH proteins showed

that acetylation of lysine140 increased enzyme activity, while the phosphorylation of Ser280 inactivated the enzyme (Fig. 4b), indicating that _Salmonella_ cells could use different

modifications to activate or inactivate the MDH enzyme to control the flux in the TAC cycle. The AcK and Sep incorporation in MDH was confirmed by LC-MS/MS analyses (Supplementary Fig. S5).

DISCUSSION In this work, we used _mj_TyrRS to incorporate pAzF and Bpa into two different native _Salmonella_ proteins. In other organisms, the _mj_TyrRS has been engineered to incorporate

many other tyrosine or phenylalanine analogs2 that enable a range of biochemical investigations1,2,9,10,14: (1) Acetyl-phenylalanine (ketone group), allyl-tyrosine (alkene group), and

propargyloxy-phenylalanine (alkyne group) can be modified by selective chemical reactions such as oxime condensation and click chemistry to allow site-specific protein labeling; (2)

Cyano-phenylalanine, amino-phenylalanine, iodo-phenylalanine, and fluoro-phenylalanine can be used as heavy atoms for X-ray structure determination and probes for IR and NMR; (3)

Photo-reactive amino acids such as bipyridyl-alanine, benzoyl-phenylalanine, and nitrobenzyl-tyrosine can be used as switches to control biological processes with light; (4) Varied

post-translation modifications such nitro-tyrosine and sulfo-tyrosine can be used to study functions of protein modifications. Since we have successfully introduced the _mj_TyrRS systems for

pAzF and Bpa incorporation in this study, the ncAAs mentioned above might also be utilized in _Salmonella_ for a wide range of biochemical and biophysical investigations. We also used the

PylRS system to install lysine acetylation into the MDH protein of _Salmonella_. This system has been widely used in both prokaryotic and eukaryotic organisms, and has been evolved for not

only lysine derivatives but also phenylalanine or even tryptophan analogs16,17. Other lysine modifications such as methylation and ubiquitylation have also be incorporated or generated by

the PylRS systems to form complete lysine modification profiles47,48. A previous study also showed that the PylRS system can incorporate pyrrolysine analogs as crosslinkers in

_Salmonella_49. Thus, the _mj_TyrRS and PylRS systems, open up a wide range of ncAA candidates with different sizes and properties for a variety of research objectives. Proteomic studies

indicate many proteins have both acetylation and phosphorylation modifications simultaneously. We have demonstrated the facile incorporation of lysine acetylation and serine phosphorylation

site-specifically into proteins, individually. Moreover, PylRS and its variants have low selectivity toward the tRNA anticodon, and can be used for incorporating ncAAs towards different

codons such as the opal stop codon (TGA), quadruple codons, and even sense codons17. Thus, combining two mutually orthogonal PylRS and SepRS systems with different stop codons or quadruplet

codons, could allow the production of simultaneously acetylated and phosphorylated proteins, providing a powerful tool to study the crosstalk between protein acetylation and phosphorylation.

Our established ncAA incorporation systems could facilitate a number of studies on _Salmonella_, such as tracking proteins, mapping protein-protein interaction networks, and characterizing

protein post-translational modifications. Together with this work, genetic code expansion has been successfully applied in several pathogens including _Salmonella, Shigella_, and

_Mycobacterium_49,50, suggesting that it is promising to extend this strategy to other pathogens for broader applications. Furthermore, all the ncAAs and chemicals mentioned in this work are

commercially available, so our systems could benefit many biological laboratories without the need for in house organic synthesis. METHODS GENERAL MOLECULAR BIOLOGY The amino acids in this

study were purchased from Sigma-Aldrich or ChemImpex. _E. coli_ TOP10 cells (Life Technologies) were used for general cloning. _Salmonella enterica_ Serovar Typhimurium LT2 was used as the

representative _Salmonella_ strain. Plasmids: The genes of AARSs and tRNAs were cloned by PCR from laboratory inventory and inserted into the pTech plasmid. The genes of target proteins with

C-terminal His6-tag were cloned into the pBAD plasmid with the arabinose promoter, or a modified pET plasmid with the _lac_ promoter. All the cloning experiments were performed by the

