Istvan Molnar

Interests

Research

Secondary metabolite biosynthesis, microbial genetics, bioinformatics

Teaching

Industrial microbiology

Courses

Scholarly Contributions

Journals/Publications

• Hu, P. F., Huang, J., Chen, L., Ding, Z., Liu, L., Molnár, I., & Zhang, B. B. (2020). Oxidative Stress Induction Is a Rational Strategy to Enhance the Productivity of Fermentations for the Antioxidant Secondary Metabolite Antrodin C. Journal of Agricultural and Food chemistry, 68(13), 3995-4004. doi:10.1021/acs.jafc.9b07965
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  Antioxidant metabolites contribute to alleviating oxidative stress caused by reactive oxygen species (ROS) in microorganisms. We utilized oxidative stressors such as hydrogen peroxide supplementation to increase the yield of the bioactive secondary metabolite antioxidant antrodin C in submerged fermentations of the medicinal mushroom . Changes in the superoxide dismutase and catalase activities of the cells indicate that ROS are critical to promote antrodin C biosynthesis, while the ROS production inhibitor diphenyleneiodonium cancels the productivity-enhancing effects of HO. Transcriptomic analysis suggests that key enzymes in the mitochondrial electron transport chain are repressed during oxidative stress, leading to ROS accumulation and triggering the biosynthesis of antioxidants such as antrodin C. Accordingly, rotenone, an inhibitor of the electron transport chain complex I, mimics the antrodin C productivity-enhancing effects of HO. Delineating the steps connecting oxidative stress with increased antrodin C biosynthesis will facilitate the fine-tuning of strategies for rational fermentation process improvement.
• Kozák, L., Szilágyi, Z., Tóth, L., Pócsi, I., & Molnár, I. (2020). Functional characterization of the idtF and idtP genes in the Claviceps paspali indole diterpene biosynthetic gene cluster. Folia Microbiologica, 65(3), 605-613. doi:10.1007/s12223-020-00777-6
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  Claviceps paspali is used in the pharmaceutical industry for the production of ergot alkaloids. This fungus also biosynthesizes paspalitrems, indole diterpene (IDT) mycotoxins that cause significant economic losses in agriculture and represent safety concerns for ergot alkaloid manufacture. Here, we use Agrobacterium-mediated transformation to replace the idtP and the idtF genes in the IDT biosynthetic gene cluster of C. paspali with a selectable marker gene. We show that the ΔidtP knockout mutant produces paspaline, the first IDT intermediate of the pathway. The ΔidtF strain produces unprenylated IDTs such as paspalinine and paspaline. These experiments validate the function of idtP as the gene encoding the cytochrome P450 monooxygenase that oxidizes and demethylates paspaline to produce 13-desoxypaxilline, and that of idtF as the gene that encodes the α-prenyltransferase that prenylates paspalinine at the C20 or the C21 positions to yield paspalitrems A and C, respectively. In addition, we also show that axenic cultures of the wild type, the ΔidtP and the ΔidtF mutant C. paspali strains fail to produce an assembly of IDTs that are present in C. paspali-Paspalum spp. associations.
• Li, M., Kang, L., Ding, X., Liu, J., Shao, Y., Molnar, I., & Chen, F. (2020). Monasone naphthoquinone biosynthesis and resistance in Monascus fungi. mBio, 11, e02676-19. doi:10.1128/mBio.02676-19
• Wang, C., Wang, X., Zhang, L., Yue, Q., Liu, Q., Xu, Y. M., Gunatilaka, A. A., Wei, X., Xu, Y., & Molnár, I. (2020). Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases. Journal of the American Chemical Society, 142(40), 17093-17104.
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  Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.
• Wu, S., Zhang, F., Xiong, W., Molnar, I., Liang, J., Ji, A., Li, Y., Wang, C., Wang, S., Liu, Z., Wu, R., & Duan, L. (2020). An unexpected oxidosqualene cyclase active site architecture in the Iris tectorum multifunctional α-amyrin synthase. ACS Catalysis, 10, 9515-9520. doi:10.1021/acscatal.0c03231
• Xie, L., Xiao, D., Wang, X., Wang, C., Bai, J., Yue, Q., Yue, H., Li, Y., Molnár, I., Xu, Y., & Zhang, L. (2020). Combinatorial Biosynthesis of Sulfated Benzenediol Lactones with a Phenolic Sulfotransferase from Fusarium graminearum PH-1. mSphere, 5(6), e00949-20. doi:10.1128/mSphere.00949-20
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  Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus PH-1. We expressed FgSULT1 in a chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics. Sulfation is an expedient strategy to increase the solubility, bioavailability, and bioactivity of nutraceuticals and clinically important drugs. However, chemical or biological synthesis of sulfoconjugates is challenging. Genome mining, heterologous expression, homology structural modeling, and site-directed mutagenesis identified FgSULT1 of PH-1 as a cytosolic sulfotransferase with the typical fold and active site architecture of characterized animal and plant sulfotransferases, despite low sequence similarity. FgSULT1 homologues are sparse in fungi but form a distinct clade with bacterial sulfotransferases. This study extends the functionally characterized sulfotransferase superfamily to the kingdom Fungi and demonstrates total biosynthetic and biocatalytic synthetic biological platforms to produce unnatural natural product (uNP) sulfoconjugates. Such uNP sulfates may be utilized for drug discovery in human and veterinary medicine and crop protection. Our synthetic biological methods may also be adapted to generate masked mycotoxin standards for food safety and environmental monitoring applications and to expose precarcinogenic xenobiotics.
