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Biological systems can directly upgrade carbon dioxide (CO 2 ) into chemicals. The CO 2 fixation rate of autotrophic organisms, however, is too slow for industrial utility, and the breadth of engineered metabolic pathways for the synthesis of value-added chemicals is too limited. Biotechnology workhorse organisms with extensively engineered metabolic pathways have recently been engineered for CO 2 fixation. Yet, their low carbon fixation rate, compounded by the fact that living organisms split their carbon between cell growth and chemical synthesis, has led to only cell growth with no chemical synthesis achieved to date. Here, we engineer a lysate-based cell-free expression (CFE)-based multienzyme biocatalyst for the carbon negative synthesis of the industrially relevant amino acids glycine and serine from CO 2 equivalents─formate and bicarbonate─and ammonia. The formate-to-serine biocatalyst leverages tetrahydrofolate (THF)-dependent formate fixation, reductive glycine synthesis, serine synthesis, and phosphite dehydrogenase-dependent NAD(P)H regeneration to convert 30% of formate into serine and glycine, surpassing the previous 22% conversion using a purified enzyme system. We find that (1) the CFE-based biocatalyst is active even after 200-fold dilution, enabling higher substrate loading and product synthesis without incurring additional cell lysate cost, (2) NAD(P)H regeneration is pivotal to driving forward reactions close to thermodynamic equilibrium, (3) balancing the ratio of the formate-to-serine pathway genes added to the CFE is key to improving amino acid synthesis, and (4) efficient THF recycling enables lowering the loading of this cofactor, reducing the cost of the CFE-based biocatalyst. To our knowledge, this is the first synthesis of amino acids that can capture CO 2 equivalents for the carbon negative synthesis of amino acids using a CFE-based biocatalyst. Looking ahead, the CFE-based biocatalyst process could be extended beyond serine to pyruvate, a key intermediate, to access a variety of chemicals from aromatics and terpenes to alcohols and polymers.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets, working as entry points for signaling pathways involved in metabolic, neurological, and cardiovascular diseases. Although small molecules remain the major GPCR drug type, biologic therapeutics, such as peptides and antibodies, are increasingly found among clinical trials and Food and Drug Administration (FDA)-approved drugs. Here, we review state-of-the-art technologies for the engineering of biologics that target GPCRs, as well as proof-of-principle technologies that are ripe for this application. Looking ahead, inexpensive DNA synthesis will enable the routine generation of computationally predesigned libraries for use in display assays for the rapid discovery of GPCR binders. Advances in synthetic biology are enabling the increased throughput of functional GPCR assays to the point that they can be used to directly identify biologics that modulate GPCR activity. Finally, we give an overview of adjacent technologies that are ripe for application to discover biologics that target human GPCRs., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Decarboxylation - the release of carbon dioxide (CO 2 ) from a substrate - reduces the carbon yield of bioproduced chemicals. When overlaid onto central carbon metabolism, carbon-conservation networks (CCNs) that reroute flux around CO 2 release can theoretically achieve higher carbon yields for products derived from intermediates that traditionally require CO 2 release, such as acetyl-CoA. Recently, CCNs have started to be implemented in model organisms to produce compounds at higher carbon yields. However, implementation of CCNs in nonmodel hosts may have the greatest impact given their ability to assimilate a larger array of feedstocks, greater environmental tolerance, and unique biosynthetic pathways, ultimately enabling access to a wider range of products. Here, we review recent advances in CCNs with a focus on their application to nonmodel organisms. The differences in central carbon metabolism among different nonmodel hosts reveal opportunities to engineer and apply new CCNs., Competing Interests: Declaration of Competing Interest Nothing declared., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histamine receptor 2 (HR H2 ) blockers are used to treat peptic ulcers and gastric reflux. Chlorquinaldol and chloroxine, which contain an 8-hydroxyquinoline (8HQ) core, have recently been identified as blocking HR H2 . To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HR H2 -based sensor in yeast to evaluate the role of key residues in the HR H2 active site on histamine and 8HQ-based blocker binding. We find that the HR H2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HR H2 :D186A and HR H2 :T190A retain residual activity. Based on molecular docking studies, this outcome correlates with the ability of the pharmacologically relevant histamine tautomers to interact with D98 via the charged amine. Docking studies also suggest that, unlike established HR H2 blockers that interact with both ends of the HR H2 binding site, 8HQ-based blockers interact with only one end, either the end framed by D98/Y250 or T190/D186. Experimentally, we find that chlorquinaldol and chloroxine still inactivate HR H2 :D186A by shifting their engagement from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. Importantly, the tyrosine interactions are supported by the intramolecular hydrogen bonding of the 8HQ-based blockers. The insight gained in this work will aid in the development of improved HR H2 therapeutics. More generally, this work demonstrates that Gprotein-coupled receptor (GPCR)-based sensors in yeast can help elucidate the mode of action of novel ligands for GPCRs, a family of receptors that bind 30% of FDA therapeutics.

