Biology subjects

Van Deventer, J. A.

Publications and source records attributed to Van Deventer, J. A..

15 recordsLinked to original sources

bioRxiv2025

Pharmacophore-driven antibody discovery on the yeast surface

Protein-small molecule hybrids are structures capable of combining the inhibitory properties of small molecules and the specificities of binding proteins. However, discovery of such synergistic conjugates is a substantial engineering challenge. Here, we describe pharmacophore-driven antibody discovery as a high throughput approach to hybrid discovery. In this approach, we use a yeast display antibody library containing reactive noncanonical amino acids (ncAAs) and further diversify it by conjugating the library to four sulfonamide pharmacophores. Yeast display binding screens with each of the resulting billion-member hybrid collections against bovine carbonic anhydrase (bCA) yielded diverse collections of hybrids. Individual hybrids exhibited double digit nanomolar binding affinities, and frequently exhibited inhibitory properties in solution, despite the fact that the screens were based solely on binding phenotypes. Deep sequencing of sorted populations revealed that enrichments were strongly pharmacophore-dependent. In particular, screens with a potent pharmacophore led to collections of hybrids varying substantially in pharmacophore attachment point and antibody sequence features, while screens with moderate or weak pharmacophores led to collections with much narrower sets of enriched attachment points and antibody sequence features. Identification of the most frequently isolated CDR-H3 sequences and clustering CDR-H3 sequences by similarity within enriched populations provided further evidence for pharmacophore-dependent sorting outcomes. Experimental binding assays in which the pharmacophore warhead used during screening was replaced by another warhead indicated that isolated clones can tolerate alternative pharmacophores, but tend to prefer the warhead used during screening. Overall, these efforts demonstrate the utility of introducing pharmacophores into antibody libraries along with several lines of evidence that screening outcomes are pharmacophore-driven. These findings advance our understanding of hybrid discovery and highlight opportunities to pursue hybrids as research tools and potential therapeutic leads. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/682880v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11c2a65org.highwire.dtl.DTLVardef@237055org.highwire.dtl.DTLVardef@a3fb4borg.highwire.dtl.DTLVardef@1c71700_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Engineered antimicrobial-derived peptides to manipulate mixed microbial systems

Due to complexity of microbial communities and coarseness of currently available manipulation techniques (e.g. transplantation, antibiotic-treatment), it is often difficult to fully elucidate the interactions between members that define community structure and function from the top-down. Thus, it is imperative to be able to observe and manipulate subpopulations within microbial communities to enable a fine-detail understanding of the full-spectrum and mechanism of community functions. However, there is a technological gap that prevents targeted manipulation of subpopulations within intact microbial mixtures and communities. In this work, we develop molecular probes to manipulate specific subpopulations within multispecies microbial populations and validate these methods using model synthetic populations in vitro. We leverage the narrow-spectrum of class II peptide bacteriocin (a bacterially-synthesized antimicrobial peptide), pediocin PA-1, as a model to develop molecular probes. We first demonstrate the narrow-spectrum activity of pediocin and quantify its potency against a panel of bacteria. Next, we conjugate chemical handles on the bacteriocin and show that the binding spectrum is largely unchanged. Finally, using truncated variants, also conjugated to chemical handles, we show functional non-bactericidal binders that largely maintain their specificity. Finally, with the unmodified and modified bacteriocins, we show that specific bacteria can be depleted through killing or cell sorting within a mixture of highly similar bacteria. While we developed the system with a single bacteriocin, we expect that the elucidated design rules may be applicable to a variety of natural bacteriocins to develop a generalized approach for manipulating specific bacterial members within a community. Development of such molecular probes would be transformative to advancing mechanistic underpinnings of microbial community and microbiota structure-function relationships.

synthetic biology↗

bioRxiv2025

High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation

Noncanonical amino acids (ncAAs) enable diversification of protein functions, but the efficiency of genetic code expansion (GCE) in eukaryotes is hindered by competition between suppressor tRNAs and release factors. Prior work has identified eukaryotic release factor 1 (eRF1) mutants that improve ncAA incorporation, suggesting that screens for improved variants may lead to further enhancements. Here, we developed a high-throughput system to screen eRF1 mutants in Saccharomyces cerevisiae where eRF1 mutants are coexpressed on a plasmid alongside genomically encoded, wild-type eRF1. This strategy enabled recovery of live cells expressing eRF1 variants that enhance ncAA incorporation, even with mutants known to severely affect cell viability in the absence of WT eRF1 expression. We prepared and screened a million-member library of randomly mutated eRF1 variants for clones exhibiting improved ncAA integration phenotypes. Deep sequencing revealed a diverse set of enriched mutations across all three major domains of eRF1. Interestingly, several enriched mutations identified here are also found in naturally occurring eRF1 homologs from species that recode canonical stop codons. When eRF1 variants were combined with yeast knockout strains also known to enhance ncAA incorporation, this resulted in further improvements to efficiency, highlighting the complementarity of release factor engineering to other GCE enhancement strategies. This work demonstrates that high-throughput engineering of the eukaryotic translational apparatus is a powerful approach to identify previously unknown solutions for enhancing ncAA incorporation, with implications for elucidating and precisely manipulating the molecular functions of essential translational machinery.