Gibson Assembly kit (New England Biolabs). The mutations of target genes were made by the QuikChange II mutagenesis kit (Agilent Life Sciences). The SDS-PAGE gel is 4–20% gradient gel

purchased from Bio-Rad. The chromosomal mutation PduA N67TAG was constructed as described previously51. DNA oligos used to construct the mutation were ordered from Integrated DNA

Technologies (Coralville, IA). The mutation was confirmed by DNA sequencing. The information of plasmids and primers was listed in Supplementary Tables S2 and S3, respectively. The primary

sequences of proteins were listed in the Supplementary information, and the locations of the ncAA substitution and the active sites of enzymes were marked as well. SFGFP READTHROUGH ASSAY

The strains harboring the genes of sfGFP as well as AARSs and tRNAs were inoculated into 2 ml LB medium. The overnight culture was diluted with fresh LB medium to an absorbance of 0.2 at 600

 nm, supplemented with ncAAs. 1 mM arabinose or 1 mM IPTG was added to induce the expression of sfGFP. 200 μL culture of each strain was transferred to a well in the 96 well plate. Cells

were shaken for 24 hours at 37 °C, with monitoring of fluorescence intensity (excitation 485 nm, emission 528 nm, bandwidths 20 nm) and optical cell density by the microplate-reader. PROTEIN

EXPRESSION AND PURIFICATION The genes of target proteins were cloned into the pBAD vector with a C-terminal His6-tag, and transformed into LT2 _ΔaraBAD_ cells together with the plasmids

harboring genes of AARSs and tRNAs for expression. The expression strain was grown on 1 L of LB medium at 37 °C to an absorbance of 0.6–0.8 at 600 nm, and protein expression was induced by

the addition of 1 mM arabinose and ncAAs (1 mM for pAzF, Bpa; 2 mM for Sep; and 5 mM for AcK). Cells were incubated at 30 °C for an additional 8 hours, and harvested by centrifugation. The

cell paste was suspended in 15 ml of 50 mM Tris (pH 7.5), 300 mM NaCl, 20 mM imidazole, and broken by sonication. The crude extract was centrifuged at 20,000 × g for 30 min at 4 °C. The

soluble fraction was loaded onto a column containing 2 ml of Ni-NTA resin (Qiagen). The column was then washed with 50 ml of 50 mM Tris (pH 7.5), 300 mM NaCl, 50 mM imidazole, and eluted

with 2 ml of 50 mM Tris (pH 7.5), 300 mM NaCl, 200 mM imidazole. MASS SPECTROSCOPY ANALYSIS The samples was loaded onto the SDS-PAGE gel. The bands with the corresponding molecular weight of

target proteins were cut and sent for MS analysis. The proteins were trypsin digested by a standard in-gel digestion protocol, and analyzed by LC-MS/MS on an LTQ Orbitrap XL (Thermo

Scientific) equipped with a nanoACQUITY UPLC system (Waters). A Symmetry C18 trap column (180 μm × 20 mm; Waters) and a nanoACQUITY UPLC column (1.7 μm, 100 μm × 250 mm, 35 °C) were used for

peptide separation. Trapping was done at 15 μL min−1, 99% buffer A (0.1% formic acid) for 1 min. Peptide separation was performed at 300 nL min−1 with buffer A and buffer B (CH3CN

containing 0.1% formic acid). The linear gradient was from 5% to 50% buffer B at 50 min, to 85% buffer B at 51 min. MS data were acquired in the Orbitrap with one microscan, and a maximum

inject time of 900 ms followed by data-dependent MS/MS acquisitions in the ion trap (through collision induced dissociation, CID). The Mascot search algorithm was used to determine the amino

acid composition at specific positions (Matrix Science, Boston, MA). PROTEIN LABELING The fluorescent labeling reagent was Click-IT® Alexa Fluor® 488 DIBO Alkyne purchased from Invitrogen.

