Technologies for the discovery of G protein-coupled receptor-targeting biologics.

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.

Insight into the Mode of Action of 8-Hydroxyquinoline-Based Blockers on the Histamine Receptor 2.

Histamine receptor 2 (HRH2) 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 HRH2. To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. We find that the HRH2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HRH2:D186A and HRH2: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 HRH2 blockers that interact with both ends of the HRH2 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 HRH2: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 HRH2 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.

Toward implementation of carbon-conservation networks in nonmodel organisms.

Decarboxylation - the release of carbon dioxide (CO2) from a substrate - reduces the carbon yield of bioproduced chemicals. When overlaid onto central carbon metabolism, carbon-conservation networks (CCNs) that reroute flux around CO2 release can theoretically achieve higher carbon yields for products derived from intermediates that traditionally require CO2 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.

Olfactory Receptors as an Emerging Chemical Sensing Scaffold.

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.

Discovery of 8-Hydroxyquinoline as a Histamine Receptor 2 Blocker Scaffold.

Histamine receptor 2 (HRH2) activation in the stomach results in gastric acid secretion, and HRH2 blockers are used for the treatment of peptidic ulcers and acid reflux. Over-the-counter HRH2 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 HRH2 blocker scaffold to serve in the development of next-generation HRH2 blockers, we developed an HRH2-based sensor in yeast by linking human HRH2 activation to cell luminescence. We used the HRH2-based sensor to screen a 403-member anti-infection chemical library and identified three HRH2 blockers, chlorquinaldol, chloroxine, and broxyquinoline, all sharing an 8-hydroxyquinoline scaffold, which is not found among known HRH2 antagonists. Critically, we validate their HRH2-blocking ability in mammalian cells. Molecular docking suggests that the HRH2 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 HRH2, 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 HRH2 blocker scaffold, and provides further evidence that antimicrobials not only target the human microbiota but also the human host.

Designing the bioproduction of Martian rocket propellant via a biotechnology-enabled in situ resource utilization strategy.

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 CO2, sunlight and water on Mars via a biotechnology-enabled in situ resource utilization (bio-ISRU) strategy. Photosynthetic cyanobacteria convert Martian CO2 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.

Screening for Serotonin Receptor 4 Agonists Using a GPCR-Based Sensor in Yeast.

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-HTR4). Serotonin receptor 4 agonists are used for the treatment of irritable bowel syndrome with constipation. Specifically, the HTR4-based screen couples activation of 5-HTR4 on the yeast cell surface to luciferase reporter expression. The HTR4-based screen has a throughput of one compound per second allowing the screening of more than a thousand compounds per day.

Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida.

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.

Membrane Augmented Cell-Free Systems: A New Frontier in Biotechnology.

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.

Fully biological production of adipic acid analogs from branched catechols.

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.

Advances in G protein-coupled receptor high-throughput screening.

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.

Identification of Three Antimicrobials Activating Serotonin Receptor 4 in Colon Cells.

The serotonin receptor 4b (5-HTR4b) 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-HTR4b agonists. Here, we developed a luciferase-based 5-HTR4b 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-HTR4b ligands: hordenine, halofuginone, proflavine, ethacridine, and revaprazan. We demonstrate that hordenine (antibiofilm), halofuginone (antiparasitic), and revaprazan (gastric acid reducer) activate 5-HTR4b in human colon epithelial cells, leading to increased cell motility or wound healing. The 5-HTR4b 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.

Rapid Deorphanization of Human Olfactory Receptors in Yeast.

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.

Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production.

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 15N-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.

Microbial synthesis of medium-chain chemicals from renewables.

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 ß-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.

Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

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.

Quantifying the efficiency of Saccharomyces cerevisiae translocation tags.

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.

Metabolic engineering strategies to bio-adipic acid production.

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.