• Zhang, L., Fasoyin, O. E., Molnár, I., & Xu, Y. (2020). Secondary metabolites from hypocrealean entomopathogenic fungi: novel bioactive compounds. Natural Product Reports, 37(9), 1181-1206. doi:10.1039/c9np00065h
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  Covering: 2014 up to the third quarter of 2019 Entomopathogens constitute a unique, specialized trophic subgroup of fungi, most of whose members belong to the order Hypocreales (class Sordariomycetes, phylum Ascomycota). These Hypocrealean Entomopathogenic Fungi (HEF) produce a large variety of secondary metabolites (SMs) and their genomes rank highly for the number of predicted, unique SM biosynthetic gene clusters. SMs from HEF have diverse roles in insect pathogenicity as virulence factors by modulating various interactions between the producer fungus and its insect host. In addition, these SMs also defend the carcass of the prey against opportunistic microbial invaders, mediate intra- and interspecies communication, and mitigate abiotic and biotic stresses. Thus, these SMs contribute to the role of HEF as commercial biopesticides in the context of integrated pest management systems, and provide lead compounds for the development of chemical pesticides for crop protection. These bioactive SMs also underpin the widespread use of certain HEF as nutraceuticals and traditional remedies, and allowed the modern pharmaceutical industry to repurpose some of these molecules as life-saving human medications. Herein, we survey the structures and biological activities of SMs described from HEF, and summarize new information on the roles of these metabolites in fungal virulence.
• Zhang, L., Yue, Q., Wang, C., Xu, Y., & Molnár, I. (2020). Secondary metabolites from hypocrealean entomopathogenic fungi: genomics as a tool to elucidate the encoded parvome. Natural Product Reports, 37(9), 1164-1180. doi:10.1039/d0np00007h
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  Covering: 2014 up to the third quarter of 2019 Hypocrealean entomopathogenic fungi (HEF) produce a large variety of secondary metabolites (SMs) that are prominent virulence factors or mediate various interactions in the native niches of these organisms. Many of these SMs show insecticidal, immune system modulatory, antimicrobial, cytotoxic and other bioactivities of clinical or agricultural significance. Recent advances in whole genome sequencing technologies and bioinformatics have revealed many biosynthetic gene clusters (BGCs) potentially involved in SM production in HEF. Some of these BGCs are now well characterized, with the structures of the cognate product congeners elucidated, and the proposed biosynthetic functions of key enzymes validated. However, the vast majority of HEF BGCs are still not linked to SM products ("orphan" BGCs), including many clusters that are not expressed (silent) under routine laboratory conditions. Thus, investigations into the encoded parvome (the secondary metabolome predicted from the genome) of HEF allows the discovery of BGCs for known SMs; uncovers novel metabolites based on the BGCs; and catalogues the predicted SM biosynthetic potential of these fungi. Herein, we summarize new developments of the field, and survey the polyketide, nonribosomal peptide, terpenoid and hybrid SM BGCs encoded in the currently available 40 HEF genome sequences. Studying the encoded parvome of HEF will increase our understanding of the multifaceted roles that SMs play in biotic and abiotic interactions and will also reveal biologically active SMs that can be exploited for the discovery of human and veterinary drugs or crop protection agents.
• Chen, W., Feng, Y., Molnár, I., & Chen, F. (2019). Nature and nurture: confluence of pathway determinism with metabolic and chemical serendipity diversifies Monascus azaphilone pigments. Natural Product Reports, 36, 561-572. doi:10.1039/C8NP00060C
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  Covering: up to June 2018Understanding the biosynthetic mechanisms that generate the astounding structural complexity and variety of fungal secondary metabolites (FSMs) remains a challenge. As an example, the biogenesis of the Monascus azaphilone pigments (MonAzPs) has remained obscure until recently despite the significant medical potential of these metabolites and their long history of widespread use as food colorants. However, a considerable progress has been made in recent years towards the elucidation of MonAzPs biosynthesis in various fungi. In this highlight, we correlate a unified biosynthetic pathway with the diverse structures of the 111 MonAzPs congeners reported until June 2018. We also discuss the origins of structural diversity amongst MonAzPs analogues and summarize new research directions towards exploring novel MonAzPs. The case of MonAzPs illuminates the various ways that FSMs metabolic complexity emerges by the interplay of biosynthetic pathway determinism with metabolic and chemical serendipity.
