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Reducing the Genetic Code Induces Massive Rearrangement of the Proteome

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Reducing the genetic code induces massive rearrangement of the proteome Patrick O’Donoghuea,b, Laure Pratc, Martin Kucklickd, Johannes G. Schäferc, Katharina Riedele, Jesse Rinehartf,g, Dieter Söllc,h,1, and Ilka U. Heinemanna,1 Departments of aBiochemistry and bChemistry, The University of Western Ontario, London, ON N6A 5C1, Canada; Departments of cMolecular Biophysics and Biochemistry, fCellular and Molecular Physiology, and hChemistry, and gSystems Biology Institute, Yale University, New Haven, CT 06520; dDepartment of Microbiology, Technical University of Braunschweig, Braunschweig 38106, Germany; and eDivision of Microbial Physiology and Molecular Biology, University of Greifswald, Greifswald 17487, Germany Contributed by Dieter Söll, October 22, 2014 (sent for review September 29, 2014; reviewed by John A. Leigh) Expanding the genetic code is an important aim of synthetic Opening codons by reducing the genetic code is highly biology, but some organisms developed naturally expanded ge- promising, but it is unknown how removing 1 amino acid from netic codes long ago over the course of evolution. Less than 1% of the genetic code might impact the proteome or cellular viability. all sequenced genomes encode an operon that reassigns the stop Many genetic code variations are found in nature (15), including codon UAG to pyrrolysine (Pyl), a genetic code variant that results stop or sense codon reassignments, codon recoding, and natural from the biosynthesis of Pyl-tRNAPyl. To understand the selective code expansion (16). Pyrrolysine (Pyl) is a rare example of nat- advantage of genetically encoding more than 20 amino acids, we ural genetic code expansion. Evidence for genetically encoded constructed a markerless tRNAPyl deletion strain of Methanosarcina Pyl is found in <1% of all sequenced genomes (17). In these acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on organisms, Pyl is encoded by the UAG codon, which requires trimethylamine. Phenotypic defects in the ΔpylT strain were evi- tRNAPyl, pyrrolysyl-tRNA synthetase (PylRS), and the products dent in minimal medium containing methanol. Proteomic analyses of three genes (pylBCD) that synthesize Pyl from two molecules of wild type (WT) M. acetivorans and ΔpylT cells identified 841 of lysine (18). The PylRS enzyme was engineered to genetically proteins from >7,000 significant peptides detected by MS/MS. Pro- encode >100 ncAAs (19). The Pyl encoding system has already tein production from UAG-containing mRNAs was verified for 19 been used to expand the genetic codes of Escherichia coli (20– EVOLUTION proteins. Translation of UAG codons was verified by MS/MS for 22), mammalian cells, and animals (23). eight proteins, including identification of a Pyl residue in PylB, Despite the use of Pyl in synthetic biology, little is known which catalyzes the first step of Pyl biosynthesis. Deletion of about the role of Pyl in its native environment or the evolu- tRNAPyl globally altered the proteome, leading to >300 differen- tionary pressures that sustain expanded genetic codes in nature. tially abundant proteins. Reduction of the genetic code from 21 The Pyl-decoding trait is found in methanogenic archaea of the to 20 amino acids led to significant down-regulation in translation orders Methanosarcinales and Methanomassiliicoccales (24) and initiation factors, amino acid metabolism, and methanogenesis certain anaerobic bacteria (17). In addition to producing 74% of from methanol, which was offset by a compensatory (100-fold) global methane emissions, methanogens are remarkable for their up-regulation in dimethyl sulfide metabolic enzymes. The data ability to survive with only the most basic carbon and energy show how a natural proteome adapts to genetic code reduction sources (25). Methanosarcina shows the greatest substrate range and indicate that the selective value of an expanded genetic code among methanogens and survives on acetate, carbon monoxide, is related to carbon source range and metabolic efficiency. methylamines, methanol, or dimethyl sulfide (DMS). Their broad substrate range depends, in part, on the presence of Pyl in the evolution | genetic code expansion | methanogenesis | pyrrolysine | active site of several methylamine methyltransferases (26). Hundreds tRNAPyl of Methanosracina genes contain in-frame TAG codons (27), but natural Pyl incorporation was only shown in methylamine methyl- ynthesizing whole genomes (1) and eliminating codons (2) transferases (17, 28) and tRNAHis guanylyltransferase (Thg1) (29). Sare novel methods for rewriting the genetic code that may dramatically alter the repertoire of genetically encoded amino Significance acids. Expansion of the