The labeling experiment was performed by following the protocol from the manufacturer. 100 μg purified PduA protein or 1 mg purified _Salmonella_ microcompartment was used for labeling. The

labeled proteins were separated by the SDS-PAGE gel and imaged by the ChemiDocTM MP System from Bio-Rad. PHOTOCROSSLINKING The reactions were performed in a 96-well plate by using 100 μl of

proteins with the concentration of 1 mg/ml. Samples were irradiated at 365 nm by using the UV crosslinker CL-1000L (Denville Scientific). Then, samples were removed from the wells and

analyzed by the SDS-PAGE gel. MDH ACTIVITY ASSAY The assays were performed by following the instruction of the EnzyChromTM Malate Dehydrogenase Assay Kit (EMDH-100) from BioAssay Systems.

This non-radioactive, colorimetric assay is based on the reduction of the tetrazolium salt MTT in a NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption

maximum at 565 nm. The increase in absorbance at 565 nm is proportional to the enzyme activity. 100 μg purified MDH and its variants were used in the assay. ADDITIONAL INFORMATION HOW TO

CITE THIS ARTICLE: Gan, Q. _et al_. Expanding the genetic code of _Salmonella_ with non-canonical amino acids. _Sci. Rep._ 6, 39920; doi: 10.1038/srep39920 (2016). PUBLISHER'S NOTE:

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES * O’Donoghue, P., Ling, J., Wang, Y. S. & Soll, D.

Upgrading protein synthesis for synthetic biology. Nat Chem Biol 9, 594–8 (2013). PubMed  PubMed Central  Google Scholar  * Liu, C. C. & Schultz, P. G. Adding new chemistries to the

genetic code. Annu Rev Biochem 79, 413–44 (2010). CAS  PubMed  Google Scholar  * Hoesl, M. G. & Budisa, N. Recent advances in genetic code engineering in Escherichia coli. Curr Opin

Biotechnol 23, 751–7 (2012). CAS  PubMed  Google Scholar  * Antonczak, A. K., Morris, J. & Tippmann, E. M. Advances in the mechanism and understanding of site-selective noncanonical

amino acid incorporation. Curr Opin Struct Biol 21, 481–7 (2011). CAS  PubMed  Google Scholar  * Terasaka, N., Iwane, Y., Geiermann, A. S., Goto, Y. & Suga, H. Recent developments of

engineered translational machineries for the incorporation of non-canonical amino acids into polypeptides. Int J Mol Sci 16, 6513–31 (2015). CAS  PubMed  PubMed Central  Google Scholar  *

Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83, 379–408 (2014). CAS  PubMed  Google Scholar  * Lemke, E. A. The exploding genetic code.

Chembiochem 15, 1691–4 (2014). CAS  PubMed  Google Scholar  * Xie, J. & Schultz, P. G. A chemical toolkit for proteins–an expanded genetic code. Nat Rev Mol Cell Biol 7, 775–82 (2006).

CAS  PubMed  Google Scholar  * Neumann, H. Rewiring translation - Genetic code expansion and its applications. FEBS Lett 586, 2057–64 (2012). CAS  PubMed  Google Scholar  * Davis, L. &

Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nat Rev Mol Cell Biol 13, 168–82 (2012). CAS  PubMed  Google Scholar  * Yanagisawa, T., Umehara, T.,

Sakamoto, K. & Yokoyama, S. Expanded genetic code technologies for incorporating modified lysine at multiple sites. Chembiochem 15, 2181–7 (2014). CAS  PubMed  Google Scholar  * Nikic,

I. & Lemke, E. A. Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr Opin Chem Biol 28, 164–73 (2015). CAS  PubMed 

Google Scholar  * Li, X. & Liu, C. C. Biological applications of expanded genetic codes. Chembiochem 15, 2335–41 (2014). CAS  PubMed  Google Scholar  * Wang, Q., Parrish, A. R. &

Wang, L. Expanding the genetic code for biological studies. Chem Biol 16, 323–36 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Wang, L., Brock, A., Herberich, B. & Schultz, P.

