Upcycling the anthracyclines: New mechanisms of action, toxicology, and pharmacology.

The anthracyclines are a family of natural products isolated from soil bacteria with over 2000 chemical representatives. Since their discovery seventy years ago by Waksman and co-workers, anthracyclines have become one of the best-characterized anticancer chemotherapies in clinical use. The anthracyclines exhibit broad-spectrum antineoplastic activity for the treatment of a variety of solid and liquid tumors, however, their clinical use is limited by their dose-limiting cardiotoxicity. In this review article, we discuss the toxicity of the anthracyclines on several organ systems, including new insights into doxorubicin-induced cardiotoxicity. In addition, we discuss new medicinal chemistry developments in the biosynthesis of new anthracycline analogs and the synthesis of new anthracycline analogs with diminished cardiotoxicity. Lastly, we review new studies that describe the repurposing of the anthracyclines, or "upcycling" of the anthracyclines, as anti-infective agents, or drugs for niche indications. Altogether, the anthracyclines remain a mainstay in the clinic with a potential new "lease on life" due to deeper insight into the mechanism underlying their cardiotoxicity and new developments into potential new clinical indications for their use. Keywords: Anthracycline, chemotherapy, toxicology, medicinal chemistry, biosynthesis.

Renewed interests in the discovery of bioactive actinomycete metabolites driven by emerging technologies.

Actinomycetes are prolific sources of bioactive molecules. Traditional workflows including bacterial isolation, fermentation, metabolite identification and structure elucidation have resulted in high rates of natural product rediscovery in recent years. Recent advancements in multi-omics techniques have uncovered cryptic gene clusters within the genomes of actinomycetes, potentially introducing vast resources for the investigation of bioactive molecules. While developments in culture techniques have allowed for the fermentation of difficult-to-culture actinomycetes, high-throughput metabolite screening has offered plenary tools to accelerate hits discovery. A variety of new bioactive molecules have been isolated from actinomycetes of unique environmental origins, such as endophytic and symbiotic actinomycetes. Synthetic biology and genome mining have also emerged as new frontiers for the discovery of bioactive molecules. This review covers the highlights of recent developments in actinomycete-derived natural product drug discovery.

Pathway Engineering of Anthracyclines: Blazing Trails in Natural Product Glycodiversification.

The anthracyclines are structurally diverse anticancer natural products that bind to DNA and poison the topoisomerase II-DNA complex in cancer cells. Rational modifications in the deoxysugar functionality are especially advantageous for synthesizing drugs with improved potency. Combinatorial biosynthesis of glycosyltransferases and deoxysugar synthesis enzymes is indispensable for the generation of glycodiversified anthracyclines. This Synopsis considers recent advances in glycosyltransferase structural biology and site-directed mutagenesis, pathway engineering, and deoxysugar combinatorial biosynthesis with a focus on the generation of "new-to-nature" anthracycline analogues.

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells.

Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson's disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism.

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31-C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g(-1) fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.

Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals.

The ability of autotrophic organisms to fix CO2 presents an opportunity to utilize this 'greenhouse gas' as an inexpensive substrate for biochemical production. Unlike conventional heterotrophic microorganisms that consume carbohydrates and amino acids, prokaryotic chemolithoautotrophs have evolved the capacity to utilize reduced chemical compounds to fix CO2 and drive metabolic processes. The use of chemolithoautotrophic hosts as production platforms has been renewed by the prospect of metabolically engineered commodity chemicals and fuels. Efforts such as the ARPA-E electrofuels program highlight both the potential and obstacles that chemolithoautotrophic biosynthetic platforms provide. This review surveys the numerous advances that have been made in chemolithoautotrophic metabolic engineering with a focus on hydrogen oxidizing bacteria such as the model chemolithoautotrophic organism (Ralstonia), the purple photosynthetic bacteria (Rhodobacter), and anaerobic acetogens. Two alternative strategies of microbial chassis development are considered: (1) introducing or enhancing autotrophic capabilities (carbon fixation, hydrogen utilization) in model heterotrophic organisms, or (2) improving tools for pathway engineering (transformation methods, promoters, vectors etc.) in native autotrophic organisms. Unique characteristics of autotrophic growth as they relate to bioreactor design and process development are also discussed in the context of challenges and opportunities for genetic manipulation of organisms as production platforms.

Triterpene hydrocarbon production engineered into a metabolically versatile host--Rhodobacter capsulatus.