genetic code has led to exciting tech- nologies, including site-directed protein labeling and production Expanding the genetic code is an important aim of synthetic of proteins with hardwired posttranslational modifications (3). biology, but some organisms developed naturally expanded The current approaches to cotranslationally insert noncanonical genetic codes over the course of evolution. To understand the amino acids (ncAAs) into proteins rely on the reassigning of one selective advantage of genetically encoding more than 20 of three stop codons (4). amino acids, we investigated the proteome-wide response to Although these approaches were highly successful in incor- reducing the genetic code of Methanosarcina acetivorans from porating over 100 ncAAs into proteins (3), they limit the ex- 21 to 20 amino acids. The data show how a natural proteome pansion of the code to no more than 2 additional amino acids at adapts to genetic code reduction and indicate that the selective a time and significantly challenge the cellular production host by value of an expanded genetic code is related to carbon source unnaturally extending proteins and reducing growth rate (5). Al- range and metabolic efficiency. ternate methods focus on quadruplet codons (6, 7) and recoding – (8) or reassigning sense codons (9 13). Attempts to reassign Author contributions: P.O., L.P., K.R., J.R., D.S., and I.U.H. designed research; P.O., M.K., asensecodoninMycoplasma capricolum were defied by tRNA J.G.S., K.R., J.R., and I.U.H. performed research; P.O., D.S., and I.U.H. analyzed data; and misacylation by endogenous aminoacyl-tRNA synthetases (9). P.O., D.S., and I.U.H. wrote the paper. This result indicates that, although extensively rewriting the ge- Reviewers included: J.A.L., University of Washington. netic code may be possible, it comes with unexpected challenges The authors declare no conflict of interest. related to cellular fitness and translation fidelity. These consid- 1To whom correspondence may be addressed. Email: [email protected] or ilka. erations will impact efforts to engineer cells to synthesize proteins [email protected]. with multiple ncAAs or create biologically contained strains that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. require an expanded code for survival (14). 1073/pnas.1420193111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1420193111 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Methanosarcina acetivorans provides an ideal model system to Table 1. Growth statistics for M. acetivorans strains identify Pyl-containing proteins and study the impact of genetic Strain Carbon source Doubling time (h) Maximum A Lag time (h) code reduction on the proteome and physiology of the cell. We 578 constructed a markerless tRNAPyl deletion (ΔpylT)strainof WT MeOH 5.9 ± 0.7 1.02 ± 0.01 39.9 ± 1.6 M. acetivorans C2A and used three independent mass spectrom- ΔpylT MeOH 7.9 ± 0.5 0.99 ± 0.02 45.2 ± 1.5 etry (MS) approaches to characterize soluble proteomes from WT TMA 7.5 ± 0.3 1.06 ± 0.40 52.4 ± 0.9 M. acetivorans grown on minimal medium containing trimethyl- amine (TMA) or methanol and ΔpylT cells grown on methanol. The data reveal previously unidentified biochemical roles for Pyl From 21 to 20—Proteome Adaptation to Genetic Code Reduction. To and Pyl-containing proteins and indicate that the expanded genetic better understand the nature of the selective value of Pyl, we code of Methanosarcina is intricately linked with cellular metabo- characterized the soluble proteomes of WT and ΔpylT strains. Of lism and the composition of the proteome. 4,721 potential protein coding genes in M. acetivorans, 841 pro- teins were identified, including ∼300 proteins identified by gel- Results based methods; the liquid chromatography (LC) -MS/MS M. acetivorans with a Reduced Genetic Code. There are 267 ORFs approach identified 583 proteins. Proteins were considered iden- in the M. acetivorans genome with one or multiple in-frame tified if two or more significant peptides (peptide score > 35) were UAG codon(s) (Figs. S1 and S2 and Table S1). Except for Thg1 detected and verified by MS/MS spectra. All peptides identified by and the methylamine methyltransferase (mtxB), it is unknown if LC-MS/MS are listed in Dataset S1. these ORFs are expressed or the resulting protein contains Pyl. The ΔpylT strain has a globally altered proteome (Fig. 2). We To uncover more Pyl-containing proteins and investigate the role identified 347 differentially regulated proteins showing more of Pyl in the M. acetivorans proteome, we constructed and than twofold change (Tables S2 and S3), most of which are Pyl characterized a tRNA deletion strain of M. acetivorans C2A proteins that do not contain Pyl. The most affected pathways (Fig. 1). We monitored the growth rate of three independently
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