Biology subjects

Kunjapur, A. M.

Publications and source records attributed to Kunjapur, A. M..

18 recordsLinked to original sources

bioRxiv2026

Air-driven aldehyde synthesis in engineered bacteria via gene deletion and aryl-alcohol oxidase profiling

Engineered bacterial routes for oxidation of non-native alcohols face three challenges: Nicotinamide-dependent enzymes are coupled to cellular redox metabolism, nicotinamide-independent aryl-alcohol oxidases (AAOs) usually express poorly in bacteria, and aldehyde products are rapidly modified by host enzymes. Here, we address these limitations by engineering aldehyde-retaining Escherichia coli for discovery and application of soluble bacterial AAOs. Screening 51 candidates revealed a high-expression sequence cluster containing enzymes that are active on diverse aromatic and furan-based alcohols. Pairing the top-performing AAO with designer pathways in aldehyde-retaining cells enabled modular C-N and C-C bond forming cascades starting from supplied alcohols. By making both the oxidase and its product compatible with the host, this work advances air-driven oxidation of diverse alcohols as a programmable entry point to aldehyde-derived chemistry in engineered bacteria.

synthetic biology↗

bioRxiv2026

Genetic code expansion enables plant-directed control of bacterial activity

Programmable control of microbial gene expression by plant hosts could enable a new generation of precision agricultural biotechnology. Here, using O-methyl-L-tyrosine (OMY) as a model compound, we establish non-canonical amino acids (ncAAs) as a tool for plant-based control of associated microbial activity. We use genetic code expansion to engineer OMY-dependent control of protein synthesis in the soil bacterium Bacillus subtilis. Then, we engineer agronomically diverse plants, including Arabidopsis, tomato and poplar, to biosynthesize OMY. We show that plant-derived OMY can stimulate gene expression in both model and wild soil bacteria while also demonstrating that inducible and tissue-specific expression of a single biosynthetic enzyme by the plant enables on-demand control over microbial activity. This work establishes ncAAs as a tool for programming plant-microbe partnerships.

synthetic biology↗

bioRxiv2025

Robust cellular transformations of PET deconstruction products by import of glycol esters

Efforts to transform polyethylene terephthalate (PET) deconstruction products using live cells have been limited by terephthalic acid (TPA) uptake. Here, we used an intracellular carboxylate reduction assay to show that apparent TPA uptake in E. coli cells that lack a dedicated TPA transporter sharply increases between pH 5-6. Furthermore, we discovered that glycol ester deconstruction products, mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), surprisingly each result in rapid pH-independent uptake. We exploited glycol ester uptake along with deletion of 22 cellular oxidoreductases to design intracellular hydrolysis routes for synthesis of upcycled reduction products from BHET at >90% yields, and from real PET wastes after tandem catalytic glycolysis and cell-based valorization at >80% combined yields. Our work has important ramifications for PET utilization by cells and adds new perspectives on the evolution of the PETase/MHETase system.

bioengineering↗

bioRxiv2025

Combinatorial mutagenesis of N-terminal sequences reveals unexpected and expanded stability determinants of the Escherichia coli N-degron pathway

Although it is known that residues near the N-terminus can influence protein stability, there has not been a comprehensive effort to document how these penultimate residues impact protein stability in prokaryotes. Here, we utilize combinatorial mutagenesis, cell sorting, and next generation sequencing to perform the deepest sequence coverage screen of the bacterial N-degron pathway of proteolysis. We present nuance and exceptions to the N-terminus (P1) functioning as a primary stability determinant. We reveal stability contributions for P2-P5 motifs, including lowered stability for clustered bulky residues and Gln, and heightened stability for negatively charged residues, Pro, and Gly. We find that P1 Cys can be an N-degron component in a sequence-specific manner. Furthermore, we employ stability-predictive machine learning to identify motifs with unexpected fates. Our work expands the stability determinants of the N-degron pathway with unprecedented granularity, serving as a resource for N-degron identification, N-degron design, and future molecular basis elucidation.

synthetic biology↗

bioRxiv2025

The influence of surfactin and glutamate on tomato root association by plant growth promoting rhizobacteria Bacillus subtilis