Triterpene hydrocarbon biosynthesis of the ancient algae Botryococcus braunii was installed into Rhodobacter capsulatus to explore the production of C30 hydrocarbon in a host capable of diverse growth habits-utilizing carbohydrate, sunlight or hydrogen (with CO2 fixation) as alternative energy feedstocks. Engineering an enhanced MEP pathway was also used to augment triterpene accumulation. Despite dramatically different sources of carbon and reducing power, nearly the same level of botryococcene or squalene (∼5 mg oil/g-dry-weight [gDW]) was achieved in small-scale aerobic heterotrophic, anaerobic photoheterotrophic, and aerobic chemoautotrophic growth conditions. A glucose fed-batch bioreactor reached 40 mg botryococcene/L (∼12 mg/gDW), while autotrophic bioreactor performance with CO2 , H2 , and O2 reached 110 mg/L (16.7 mg/gDW) during batch and 60 mg/L (23 mg/gDW) during continuous operation at a dilution rate corresponding to about 10% of µ(max). Batch and continuous autotrophic specific productivity was found to reach 0.5 and 0.32 mg triterpene/g DW/h, comparable to prior reports for terpene production driven by heterotrophic growth conditions. This demonstrates the feasibility of alternative feedstocks and trophic modes to provide comparable routes to biochemicals that do not rely on sugar.

Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins.

Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2',3',4'-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4'-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.

Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis.

Covering: 1997 to 2010. The angucycline group is the largest group of type II PKS-engineered natural products, rich in biological activities and chemical scaffolds. This stimulated synthetic creativity and biosynthetic inquisitiveness. The synthetic studies used five different strategies, involving Diels-Alder reactions, nucleophilic additions, electrophilic additions, transition-metal mediated cross-couplings and intramolecular cyclizations to generate the angucycline frames. Biosynthetic studies were particularly intriguing when unusual framework rearrangements by post-PKS tailoring oxidoreductases occurred, or when unusual glycosylation reactions were involved in decorating the benz[a]anthracene-derived cores. This review follows our previous reviews, which were published in 1992 and 1997, and covers new angucycline group antibiotics published between 1997 and 2010. However, in contrast to the previous reviews, the main focus of this article is on new synthetic approaches and biosynthetic investigations, most of which were published between 1997 and 2010, but go beyond, e.g. for some biosyntheses all the way back to the 1980s, to provide the necessary context of information.

Ketoolivosyl-tetracenomycin C: a new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT.

A new tetracenomycin analog, 8-demethyl-8-(4'-keto)-α-L-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a 'sugar plasmid' (pKOL) directing the biosynthesis of NDP-4-keto-L-olivose. This strain harbors all of the genes necessary for production of 8-demethyl-tetracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-L-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways.

A BioBricks toolbox for metabolic engineering of the tetracenomycin pathway.

BACKGROUND/GOAL/AIM: The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. MAIN METHODS AND MAJOR RESULTS: We developed a BioBricks genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: (1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to ß-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. CONCLUSIONS AND IMPLICATIONS: Altogether, this system is compatible with the BioBricks [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE. This production platform improves access to potent analogs, such as tetracenomycin X, and sets the stage for the production of new tetracenomycins via combinatorial biosynthesis.

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor.

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Cloning and characterization of the ravidomycin and chrysomycin biosynthetic gene clusters.

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1-1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D-ravidosamine and D-virenose biosynthetic genes, post-PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross-complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino- and neutral sugars, exemplified through the structurally distinct 6-membered D-ravidosamine and 5-membered D-fucofuranose, to the coumarin-based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.

Design of a disaster preparedness escape room for first and second-year pharmacy students.

BACKGROUND AND PURPOSE: Educational escape rooms assist students with the development of teamwork, augmentation of problem-solving skills, and reinforcement of key course concepts. In this report, we examined the feasibility of creating a bioterror preparedness escape room in a small enrollment pharmacy public health elective course. EDUCATIONAL ACTIVITY AND SETTING: A bioterror preparedness escape room was developed for pharmacy students in a health elective course. The instructional objectives of training students in disaster preparedness were assessed via group readiness assessment tests in the scenario and individual readiness assessment tests after the completion of the activity. FINDINGS: Twenty-eight students participated in the escape room activity in groups of 6 to 8 students (n = 4 observations) and all groups escaped. Student performance was higher on the initial attempts of three group readiness assessment tests (88 ± 16.0%, 82 ± 7.1%, 78 ± 12.0%) than in the final individual readiness assessment test (73.4 ± 20.4%). Students indicated that they found the educational escape room to be enjoyable (95.7%) and felt that all members of the team were involved in solving the problems (86.9%). SUMMARY: A disaster preparedness educational escape room was designed and implemented in a public health elective for pharmacy students. Findings indicate that the educational escape room format is an effective method for reinforcing course content, however additional improvements could be made to the instructional design to enhance individual student knowledge retention.