synthetic biology↗

bioRxiv2024

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

synthetic biology↗

bioRxiv2024

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/596443v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1024b0eorg.highwire.dtl.DTLVardef@18da36corg.highwire.dtl.DTLVardef@1e4397dorg.highwire.dtl.DTLVardef@7a4ad2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Systemic evaluation of protein-small molecule hybrids on the yeast surface

Protein-small molecule hybrids are structures that have the potential to combine the inhibitory properties of small molecules and the specificities of binding proteins. However, achieving such synergies is a substantial engineering challenge, with fundamental principles yet to be elucidated. Recent work has demonstrated the power of yeast display-based discovery of hybrids using a combination of fibronectin binding domains and thiol-mediated conjugations to introduce small molecule warheads. Here, we systematically study the effects of expanding the chemical diversity of these hybrids on the yeast surface, investigating a combinatorial set of fibronectins, noncanonical amino acid (ncAA) substitutions, and small molecule pharmacophores. Our results show that previously discovered thiol-fibronectin hybrids are generally tolerant of a range of ncAA substitutions and retain binding to carbonic anhydrases following click chemistry-mediated assembly of hybrids with diverse linker structures. Most surprisingly, we identified several cases where replacement of a potent acetazolamide warhead with a substantially weaker benzenesulfonamide warhead still resulted in the assembly of functional hybrids. In addition to these unexpected findings, we expanded the throughput of our system by validating a 96-well plate-based format to produce yeast-displayed hybrid conjugates in parallel. These efficient explorations of hybrid chemical diversity demonstrate that there are abundant opportunities to expand the functions of protein-small molecule hybrids and elucidate principles that dictate their efficient discovery and design.

bioengineering↗

bioRxiv2022

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Incorporation of more than one non-canonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent crosslinking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wildtype protein controls. The successful display of doubly-substituted proteins enabled the exploration of two critical applications on the yeast surface - A) antigen-binding functionality; and B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly-substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/512873v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@188f6d6org.highwire.dtl.DTLVardef@1056391org.highwire.dtl.DTLVardef@cbfa74org.highwire.dtl.DTLVardef@1a80751_HPS_FORMAT_FIGEXP M_FIG C_FIG Herein we report the establishment of dual noncanonical amino acid incorporation in yeast to support expression and site-selective labelling of doubly substituted proteins in solution and on the yeast surface.

synthetic biology↗

bioRxiv2022

Genome-wide screen for enhanced noncanonical amino acid incorporation in yeast

Numerous applications of noncanonical amino acids (ncAAs) in basic biology and therapeutic development require efficient protein biosynthesis using an expanded genetic code. However, achieving such incorporation at repurposed stop codons in cells is generally inefficient and limited by complex cellular processes that preserve the fidelity of protein synthesis. A more comprehensive understanding of the processes that contribute to ncAA incorporation would aid in the development of genomic engineering strategies for augmenting genetic code manipulation. In this work, we screened a pooled Saccharomyces cerevisiae molecular barcoded yeast knockout (YKO) collection to identify single-gene knockout strains exhibiting improved ncAA incorporation efficiency in response to the amber (TAG) stop codon. We used a series of intracellular fluorescent reporters in tandem with fluorescence activated cell sorting (FACS) to identify 55 unique candidate deletion strains. Identified genes encode for proteins that participate in diverse cellular processes; many of the genes have no known connection with protein translation. We then verified that two knockouts, yil014c-a{Delta} and alo1{Delta}, had improved incorporation efficiency using independently acquired strains possessing the knockouts. Characterizations of the activity of yil014c-a{Delta} and alo1{Delta} with additional orthogonal translation systems and ncAAs indicate that deletion of each of these genes enhances ncAA incorporation efficiency without loss of fidelity over a wide range of conditions. Our findings highlight opportunities for further modulating gene expression with genetic, genomic, and synthetic biology approaches to improve ncAA incorporation efficiency. In addition, these discoveries have the potential to enhance our fundamental understanding of protein translation. Ultimately, this study provides a foundation for future efforts to engineer cells to incorporate ncAA at greater efficiencies, which in turn will streamline the realization of applications utilizing expanded genetic codes ranging from basic biology to drug discovery. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