Chemical biosensors are an increasingly ubiquitous part of our lives. Beyond enzyme-coupled assays, recent synthetic biology advances now allow us to hijack more complex biosensing systems to respond to difficult to detect analytes, such as chemical small molecules. Here, we briefly overview recent advances in the biosensing of small molecules, including nucleic acid aptamers, allosteric transcription factors, and two-component systems. We then look more closely at a recently developed chemical sensing system, G protein-coupled receptor (GPCR)-based sensors. Finally, we consider the chemical sensing capabilities of the largest GPCR subfamily, olfactory receptors (ORs). We examine ORs' role in nature, their potential as a biomedical target, and their ability to detect compounds not amenable for detection using other biological scaffolds. We conclude by evaluating the current challenges, opportunities, and future applications of GPCR- and OR-based sensors.

Histamine receptor 2 (HR H2 ) activation in the stomach results in gastric acid secretion, and HR H2 blockers are used for the treatment of peptidic ulcers and acid reflux. Over-the-counter HR H2 blockers carry a five-membered aromatic heterocycle, with two of them additionally carrying a tertiary amine that decomposes to N-nitrosodimethylamine, a human carcinogen. To discover a novel HR H2 blocker scaffold to serve in the development of next-generation HR H2 blockers, we developed an HR H2 -based sensor in yeast by linking human HR H2 activation to cell luminescence. We used the HR H2 -based sensor to screen a 403-member anti-infection chemical library and identified three HR H2 blockers, chlorquinaldol, chloroxine, and broxyquinoline, all sharing an 8-hydroxyquinoline scaffold, which is not found among known HR H2 antagonists. Critically, we validate their HR H2 -blocking ability in mammalian cells. Molecular docking suggests that the HR H2 blockers bind the histamine binding pocket and structure-activity data point toward these blockers acting as competitive antagonists. Chloroxine and broxyquinoline are antimicrobials that can be found in the gastrointestinal tract at concentrations that would block HR H2 , thus likely modulating gastric acid secretion. Taken together, this work demonstrates the utility of GPCR-based sensors for rapid drug discovery applications, identifies a novel HR H2 blocker scaffold, and provides further evidence that antimicrobials not only target the human microbiota but also the human host.

Mars colonization demands technological advances to enable the return of humans to Earth. Shipping the propellant and oxygen for a return journey is not viable. Considering the gravitational and atmospheric differences between Mars and Earth, we propose bioproduction of a Mars-specific rocket propellant, 2,3-butanediol (2,3-BDO), from CO 2 , sunlight and water on Mars via a biotechnology-enabled in situ resource utilization (bio-ISRU) strategy. Photosynthetic cyanobacteria convert Martian CO 2 into sugars that are upgraded by engineered Escherichia coli into 2,3-BDO. A state-of-the-art bio-ISRU for 2,3-BDO production uses 32% less power and requires a 2.8-fold higher payload mass than proposed chemical ISRU strategies, and generates 44 tons of excess oxygen to support colonization. Attainable, model-guided biological and materials optimizations result in an optimized bio-ISRU that uses 59% less power and has a 13% lower payload mass, while still generating 20 tons excess oxygen. Addressing the identified challenges will advance prospects for interplanetary space travel., (© 2021. The Author(s).)

CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for metabolic pathway engineering. Here we implement CRISPR-Cas transcriptional activation (CRISPRa) in P. putida using a system previously developed in E. coli. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of promoter features. Using this optimized system, we regulate biosynthesis in the biopterin and mevalonate pathways. We demonstrate that multiple genes can be activated simultaneously by targeting multiple promoters or by targeting a single promoter in a multi-gene operon. This work will enable new metabolic engineering strategies in P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Membrane proteins are present in a wide array of cellular processes from primary and secondary metabolite synthesis to electron transport and single carbon metabolism. A key barrier to applying membrane proteins industrially is their difficult functional production. Beyond expression, folding, and membrane insertion, membrane protein activity is influenced by the physicochemical properties of the associated membrane, making it difficult to achieve optimal membrane protein performance outside the endogenous host. In this review, we highlight recent work on production of membrane proteins in membrane augmented cell-free systems (CFSs) and applications thereof. CFSs lack membranes and can thus be augmented with user-specified, tunable, mimetic membranes to generate customized environments for production of functional membrane proteins of interest. Membrane augmented CFSs would enable the synthesis of more complex plant secondary metabolites, the growth and division of synthetic cells for drug delivery and cell therapeutic applications, as well as enable green energy applications including methane capture and artificial photosynthesis.

More than 30% of all pharmaceuticals target G-protein-coupled receptors (GPCRs). Here, we present a GPCR-based screen in yeast to identify ligands for human serotonin receptor 4 (5-HTR 4 ). Serotonin receptor 4 agonists are used for the treatment of irritable bowel syndrome with constipation. Specifically, the HTR 4 -based screen couples activation of 5-HTR 4 on the yeast cell surface to luciferase reporter expression. The HTR 4 -based screen has a throughput of one compound per second allowing the screening of more than a thousand compounds per day.

Microbial production of adipic acid from lignin-derived monomers, such as catechol, is a greener alternative to the petrochemical-based process. Here, we produced adipic acid from catechol using catechol 1,2-dioxygenase (CatA) and a muconic acid reductase (MAR) in Escherichia coli. As the reaction progressed, the pH of the media dropped from 7 to 4-5 and the muconic acid isomerized from the cis,cis (ccMA) to the cis,trans (ctMA) isomer. Feeding experiments suggested that cells preferentially uptook ctMA and that MAR efficiently reduced all muconic isomers to adipic acid. Intrigued by the substrate promiscuity of MAR, we probed its utility to produce branched chiral diacids. Using branched catechols likely found in pretreated lignin, we found that while MAR fully reduced 2-methyl-muconic acid to 2-methyl-adipic acid, MAR reduced only one double bond in 3-substituted muconic acids. In the future, MAR's substrate promiscuity could be leveraged to produce chiral-branched adipic acid analogs to generate branched, nylon-like polymers with reduced crystallinity.