The influence of microbially and plant synthesized compounds on attachment of plant growth promoting rhizobacteria (PGPR) to various regions of the plant root are underexplored. Here, we examine the influence of surfactin and glutamate on the level and specificity of attachment of Bacillus subtilis along different regions of tomato root using chemical and genetic perturbations. First, we modify Bacillus subtilis PY79 to express a full-length phosphopantetheinyl transferase (sfp) native to other B. subtilis strains, and we observe surfactin biosynthesis under static incubation at 25{degrees}C in plant culturing media. We then perform microscopy using the wild-type PY79 strain, the sfp+ strain, and the wild isolate B. subtilis UD1022 to map the attachment of each strain along the entire root of young tomato plants incubated at 25{degrees}C, including when exogenously supplementing surfactin or glutamate. Through root mapping, we show that these strains prefer to attach near the mature region (MR) of the root and that colonization patterns vary based on exogenous metabolite concentration. Inclusion of glutamate in the media or through transient priming of the plant prior to bacterial inoculation strongly promoted root colonization by B. subtilis strains (both surfactin null and producers). In addition, the data shows that the domesticated strains were less efficient in binding compared to the wild B. subtilis strain. Interestingly, in the presence of glutamate, microbes lost their preference for association at the mature region, instead colonizing along the entire root. Overall, our work reveals a preference for association of these B. subtilis strains to the mature region of tomato in the absence of glutamate supplementation, confirms a strong influence of glutamate on root association, and demonstrates a smaller than anticipated role of biosynthesized or supplemented surfactin on root association, at least in a hydroponic culturing format.

plant biology↗

bioRxiv2025

Design of an exclusive obligate symbiosis for organism-based biological containment

Biological containment is a critical safeguard for genetically engineered microbes prior to their environmental release to prevent proliferation in unintended regions. However, few biocontainment strategies can support the longer-term microbial survival that may be desired in a target environment without repeated human intervention. Here, we introduce the concept of an orthogonal obligate commensalism for the autonomous creation of environments that are permissive for survival of a biocontained microbe. We engineer one microbe to produce a non-standard amino acid (nsAA), and we engineer synthetic auxotrophy in a second microbe via reliance on this nsAA for growth. We show that this obligate commensalism is highly effective, with the survival of our commensal organism during co-culture dependent on the presence of our producer strain. We also show that this commensalism is orthogonal to a small microbial consortium isolated from maize roots, with survival of the synthetic auxotroph conditional upon the presence of the nsAA-producing strain in the consortium. Overall, our study demonstrates a transition from a chemical to a biological dependence for biocontained organisms that could lay the groundwork for biocontained synthetic ecologies.

synthetic biology↗

bioRxiv2024

Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids in engineered bacteria

Applications of genetic code expansion in live cells are widespread and continually emerging, yet they have been limited by their reliance on the supplementation of non-standard amino acids (nsAAs) to cell culturing media. While advances in cell-free biocatalysis are improving nsAA synthesis cost and sustainability, such processes remain reliant on multi-step processes of product isolation followed by supplementation to engineered cells. Here, we report the design of a modular and genetically encoded system that combines the steps of biosynthesis of diverse phenylalanine derivatives, which are the most frequently used family of nsAAs for genetic code expansion, and their site-specific incorporation within target proteins using a single engineered bacterial host. Unlike previous demonstrations in which individual nsAAs were targeted for biosynthesis and site-specific incorporation, our system serves as a platform that exhibits broad substrate specificity towards commercially ubiquitous, achiral building blocks of aryl aldehydes or carboxylic acids. We demonstrate that this modular system enables high fidelity biosynthesis and incorporation of nsAAs for multiple industrially relevant nsAAs, such as the incorporation of 4,4-L-biphenylalanine within proteins after supplementation with biphenylaldehyde and the incorporation of 4-azido-L-phenylalanine within proteins after supplementation with 4-azido-benzoic acid. Finally, we show that the combination of nsAA biosynthesis and incorporation steps can extend the chemical reach of the intrinsic biological containment strategy of synthetic auxotrophy from nsAAs to low-cost and achiral building blocks. We anticipate that our system will aid industrial-scale manufacturing of proteins that contain nsAAs and democratize access to expensive or commercially unavailable chemistries for labs that lack separations or synthesis expertise.

synthetic biology↗

bioRxiv2024

A One-Pot Biocatalytic Cascade to Access Diverse L-Phenylalanine Derivatives from Aldehydes or Carboxylic Acids