synthetic biology↗

bioRxiv2022

Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface

Display technologies are powerful tools for discovering antibodies and other binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of such targets because the phenotypic readout during display screens is binding. The goal of this work is to investigate approaches for discovering inhibitory antibodies in yeast display format using a well-defined series of constructs and the target matrix metalloproteinase-9 (MMP-9). Three previously reported antibodies (DX-2802, M0076 and FAPB2.3.6) were used to create model libraries that are representative of protein libraries consisting of inhibitory binders, non-inhibitory binders, and non-binding constructs. Conditions that preferentially enrich for inhibitory clones were identified for both magnetic bead-based enrichments and fluorescence-activated cell sorting (FACS). Finally, we used direct titration of yeast to estimate inhibitor IC50 values with yeast-displayed and soluble constructs and found that the IC50 obtained for DX-2802 in yeast display format (20.01 {+/-} 9.01 nM) falls within the confidence interval of IC50 the soluble scFv-Fc form of DX-2802 (17.56 {+/-} 6.16 nM). Thus, it is possible to obtain IC50 values on the yeast surface, which greatly streamlines initial characterizations of inhibitory properties. Overall, we used these well-defined constructs to identify strategies for the discovery and characterization of inhibitory clones directly in surface display format.

bioengineering↗

bioRxiv2022

Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/475408v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@606cceorg.highwire.dtl.DTLVardef@874cadorg.highwire.dtl.DTLVardef@bb2c99org.highwire.dtl.DTLVardef@6cf963_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Flow Cytometric Evaluation of Yeast-Bacterial Cell-Cell Interactions

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting (FACS) to enrich for a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic E. coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high throughput discovery of bacterial surface-binding molecules.

synthetic biology↗

bioRxiv2021

Intracellular Delivery of Antibodies for Selective Cell Signaling Interference

Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with "undruggable" targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Here, we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. To enable such investigations, we first produced a series of antibody constructs, known as scFv-Fcs, containing additional, genetically encoded negative charges located at the C-termini of the constructs. Preparing proteins with negatively charged motifs has previously been shown to enhance intracellular protein delivery with cationic lipids, but usually for the purpose of genome editing or targeted cell death. We started by generating derivatives of scFv-Fc17, an antibody construct previously reported to bind specifically to signal transducer and activator of transcription 3 (STAT3) phosphorylated at Tyr705 (pYSTAT3). We screened a small number of lipids from our combinatorial lipid library with flow cytometry and found that PBA-Q76-O16B facilitated the most efficient delivery of scFv-Fcs under the conditions tested. In HepG2 cells, we observed up to 60.5% delivery efficacy, while in a STAT3-luciferase reporter cell line up to 71.5% delivery efficacy was observed. These results demonstrated the feasibility of accessing the intracellular space with scFv-Fcs. However, we also note that no more than modest changes were observed upon changing the numbers of negative charges in these constructs during delivery. Characterization of the cytotoxicity, size, and encapsulation efficiency of scFv-Fcs with PBA-Q76-O16B revealed that the constructs were generally well-behaved, with addition of differing quantities of negative charge resulting in at most modest effects. Importantly, functional assays monitoring transcriptional activity in luciferase reporter cell lines and HepG2 cells demonstrated significant reduction of gene expression downstream of pYSTAT3 following delivery of scFv-Fc17 constructs. Together, our results establish the use of recombinantly produced antibodies to selectively interfere with cell signaling events driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously "undruggable" targets, including for potential therapeutic applications.

bioengineering↗

bioRxiv2021

Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to lack of convenient selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to or improved relative to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS led to enrichment of a strain known to support higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well-behaved in yeast and provide a convenient alternative to DPSs. In particular, SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/454837v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@25c980org.highwire.dtl.DTLVardef@cfffe9org.highwire.dtl.DTLVardef@1aa2de7org.highwire.dtl.DTLVardef@1aba9f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific or polyspecific properties is invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering. SynopsisA range of flow cytometry-based screens yielded diverse translational machinery for genetic code expansion in yeast, facilitating access to new chemistries and tunable specificity profiles. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

synthetic biology↗

bioRxiv2019

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N-and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a \"drop-in\" version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems--and protein translation apparatuses--to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/737197v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@342d13org.highwire.dtl.DTLVardef@17b6ab6org.highwire.dtl.DTLVardef@19aed21org.highwire.dtl.DTLVardef@4648b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such \"noncanonical amino acids\" (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.

synthetic biology↗