G protein-coupled receptors (GPCRs) detect compounds on the cell surface and are the starting point of a number of medically relevant signaling cascades. Indeed, over 30% of FDA approved drugs target GPCRs, making them a primary target for drug discovery. Computational and experimental high-throughput screening (HTS) approaches of clinically relevant GPCRs are a first-line drug discovery effort in biomedical research. In this opinion, we review recent advances in GPCR HTS. We focus primarily on cell-based assays, and highlight recent advances in in vitro assays using purified receptors, and computational approaches for GPCR HTS. To date, GPCR HTS has led to the identification of new and repurposing of existing drugs, and the deorphanization of GPCRs with unknown ligands. As automation equipment becomes more common, GPCR HTS will move beyond a drug discovery tool to a key technology to probe basic biological processes that will have an outsized impact on personalized medicine., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The serotonin receptor 4b (5-HTR 4b ) is expressed throughout the gastrointestinal tract, and its agonists are used in the treatment of irritable bowel syndrome with constipation (IBS-C). Today, there are no rapid assays for the identification of 5-HTR 4b agonists. Here, we developed a luciferase-based 5-HTR 4b assay capable of assessing one compound per second with a 38-fold dynamic range and nM limit of detection for serotonin. We used the assay to screen more than 1000 natural products and anti-infection agents and identified five new 5-HTR 4b ligands: hordenine, halofuginone, proflavine, ethacridine, and revaprazan. We demonstrate that hordenine (antibiofilm), halofuginone (antiparasitic), and revaprazan (gastric acid reducer) activate 5-HTR 4b in human colon epithelial cells, leading to increased cell motility or wound healing. The 5-HTR 4b assay can be used to screen larger pharmaceutical libraries to identify novel treatments for IBS-C. This work shows that antimicrobials interact not only with the gut microbiota, but also with the human host.

Olfactory receptors are ectopically expressed (exORs) in more than 16 different tissues. Studying the role of exORs is hindered by the lack of known ligands that activate these receptors. Of particular interest are exORs in the colon, the section of the gastrointestinal tract with the greatest diversity of microbiota where ORs may be participating in host-microbiome communication. Here, we leverage a G-protein-coupled receptor (GPCR)-based yeast sensor strain to generate sensors for seven ORs highly expressed in the colon. We screen the seven colon ORs against 57 chemicals likely to bind ORs in olfactory tissue. We successfully deorphanize two colon exORs for the first time, OR2T4 and OR10S1, and find alternative ligands for OR2A7. The same OR deorphanization workflow can be applied to the deorphanization of other ORs and GPCRs in general. Identification of ligands for OR2T4, OR10S1, and OR2A7 will enable the study of these ORs in the colon. Additionally, the colon OR-based sensors will enable the elucidation of endogenous colon metabolites that activate these receptors. Finally, deorphanization of OR2T4 and OR10S1 supports studies of the neuroscience of olfaction.

Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chain fatty acyl-ACP thioesterase for improved MCFA production in Escherichia coli. Electrostatically matching the interface between the heterologous medium-chain Acinetobacter baylyi fatty acyl-ACP thioesterase (AbTE) and the endogenous E. coli fatty acid ACP ( E. coli AcpP) by replacing small nonpolar amino acids on the AbTE surface for positively charged ones increased secreted MCFA titers more than 3-fold. Nuclear magnetic resonance titration of E. coli 15 N-octanoyl-AcpP with a single AbTE point mutant and the best double mutant showed a progressive and significant increase in the number of interactions when compared to AbTE wildtype. The best AbTE mutant produced 131 mg/L of MCFAs, with MCFAs being 80% of all secreted fatty acid chain lengths after 72 h. To enable the future screening of larger numbers of AbTE variants to further improve MCFA titers, we show that a previously developed G-protein coupled receptor (GPCR)-based MCFA sensor differentially detects MCFAs secreted by E. coli expressing different AbTE variants. This work demonstrates that engineering the interface of heterologous enzymes to better couple with endogenous host proteins is a useful strategy to increase the titers of microbially produced chemicals. Further, this work shows that GPCR-based sensors are producer microbe agnostic and can detect chemicals directly in the producer microbe supernatant, setting the stage for the sensor-guided engineering of MCFA producing microbes.

