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.

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.

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.

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.

Similarities in Recalcitrant Structures of Industrial Non-Kraft and Kraft Lignin.

This work compares the structure of industrially isolated lignin samples from kraft pulping and three alternative processes: butanol organosolv, supercritical water hydrolysis, and sulfur dioxide/ethanol/water fractionation. Kraft processes are known to produce highly condensed lignin, with reduced potential for catalytic depolymerization, whereas the alternative processes have been hypothesized to impact the lignin less. The structural properties most relevant to catalytic depolymerization are characterized by elemental analysis, quantitative 13 C and 2 D HQSC NMR spectroscopy, gel permeation chromatography, and thermogravimetric analysis. Quantification of the ß-O-4 ether bond content shows partial depolymerization, with all samples having less than 12 bonds per 100 aromatic units. This results in theoretical monomer yields of less than 5 %, strongly suggesting the alternative fractionation processes generate highly condensed lignin structures that are no more suitable for catalytic depolymerization than kraft lignin. However, the different thermal degradation profiles suggest there are physicochemical differences that could be leveraged in other valorization strategies.

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.

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.