Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.

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Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

10.1101/371757 ◽

2018 ◽

Author(s):

Birthe Meineke ◽

Johannes Heimgärtner ◽

Lorenzo Lafranchi ◽

Simon J Elsässer

Keyword(s):

Genetic Code ◽

Cell Biology ◽

Mammalian Cells ◽

Stop Codon ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Human Gut ◽

Methanosarcina Mazei ◽

Genetic Code Expansion

ABSTRACTGenetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

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Adaptive landscape flattening allows the design of both enzyme:substrate binding and catalytic power

10.1101/771824 ◽

2019 ◽

Cited By ~ 1

Author(s):

Vaitea Opuu ◽

Giuliano Nigro ◽

Emmanuelle Schmitt ◽

Yves Mechulam ◽

Thomas Simonson

Keyword(s):

Transition State ◽

Directed Evolution ◽

Bound State ◽

Reaction Rates ◽

Trna Synthetase ◽

Adaptive Landscape ◽

Enzyme Design ◽

Computational Enzyme Design ◽

Genetic Code Expansion ◽

Catalytic Power

AbstractDesigned enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are complementary. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We extend the method to the design of an enzyme for specific transition state binding, i.e., for catalytic power. We consider methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. MetRS and other synthetases have been extensively redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. We redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered, and mutants predicted to bind MetAMP were characterized experimentally and found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We expect the present method will become the paradigm for computational enzyme design.

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Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811438115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12388-E12397 ◽

Cited By ~ 9

Author(s):

Elizabeth S. Maywood ◽

Thomas S. Elliott ◽

Andrew P. Patton ◽

Toke P. Krogager ◽

Johanna E. Chesham ◽

...

Keyword(s):

Stop Codon ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Adeno Associated Virus ◽

Biologically Relevant ◽

Negative Feedback Loops ◽

Translational Readthrough ◽

Genetic Code Expansion ◽

And Behavior ◽

Circadian Behavior

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional–translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

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Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette

Archaea ◽

10.1155/2014/374146 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 36

Author(s):

Guillaume Borrel ◽

Nadia Gaci ◽

Pierre Peyret ◽

Paul W. O'Toole ◽

Simonetta Gribaldo ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Genetic Code ◽

Stop Codon ◽

Trna Synthetase ◽

Coding System ◽

Genomic Features ◽

Close Relationship ◽

Seventh Order ◽

Genetic Code Expansion

Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known fromBacteriaandArchaeaand analyzed the phylogeny of each component. This shows unique peculiarities, notably anamber tRNAPylwith an imperfect anticodon stem and a shortenedtRNAPylsynthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.Corrigendum to “Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette”

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Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway

Nature Communications ◽

10.1038/ncomms10005 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 40

Author(s):

Wai Shun Mak ◽

Stephen Tran ◽

Ryan Marcheschi ◽

Steve Bertolani ◽

James Thompson ◽

...

Keyword(s):

Catalytic Efficiency ◽

Fold Increase ◽

Enzyme Function ◽

Enzyme Design ◽

Alcohol Production ◽

Computational Enzyme Design ◽

Total Alcohol ◽

Sequence Databases ◽

Function Discovery

Abstract The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5–C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.

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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

10.1101/2021.02.19.432053 ◽

2021 ◽

Author(s):

Devon A. Stork ◽

Georgia R. Squyres ◽

Erkin Kuru ◽

Katarzyna A. Gromek ◽

Jonathan Rittichier ◽

...

Keyword(s):

Bacillus Subtilis ◽

Genetic Code ◽

Cell Biology ◽

Stop Codon ◽

Positive Bacterium ◽

E Coli ◽

Binding Interface ◽

Genetic Code Expansion ◽

And Control

AbstractBacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Nature Communications ◽

10.1038/s41467-021-25691-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Devon A. Stork ◽

Georgia R. Squyres ◽

Erkin Kuru ◽

Katarzyna A. Gromek ◽

Jonathan Rittichier ◽

...

Keyword(s):

Bacillus Subtilis ◽

Genetic Code ◽

Cell Biology ◽

Stop Codon ◽

Positive Bacterium ◽

E Coli ◽

Binding Interface ◽

Genetic Code Expansion ◽

And Control

AbstractBacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

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Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

10.1101/759662 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer C. Peeler ◽

Rachel E. Kelemen ◽

Masahiro Abo ◽

Laura C. Edinger ◽

Jingjia Chen ◽

...

Keyword(s):

Genetic Code ◽

Mammalian Cells ◽

Stop Codon ◽

Biological Evaluation ◽

Trna Synthetase ◽

Secis Element ◽

E Coli ◽

Domains Of Life ◽

Translation Mechanism ◽

Genetic Code Expansion

ABSTRACTSelenoproteins contain the amino acid selenocysteine and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence (SECIS) element in the 3’ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element, and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

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