Non-standard amino acids (nsAAs) that are L-phenylalanine derivatives with aryl ring functionalization have long been harnessed in natural product synthesis, therapeutic peptide synthesis, and diverse applications of genetic code expansion. Yet, to date these chiral molecules have often been the products of poorly enantioselective and environmentally harsh organic synthesis routes. Here, we reveal the broad specificity of multiple natural pyridoxal 5-phosphate (PLP)-dependent enzymes, specifically an L-threonine transaldolase, a phenylserine dehydratase, and an aminotransferase, towards substrates that contain aryl side chains with diverse substitutions. We exploit this tolerance to construct a one-pot biocatalytic cascade that achieves high-yield synthesis of 18 diverse L-phenylalanine derivatives from aldehydes under mild aqueous reaction conditions. We demonstrate addition of a carboxylic acid reductase module to this cascade to enable the biosynthesis of L-phenylalanine derivatives from carboxylic acids that may be less expensive or less reactive than the corresponding aldehydes. Finally, we investigate the scalability of the cascade by developing a lysate-based route for preparative-scale synthesis of 4-formyl-L-phenylalanine, a nsAA with a bio-orthogonal handle that is not readily market-accessible. Overall, this work offers an efficient, versatile, and scalable route with the potential to lower manufacturing cost and democratize synthesis for many valuable nsAAs.

synthetic biology↗

bioRxiv2023

Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products

The selective introduction of amine groups within deconstruction products of lignin could provide an avenue for valorizing waste biomass while achieving a green synthesis of industrially relevant building blocks from sustainable sources. Here, we built and characterized enzyme cascades that create aldehydes and subsequently primary amines from diverse lignin-derived carboxylic acids using a carboxylic acid reductase (CAR) and an {omega}-transaminase (TA). Unlike previous studies that have paired CAR and TA enzymes, here we examine multiple homologs of each of these enzymes and a broader set of candidate substrates. In addition, we compare the performance of these systems in cell-free and resting whole-cell biocatalysis formats using the conversion of vanillate to vanillyl amine as model chemistry. We also demonstrate that resting whole cells can be recycled for multiple batch reactions. We used the knowledge gained from this study to produce several amines from carboxylic acid precursors using one-pot biocatalytic reactions, several of which we report for the first time. These results expand our knowledge of these industrially relevant enzyme families to new substrates and contexts for environmentally friendly and potentially low-cost synthesis of diverse aryl aldehydes and amines.

synthetic biology↗

bioRxiv2023

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild-type strain MG1655 and an engineered strain for reduced aromatic aldehyde rection (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24h. We applied this new strain for the production of para-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared to RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

synthetic biology↗

bioRxiv2023

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by resting cells of E. coli RARE strains

Aldehydes are attractive chemical targets given applications as end products in the flavors and fragrances industry and as intermediates due to their propensity for C-C bond formation. While biosynthetic routes to diverse aldehydes have been designed, a common challenge is the stability of these aldehydes in the presence of microbial hosts of engineered pathways. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation, in the presence of Escherichia coli strains that were engineered to minimize aldehyde reduction. Of heightened interest to us were resting cell conditions as they offer numerous advantages for the bioconversion of toxic metabolites. Surprisingly, when diverse aldehydes are supplemented to E. coli RARE cells grown under aerobic conditions, they remain stabilized on the timescale of days, whereas when these same aldehydes are supplemented to resting cell preparations of E. coli RARE that had been grown under the same conditions, we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 hours after their addition. Given that our newly engineered strain exhibits Reduced Oxidation And Reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. Seeking to apply this new strain to resting cell biocatalysis, we compared the capability to synthesis the aldehyde furfural from 2-furoic acid via the carboxylic acid reductase enzyme from Nocardia iowensis. Here, we found that use of ROAR resting cells achieved 2-fold enhancement in furfural titer after 4 h and nearly 9-fold enhancement after 20 h as compared to resting cells of the RARE strain. Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity. HighlightsO_LIWhen genes that encode aldehyde reductases are knocked out in Escherichia coli strains, supplemented aldehydes can experience oxidation instead of reduction, which is catalyzed by a different set of endogenous enzymes. C_LIO_LIInterestingly, we show for a collection of aromatic aldehydes that this oxidation is far more substantial when using resting cell preparations than during aerobic fermentation. C_LIO_LIWe investigate the identity of the responsible genes by performing combinatorial gene inactivation using multiplex automatable genome engineering. C_LIO_LIThe strain that we engineer exhibits Reduced Oxidation And Reduction (the E. coli ROAR strain) and thereby enables design of more efficient aldehyde bioconversion processes under diverse formats. C_LI