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Compartmentalization of metabolic pathways into organelles of the yeast Saccharomyces cerevisiae has been used to improve chemical production. Pathway compartmentalization aids chemical production by bringing enzymes into close proximity to one another, placing enzymes near key starting metabolites or essential co-factors, increasing the effective concentration of metabolic intermediates, and providing a more suitable chemical environment for enzymatic activity. Although several translocation tags have been used to localize enzymes to different yeast organelles, their translocation efficiencies have not been quantified. Here, we systematically quantify the translocation efficiencies of 10 commonly used S. cerevisiae tags by localizing green fluorescent protein (GFP) into three yeast organelles: the mitochondrion (4 tags), the vacuole (3 tags), and the peroxisome (3 tags). Further, we investigate whether plasmid copy number or mRNA levels vary with tag translocation efficiency. Quantification of the efficiencies of S. cerevisiae translocation tags provides an important resource for bioengineering practitioners when choosing a tag to compartmentalize their desired protein. Finally, these efficiencies can be used to determine the percentage of enzyme compartmentalization and, thus, help better quantify effects of compartmentalization on metabolic pathway efficiency., (© 2017 Wiley Periodicals, Inc.)

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

Adipic acid is the most industrially important dicarboxylic acid as it is a key monomer in the synthesis of nylon. Today, adipic acid is obtained via a chemical process that relies on petrochemical precursors and releases large quantities of greenhouse gases. In the last two years, significant progress has been made in engineering microbes for the production of adipic acid and its immediate precursors, muconic acid and glucaric acid. Not only have the microbial substrates expanded beyond glucose and glycerol to include lignin monomers and hemicellulose components, but the number of microbial chassis now goes further than Escherichia coli and Saccharomyces cerevisiae to include microbes proficient in aromatic degradation, cellulose secretion and degradation of multiple carbon sources. Here, we review the metabolic engineering and nascent protein engineering strategies undertaken in each of these chassis to convert different feedstocks to adipic, muconic and glucaric acid. We also highlight near term prospects and challenges for each of the metabolic routes discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Alkaloid-derived pharmaceuticals are commonly semisynthesized from plant-extracted starting materials, which often limits their availability and final price. Recent advances in synthetic biology have enabled the introduction of complete plant pathways into microbes for the production of plant alkaloids. Microbial production of modified alkaloids has the potential to accelerate the semisynthesis of alkaloid-derived drugs by providing advanced intermediates that are structurally closer to the final pharmaceuticals and could be used as advanced intermediates for the synthesis of novel drugs. Here, we analyze the scientific and engineering challenges that must be overcome to generate microbes to produce modified plant alkaloids that can provide more suitable intermediates to US Food and Drug Administration-approved pharmaceuticals. We highlight modified alkaloids that currently could be produced by leveraging existing alkaloid microbial platforms with minor variations to accelerate the semisynthesis of seven pharmaceuticals on the market.

A key limitation to engineering microbes for chemical production is a reliance on low-throughput chromatography-based screens for chemical detection. While colorimetric chemicals are amenable to high-throughput screens, many value-added chemicals are not colorimetric and require sensors for high-throughput screening. Here, we use G-protein coupled receptors (GPCRs) known to bind medium-chain fatty acids in mammalian cells to rapidly construct chemical sensors in yeast. Medium-chain fatty acids are immediate precursors to the advanced biofuel fatty acid methyl esters, which can serve as a "drop-in" replacement for D2 diesel. One of the sensors detects even-chain C8-C12 fatty acids with a 13- to 17-fold increase in signal after activation, with linear ranges up to 250 μM. Introduction of a synthetic response unit alters both dynamic and linear range, improving the sensor response to decanoic acid to a 30-fold increase in signal after activation, with a linear range up to 500 μM. To our knowledge, this is the first report of a whole-cell medium-chain fatty acid biosensor, which we envision could be applied to the evolutionary engineering of fatty acid-producing microbes. Given the affinity of GPCRs for a wide range of chemicals, it should be possible to rapidly assemble new biosensors by simply swapping the GPCR sensing unit. These sensors should be amenable to a variety of applications that require different dynamic and linear ranges, by introducing different response units.