Drugs for Gram-Negative Bugs From 2010-2019: A Decade in Review.

A literature review spanning January 1, 2010, to December 31, 2019, was conducted using the PubMed and ISI Web of Science databases to determine the breadth of publication activity in the area of gram-negative bacteria antimicrobial therapy. The number of articles was used as a reflection of scholarly activity. First, PubMed was searched using the following Medical Subject Headings (MeSH): antibacterial agents, Enterobacteriaceae, Acinetobacter, and Pseudomonas. A total of 12 643 articles were identified within PubMed, and 77 862 articles were identified within ISI Web of Science that included these terms. Second, these articles were categorized by antibiotic class to identify relative contributions to the literature by drug category. Third, these studies were used to identify key trends in the treatment of gram-negative bacterial infections from the past decade. This review highlights advances made in the past 10 years in antibacterial pharmacotherapy and some of the challenges that await the next decade of practice.

Metabolic engineering of Escherichia coli for production of valerenadiene.

Valeriana officinalis is a medicinal herb which produces a suite of compounds in its root tissue useful for treatment of anxiety and insomnia. The sesquiterpene components of the root extract, valerenic acid and valerena-1,10-diene, are thought to contribute to most of the observed anxiolytic of Valerian root preparations. However, valerenic acid and its biosynthetic intermediates are only produced in low quantities in the roots of V. officinalis. Thus, in this report, Escherichia coli was metabolically engineered to produce substantial quantities of valerena-1,10-diene in shake flask fermentations with decane overlay. Expression of the wildtype valerenadiene synthase gene (pZE-wvds) resulted in production of 12µg/mL in LB cultures using endogenous FPP metabolism. Expression of a codon-optimized version of the valerenadiene synthase gene (pZE-cvds) resulted in 3-fold higher titers of valerenadiene (32µg/mL). Co-expression of pZE-cvds with an engineered methyl erythritol phosphate (MEP) pathway improved valerenadiene titers 65-fold to 2.09mg/L valerenadiene. Optimization of the fermentation medium to include glycerol supplementation enhanced yields by another 5.5-fold (11.0mg/L valerenadiene). The highest production of valerenadiene resulted from engineering the codon-optimized valerenadiene synthase gene under strong Ptrc and PT7 promoters and via co-expression of an exogenous mevalonate (MVA) pathway. These efforts resulted in an E. coli production strain that produced 62.0mg/L valerenadiene (19.4mg/L/OD600 specific productivity). This E. coli production platform will serve as the foundation for the synthesis of novel valerenic acid analogues potentially useful for the treatment of anxiety disorders.

Tavaborole, Efinaconazole, and Luliconazole: Three New Antimycotic Agents for the Treatment of Dermatophytic Fungi.

Fungal diseases of the nail bed (onychomycosis) and epidermis are recurrent illnesses in the elderly and immunocompromised patients, which have few efficacious treatment options. Current treatment options for onychomycosis are limited to topical agents, laser treatment, and oral antifungals. Previous generations of topical agents were not efficacious, owing to poor penetration of the nail bed. Oral antifungal drugs, such as itraconazole, terbinafine, and fluconazole, not only give better response rates but also inhibit a host of CYP450 enzymes. Oral antifungals can exacerbate drug-drug interactions for patients taking other medications concurrently. Newer topical agents might recognize improved efficacy and provide therapeutic alternatives when the use of oral antifungal agents is contraindicated. Recently, the Food and Drug Administration (FDA) approved efinaconazole and tavaborole for the treatment of onychomycosis. Additionally, the FDA approved luliconazole for the treatment of tinea pedis, tinea cruris, and tinea corporis. This review examines the mechanism of action, spectrum of activity, pharmacokinetics, and clinical trials data and considers the place in therapy for these 3 new antimycotic agents.