Discovery of Small Molecule Interleukin 17A Inhibitors with Novel Binding Mode and Stoichiometry: Optimization of DNA-Encoded Chemical Library Hits to In Vivo Active Compounds.

Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.

Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection.

Horseshoe crabs have been integral to the safe production of vaccines and injectable medications for the past 40 years. The bleeding of live horseshoe crabs, a process that leaves thousands dead annually, is an ecologically unsustainable practice for all four species of horseshoe crab and the shorebirds that rely on their eggs as a primary food source during spring migration. Populations of both horseshoe crabs and shorebirds are in decline. This study confirms the efficacy of recombinant Factor C (rFC), a synthetic alternative that eliminates the need for animal products in endotoxin detection. Furthermore, our findings confirm that the biomedical industry can achieve a 90% reduction in the use of reagents derived from horseshoe crabs by using the synthetic alternative for the testing of water and other common materials used in the manufacturing process. This represents an extraordinary opportunity for the biomedical and pharmaceutical industries to significantly contribute to the conservation of horseshoe crabs and the birds that depend on them.

Advancing a New Toolkit for Conservation: From Science to Policy.

Climate change and non-native wildlife diseases are exacerbating persistent challenges to biodiversity such as habitat destruction, invasive species and over-harvesting. With these increasing threats there is a pressing need to expand the conservationists' toolbox. CRISPR-Cas9 genome editing (GE) offers a precise and potentially transformative tool to confront these challenges. Researchers, regulators, conservationists and the public are all needed to engage proactively in the thoughtful and responsible development and application of these new tools.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway.

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae.

Streptomyces have a rich history as producers of important natural products and this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate its use in producing the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts.

Is It Time for Synthetic Biodiversity Conservation?

Evidence indicates that, despite some critical successes, current conservation approaches are not slowing the overall rate of biodiversity loss. The field of synthetic biology, which is capable of altering natural genomes with extremely precise editing, might offer the potential to resolve some intractable conservation problems (e.g., invasive species or pathogens). However, it is our opinion that there has been insufficient engagement by the conservation community with practitioners of synthetic biology. We contend that rapid, large-scale engagement of these two communities is urgently needed to avoid unintended and deleterious ecological consequences. To this point we describe case studies where synthetic biology is currently being applied to conservation, and we highlight the benefits to conservation biologists from engaging with this emerging technology.

Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols.

The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.

Divergent mechanistic routes for the formation of gem-dimethyl groups in the biosynthesis of complex polyketides.

The gem-dimethyl groups in polyketide-derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem-dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem-dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem-dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl-ACP appeared to be the sole route to form a gem-dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.

Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene.

The past decade has witnessed a large influx of research toward the creation of sustainable, biologically derived fuels. While significant effort has been exerted to improve production capacity in common hosts, such as Escherichia coli or Saccharomyces cerevisiae, studies concerning alternate microbes comparatively lag. In an effort to expand the breadth of characterized hosts for fuel production, we map the terpene biosynthetic pathway in a model actinobacterium, Streptomyces venezuelae, and further alter secondary metabolism to afford the advanced biofuel precursor bisabolene. Leveraging information gained from study of the native isoprenoid pathway, we were able to increase bisabolene titer nearly 5-fold over the base production strain, more than 2 orders of magnitude greater than the combined terpene yield in the wild-type host. We also explored production on carbon sources of varying complexity to, notably, define this host as one able to perform consolidated bioprocessing.

Mechanistic insights into the bifunctional non-heme iron oxygenase carbapenem synthase by active site saturation mutagenesis.

The carbapenem class of ß-lactam antibiotics is known for its remarkable potency, antibacterial spectrum, and resistance to ß-lactamase-mediated inactivation. While the biosynthesis of structurally "complex" carbapenems, such as thienamycin, share initial biochemical steps with carbapenem-3-carboxylate ("simple" carbapenem), the requisite inversion at C5 and formation of the characteristic α,ß-unsaturated carboxylate are different in origin between the two groups. Here, we consider carbapenem synthase, a mechanistically distinct bifunctional non-heme iron α-ketoglutarate-dependent enzyme responsible for the terminal reactions, C5 epimerization and desaturation, in simple carbapenem production. Interestingly, this enzyme accepts two stereoisomeric substrates and transforms each to a common active antibiotic. Owing both to enzyme and product instability, resorting to saturation mutagenesis of active site and selected second-sphere residues gave clearly differing profiles of CarC tolerance to structural modification. Guided by a crystal structure and the mutational data, in silico docking was used to suggest the positioning of each disastereomeric substrate in the active site. The two orientations relative to the reactive iron-oxo center are manifest in the two distinct reactions, C5-epimerization and C2/3-desaturation. These observations favor a two-step reaction scheme involving two complete oxidative cycles as opposed to a single catalytic cycle in which an active site tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring inversion, is further hypothesized to serve as a radical carrier.

A high-throughput screen for the engineered production of ß-lactam antibiotics.

High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of ß-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of ß-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.

Definition of the common and divergent steps in carbapenem ß-lactam antibiotic biosynthesis.

Approximately 50 naturally occurring carbapenem ß-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.

Non-heme iron oxygenases generate natural structural diversity in carbapenem antibiotics.

Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.

A catalytic asymmetric route to carbapenems.

Efficient syntheses of N-acetyl thienamycin and epithienamycin A in their readily deprotected form are reported where three contiguous stereocenters are established in a single catalytic asymmetric azetidinone-forming reaction. These examples are a template for synthesizing C-5/C-6 cis or trans carbapenems with independent control of the C-8 stereocenter. A library of oxidatively and sterochemically defined azetidinone precursors to a variety of naturally occurring carbapenems and potential biosynthetic intermediates has been prepared to facilitate studies of carbapenem antibiotic biosynthesis.

An externally tunable bacterial band-pass filter.

The current paradigm for tuning synthetic biological systems is through re-engineering system components. Biological systems designed with the inherent ability to be tuned by external stimuli will be more versatile. We engineered Escherichia coli cells to behave as an externally tunable band-pass filter for enzyme activity and small molecules. The band's location can be positioned within a range of 4 orders of magnitude simply by the addition of compounds to the growth medium. Inclusion in the genetic network of an enzyme-substrate pair that functions as an attenuator is a generalizable strategy that enables this tunability. The genetic circuit enabled bacteria growth to be patterned in response to chemical gradients in nonintuitive ways and facilitated the isolation of engineered allosteric enzymes. The application of this strategy to other biological systems will increase their utility for biotechnological applications and their usefulness as a tool for gaining insight into nature's underlying design principles.

Design and synthesis of a beta-lactamase activated 5-fluorouracil prodrug.

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by beta-lactamase (betaL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 betaL (ECl betaL) revealing a K(m)=95.4 microM and V(max)=3.21 microMol min(-1) mg(-1). The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.

Letter to the Editor.

In response to: Howard HC, Borry P: Direct-to-consumer genetic testing: more questions than benefits? Personalized Med. 5(4), 317-320 (2008).