G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001). ADS  CAS  PubMed  Google Scholar  * Fekner, T. & Chan, M. K. The pyrrolysine translational machinery as a

genetic-code expansion tool. Curr Opin Chem Biol 15, 387–91 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Wan, W., Tharp, J. M. & Liu, W. R. Pyrrolysyl-tRNA synthetase: an

ordinary enzyme but an outstanding genetic code expansion tool. Biochim Biophys Acta 1844, 1059–70 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Park, H. S. et al. Expanding the

genetic code of Escherichia coli with phosphoserine. Science 333, 1151–4 (2011). ADS  CAS  PubMed  PubMed Central  Google Scholar  * Rogerson, D. T. et al. Efficient genetic encoding of

phosphoserine and its nonhydrolyzable analog. Nat Chem Biol 11, 496–503 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C.

& Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24, 79–88 (2006). CAS  PubMed  Google Scholar  * Amiram, M. et al. Evolution of

translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33, 1272–1279 (2015). CAS  PubMed  PubMed Central  Google Scholar  *

Fan, C., Xiong, H., Reynolds, N. M. & Soll, D. Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids. Nucleic Acids Res 43, e156 (2015). PubMed  PubMed

Central  Google Scholar  * Lee, S. et al. A facile strategy for selective incorporation of phosphoserine into histones. Angew Chem Int Ed Engl 52, 5771–5 (2013). CAS  PubMed  PubMed Central

  Google Scholar  * Fan, C., Ho, J. M., Chirathivat, N., Soll, D. & Wang, Y. S. Exploring the substrate range of wild-type aminoacyl-tRNA synthetases. Chembiochem 15, 1805–9 (2014). CAS

  PubMed  PubMed Central  Google Scholar  * Young, D. D. et al. An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity. Biochemistry 50, 1894–900 (2011). CAS  PubMed 

Google Scholar  * Tsien, R. Y. The green fluorescent protein. Annu Rev Biochem 67, 509–44 (1998). CAS  PubMed  Google Scholar  * Bobik, T. A., Lehman, B. P. & Yeates, T. O. Bacterial

microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways. Mol Microbiol 98, 193–207 (2015). CAS  PubMed  PubMed Central  Google Scholar  *

Crowley, C. S. et al. Structural insight into the mechanisms of transport across the _Salmonella_ enterica Pdu microcompartment shell. J Biol Chem 285, 37838–46 (2010). CAS  PubMed  PubMed

Central  Google Scholar  * Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed Engl 40, 2004–2021 (2001).

CAS  PubMed  Google Scholar  * Fan, C. et al. Short N-terminal sequences package proteins into bacterial microcompartments. Proc Natl Acad Sci USA 107, 7509–14 (2010). ADS  CAS  PubMed 

PubMed Central  Google Scholar  * Pham, N. D., Parker, R. B. & Kohler, J. J. Photocrosslinking approaches to interactome mapping. Curr Opin Chem Biol 17, 90–101 (2013). CAS  PubMed 

Google Scholar  * Wang, N., Ju, T., Niu, W. & Guo, J. Fine-tuning interaction between aminoacyl-tRNA synthetase and tRNA for efficient synthesis of proteins containing unnatural amino

acids. ACS Synth Biol 4, 207–12 (2015). CAS  PubMed  Google Scholar  * Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to

the genetic code of Escherichiacoli. Proc Natl Acad Sci USA 99, 11020–4 (2002). ADS  CAS  PubMed  PubMed Central  Google Scholar  * Domingues, L., Holden, D. W. & Mota, L. J. The

_Salmonella_ effector SteA contributes to the control of membrane dynamics of _Salmonella_-containing vacuoles. Infect Immun 82, 2923–34 (2014). PubMed  PubMed Central  Google Scholar  *

Cardenal-Munoz, E., Gutierrez, G. & Ramos-Morales, F. Global impact of _Salmonella_ type III secretion effector SteA on host cells. Biochem Biophys Res Commun 449, 419–24 (2014). CAS 

PubMed  Google Scholar  * Saxholm, H. J., Pestana, A., O’Connor, L., Sattler, C. A. & Pitot, H. C. Protein acetylation. Mol Cell Biochem 46, 129–53 (1982). CAS  PubMed  Google Scholar  *

Rubin, C. S. & Rosen, O. M. Protein phosphorylation. Annu Rev Biochem 44, 831–87 (1975). CAS  PubMed  Google Scholar  * Wang, Q. et al. Acetylation of metabolic enzymes coordinates

carbon source utilization and metabolic flux. Science 327, 1004–7 (2010). ADS  CAS  PubMed  PubMed Central  Google Scholar  * Starai, V. J., Celic, I., Cole, R. N., Boeke, J. D. &

Escalante-Semerena, J. C. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298, 2390–2 (2002). ADS  CAS  PubMed  Google Scholar  * Starai, V. J.