synthetic biology↗

bioRxiv2022

Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

To better incentivize the collection of plastic wastes, new chemical transformations must be developed that add value to plastic deconstruction products. Polyethylene terephthalate (PET) is a common plastic whose deconstruction through chemical or biological means has received much attention. However, a limited number of alternative products have been formed from PET deconstruction, and only a small share could serve as building blocks for alternative materials or therapeutics. Here, we demonstrate the production of useful mono-amine and diamine building blocks from known PET deconstruction products. We achieve this by designing one-pot biocatalytic transformations that are informed by the substrate specificity of an {omega}-transaminase and diverse carboxylic acid reductases (CAR) towards PET deconstruction products. We first establish that an {omega}-transaminase from Chromobacterium violaceum (cvTA) can efficiently catalyze amine transfer to potential PET-derived aldehydes to form the mono-amine para-(aminomethyl)benzoic acid (pAMBA) or the diamine para-xylylenediamine (pXYL). We then identified CAR orthologs that could perform the bifunctional reduction of TPA to terephthalaldehyde (TPAL) or the reduction of mono-(2-hydroxyethyl) terephthalic acid (MHET) to its corresponding aldehyde. After characterizing 17 CARs in vitro, we show that the CAR from Segniliparus rotundus (srCAR) had the highest observed activity on TPA. Given these newly elucidated substrate specificity results, we designed modular enzyme cascades based on coupling srCAR and cvTA in one-pot with enzymatic co-factor regeneration. When we supply TPA, we achieve a 69 {+/-} 1% yield of pXYL, which is useful as a building block for materials. When we instead supply MHET and subsequently perform base-catalyzed ester hydrolysis, we achieve 70 {+/-} 8% yield of pAMBA, which is useful for therapeutic applications and as a pharmaceutical building block. This work expands the breadth of products derived from PET deconstruction and lays the groundwork for eventual valorization of waste PET to higher-value chemicals and materials. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/520786v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1bb7386org.highwire.dtl.DTLVardef@19d8c55org.highwire.dtl.DTLVardef@68569eorg.highwire.dtl.DTLVardef@ed398_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli

Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 {micro}M in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block. HighlightsO_LIPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion. C_LIO_LIWe establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid. C_LIO_LIWe show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product. C_LIO_LIScreening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 {+/-} 130 {micro}M in shake flask experiments with rich defined media. C_LI

synthetic biology↗

bioRxiv2021

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

Bacillus 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.

synthetic biology↗

bioRxiv2020

Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications. One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

synthetic biology↗

bioRxiv2018

Development of a vanillate biosensor for the vanillin biosynthesis pathway in E. coli

Genetically encoded small molecule sensors can facilitate metabolic engineering by enabling high-throughput detection of metabolite concentrations, directed evolution of host and pathway enzymes, and dynamic regulation. The engineered de novo vanillin biosynthesis pathway assembled in Escherichia coli is industrially relevant and ideal for biosensor deployment given that the pathway requires only three heterologous enzyme-catalyzed reactions, generates naturally occurring metabolites, and may benefit from dynamic regulation. However, pathway flux is stalled and diverted by the activity of the Homo sapiens catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To confront this challenge, we constructed and applied a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using components from a previously characterized E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We characterized sensor substrate specificity and found that this construct and an evolved variant are remarkably selective, exhibiting no detectable response to the regioisomer byproduct isovanillate. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases. We identified eight that appear to have greater desired activity than the originally used variant, including three previously uncharacterized O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

synthetic biology↗

bioRxiv2017

Long-term adaptive evolution of genomically recoded Escherichia coli

Efforts are underway to construct several recoded genomes anticipated to exhibit multi-virus resistance, enhanced non-standard amino acid (NSAA) incorporation, and capability for synthetic biocontainment. Though we succeeded in pioneering the first genomically recoded organism (Escherichia coli strain C321.{Delta}A), its fitness is far lower than that of its non-recoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for NSAA-linked applications requiring defined media such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.{Delta}A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceed the growth rates of both the ancestral C321.{Delta}A and non-recoded strains, permitting use of the recoded chassis in several new contexts. We use next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstruct individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occur only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.{Delta}A strain.\n\nSignificance StatementAfter demonstrating construction of an organism with an altered genetic code, we sought to evolve this organism for many generations to improve its fitness and learn what unique changes natural selection would bestow upon it. Although this organism initially had impaired fitness, we observed that adaptive laboratory evolution resulted in several selective mutations that corrected for insufficient translation termination and for unintended mutations that occurred when originally altering the genetic code. This work further bolsters our understanding of the pliability of the genetic code, it will help guide ongoing and future efforts seeking to recode genomes, and it results in a useful strain for non-standard amino acid incorporation in numerous contexts relevant for research and industry.

evolutionary biology↗

bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed \"Post-Translational Proofreading\" (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

synthetic biology↗