Monoterpene indole alkaloids (MIAs) have important therapeutic value, including as anticancer and antimalarial agents. Because of their chemical complexity, therapeutic MIAs, or advanced intermediates thereof, are often isolated from the native plants. The microbial synthesis of MIAs would allow for the rapid and scalable production of complex MIAs and MIA analogues for therapeutic use. Here, we produce the modified MIA hydroxystrictosidine from glucose and the monoterpene secologanin via a pterin-dependent mono-oxidation strategy. Specifically, we engineered the yeast Saccharomyces cerevisiae for the high-level synthesis of tetrahydrobiopterin to mono-oxidize tryptophan to 5-hydroxytryptophan, which, after decarboxylation to serotonin, is coupled to exogenously fed secologanin to produce 10-hydroxystrictosidine in an eight-enzyme pathway. We selected hydroxystrictosidine as our synthetic target because hydroxylation at the 10' position of the alkaloid core strictosidine provides a chemical handle for the future chemical semisynthesis of therapeutics. We show the generality of the pterin-dependent mono-oxidation strategy for alkaloid synthesis by hydroxylating tyrosine to L-DOPA-a key intermediate in benzylisoquinoline alkaloid (BIA) biosynthesis-and, thereafter, further converting it to dopamine. Together, these results present the first microbial synthesis of a modified alkaloid, the first production of tetrahydrobiopterin in yeast, and the first use of a pterin-dependent mono-oxidation strategy for the synthesis of L-DOPA. This work opens the door to the scalable production of MIAs as well as the production of modified MIAs to serve as late intermediates in the semisynthesis of known and novel therapeutics. Further, the microbial strains in this work can be used as plant pathway discovery tools to elucidate known MIA biosynthetic pathways or to identify pathways leading to novel MIAs.

The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology.

The directed evolution of biomolecules with new functions is largely performed in vitro, with PCR mutagenesis followed by high-throughput assays for desired activities. As synthetic biology creates impetus for generating biomolecules that function in living cells, new technologies are needed for performing mutagenesis and selection for directed evolution in vivo. Homologous recombination, routinely exploited for targeted gene alteration, is an attractive tool for in vivo library mutagenesis, yet surprisingly is not routinely used for this purpose. Here, we report the design and characterization of a yeast-based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co-transformed with single-stranded mutagenic oligonucleotides. Using repair of nonsense codons engineered in three different active-site loops in the selectable marker TRP1 as a model system, we first optimized the recombination efficiency. Single-loop recombination was highly efficient, averaging 5%, or 4.0×10(5) recombinants. Multiple loops could be simultaneously mutagenized, although the efficiencies dropped to 0.2%, or 6.0×10(3) recombinants, for two loops and 0.01% efficiency, or 1.5×10(2) recombinants, for three loops. Finally, the utility of this system for directed evolution was tested explicitly by selecting functional variants from a mock library of 1:10(6) wild-type:nonsense codons. Sequencing showed that oligonucleotide recombination readily covered this large library, mutating not only the target codon but also encoded silent mutations on either side of the library cassette. Together these results establish oligonucleotide recombination as a simple and powerful library mutagenesis technique and advance efforts to engineer the cell for fully in vivo directed evolution., (Copyright © 2010 The Protein Society.)

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

Glycosynthases catalyze the formation of a glycosidic bond between a glycosyl fluoride donor substrate and a glycosyl acceptor substrate with high yield, thus providing a valuable approach for the synthesis of carbohydrates and glycoconjugates. Chemical complementation can be used to link glycosynthase activity to the transcription of a reporter gene in vivo, providing a selection for the directed evolution of glycosynthase enzymes with improved properties. In this approach, glycosynthase activity is detected as covalent coupling between a small molecule disaccharide acceptor substrate and a small molecule disaccharide alpha-fluoro donor substrate. Here we report the optimized design and synthesis of these small molecule substrates. These optimized substrates are shown to give a robust, glycosynthase-dependent transcriptional read-out in the chemical complementation assay. The full synthesis and characterization of these substrates are reported for the first time. These optimized chemical dimerizer substrates should allow the potential of chemical complementation for the directed evolution of glycosynthases with diverse substrate specificities and improved properties to be fully realized.