& Escalante-Semerena, J. C. Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in _Salmonella_ enterica. J Mol Biol 340, 1005–12 (2004).

CAS  PubMed  Google Scholar  * Macek, B. & Mijakovic, I. Site-specific analysis of bacterial phosphoproteomes. Proteomics 11, 3002–11 (2011). CAS  PubMed  Google Scholar  * Bernal, V. et

al. Regulation of bacterial physiology by lysine acetylation of proteins. N Biotechnol 31, 586–95 (2014). CAS  PubMed  Google Scholar  * Musrati, R. A., Kollarova, M., Mernik, N. &

Mikulasova, D. Malate dehydrogenase: distribution, function and properties. Gen Physiol Biophys 17, 193–210 (1998). CAS  PubMed  Google Scholar  * Kim, E. Y. et al. Acetylation of malate

dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity. J Lipid Res 53, 1864–76 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Kim, E. Y., Han, B.

S., Kim, W. K., Lee, S. C. & Bae, K. H. Acceleration of adipogenic differentiation via acetylation of malate dehydrogenase 2. Biochem Biophys Res Commun 441, 77–82 (2013). CAS  PubMed 

Google Scholar  * Macek, B. et al. Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol Cell Proteomics 7, 299–307 (2008). CAS

  PubMed  Google Scholar  * Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically encoding N(epsilon)-methyl-L-lysine in recombinant histones. J Am

Chem Soc 131, 14194–5 (2009). CAS  PubMed  Google Scholar  * Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem

Int Ed Engl 48, 9184–7 (2009). CAS  PubMed  Google Scholar  * Lin, S. et al. Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens.

J Am Chem Soc 133, 20581–7 (2011). CAS  PubMed  Google Scholar  * Wang, F., Robbins, S., Guo, J., Shen, W. & Schultz, P. G. Genetic incorporation of unnatural amino acids into proteins

in Mycobacterium tuberculosis. PLoS One 5, e9354 (2010). ADS  PubMed  PubMed Central  Google Scholar  * Sinha, S. et al. Alanine scanning mutagenesis identifies an asparagine-arginine-lysine

triad essential to assembly of the shell of the Pdu microcompartment. J Mol Biol 426, 2328–45 (2014). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We

thank Professor Dieter Söll (Yale University, USA) for generous supports and suggestions. This work was financially supported by the start-up fund of University of Arkansas, the grants from

the National Institutes of Allergy and Infectious Diseases (AI119813 and AI081146), and the grant from the National Institute of General Medical Sciences (GM22854). AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, 72701, AR, USA Qinglei Gan & Chenguang Fan * Roy J. Carver Department of

Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, 50011, IA, USA Brent P. Lehman & Thomas A. Bobik Authors * Qinglei Gan View author publications You can also

search for this author inPubMed Google Scholar * Brent P. Lehman View author publications You can also search for this author inPubMed Google Scholar * Thomas A. Bobik View author

publications You can also search for this author inPubMed Google Scholar * Chenguang Fan View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

C.F. directed the studies, designed the experiments, and interpreted data. Q.G. and C.F. performed the experiments. B.P.L. and T.A.B. constructed the _Salmonella_ strains. C.F. and T.A.B.

wrote the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND

PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s

Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the

license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Gan,

Q., Lehman, B., Bobik, T. _et al._ Expanding the genetic code of _Salmonella_ with non-canonical amino acids. _Sci Rep_ 6, 39920 (2016). https://doi.org/10.1038/srep39920 Download citation

* Received: 02 September 2016 * Accepted: 29 November 2016 * Published: 23 December 2016 * DOI: https://doi.org/10.1038/srep39920 SHARE THIS ARTICLE Anyone you share the following link with

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