• Duong, D. A., Espinosa-Artiles, P., Orozco, R. A., Molnár, I., & Stock, S. P. (2019). Draft Genome Assembly of the Entomopathogenic Bacterium Photorhabdus luminescens subsp. sonorensis Caborca. Microbiology resource announcements, 8(36).
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  subsp. strain Caborca is an entomopathogenic bacterium with a dual lifestyle, namely, as a mutualist of the nematode and a pathogen to a wide range of insect species. The genome assembly, in 231 contigs, is 5.2 Mbp long and includes 25 putative gene clusters for secondary metabolism.
• Kozák, L., Szilágyi, Z., Tóth, L., Pócsi, I., & Molnár, I. (2019). Tremorgenic and neurotoxic paspaline-derived indole-diterpenes: biosynthetic diversity, threats and applications. Applied Microbiology and Biotechnology, 103, 1599-1616. doi:10.1007/s00253-018-09594-x
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  Indole-diterpenes (IDTs) such as the aflatrems, janthitrems, lolitrems, paspalitrems, penitrems, shearinines, sulpinines, and terpendoles are biogenetically related but structurally varied tremorgenic and neurotoxic mycotoxins produced by fungi. All these metabolites derive from the biosynthetic intermediate paspaline, a frequently occurring IDT on its own right. In this comprehensive review, we highlight the similarities and differences of the IDT biosynthetic pathways that lead to the generation of the main paspaline-derived IDT subgroups. We survey the taxonomic distribution and the regulation of IDT production in various fungi and compare the organization of the known IDT biosynthetic gene clusters. A detailed assessment of the highly diverse biological activities of these mycotoxins leads us to emphasize the significant losses that paspaline-derived IDTs cause in agriculture, and compels us to warn about the various hazards they represent towards human and livestock health. Conversely, we also describe the potential utility of these versatile molecules as lead compounds for pharmaceutical drug discovery, and examine the prospects for their industrial scale manufacture in genetically manipulated IDT producers or domesticated host microorganisms in synthetic biological production systems.
• Wang, X., Wang, C., Duan, L., Zhang, L., Liu, H., Xu, Y., Liu, Q., Mao, T., Zhang, W., Chen, M., Lin, M., Gunatilaka, L., Xu, Y., & Molnar, I. (2019). Rational reprogramming O-methylation regioselectivity for combinatorial biosynthetic tailoring of benzenediol lactone scaffolds. Journal of the American Chemical Society, 141, 4355-4364. doi:DOI: 10.1021/jacs.8b12967
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  O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery and optimization of known bioactive scaffolds during drug development both demand efficient O-methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone polyketides. We show that rational, structure-guided active-site cavity engineering can reprogram the regioselectivity of these enzymes. We also characterize the interplay of engineered regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic tailoring of unnatural products toward the generation of diverse chemical matter for drug discovery and the PK/PD optimization of bioactive scaffolds for drug development.
• Kozak, L., Szilagyi, Z., Vago, B., Kakuk, A., Toth, L., Molnar, I., & Pocsi, I. (2018). Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement. Applied Microbiology and Biotechnology, 102, 3255-3266. doi:DOI: 10.1007/s00253-018-8807-x
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  Article featured in the trade publication of the pharmaceutical company TEVA Industries, and widely publicized in the Hungarian online news media.
• Xie, L., Zhang, L., Wang, C., Wang, X., Xu, Y. M., Yu, H., Wu, P., Li, S., Han, L., Gunatilaka, A. A., Wei, X., Lin, M., Molnár, I., & Xu, Y. (2018). Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 115(22), E4980-E4989.
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  Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both - and -glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4--methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.
• Chen, W., Chen, R., Liu, Q., He, Y., He, K., Ding, X., Kang, L., Guo, X., Xie, N., Zhou, Y., Lu, Y., Cox, R. J., Molnar, I., Li, M., Shao, Y., & Chen, F. (2017). Orange, red and yellow: Biosynthesis of azaphilone pigments in Monascus fungi. Chemical Science, 8, 4917-4925. doi:10.1039/C7SC00475C
• Unkefer, C., Unkefer, C., Unkefer, C., Molnar, I., Molnar, I., Molnar, I., Ogden, K. L., Ogden, K. L., Ogden, K. L., Olivares, J., Olivares, J., Olivares, J., Brown, J. K., Brown, J. K., Brown, J. K., 15 other cauthors, ., 15 other cauthors, ., & 15 other cauthors, . (2017). Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts. Algal Research, 22, 187-215. doi:10.1016/j.algal.2016.06.002
• Xu, L., Wu, P., Xue, J., Molnar, I., & Wei, X. (2017). Antifungal and cytotoxic beta-resorcylic acid lactones from a Paecilomyces species. Journal of Natural Products, 80, 2215-2223. doi:10.1021/acs.jnatprod.7b00066
• Molnar, I., & 21 Authors, C. (2016). Insights into adaptations to a near-obligate nematode endoparasitic lifestyle from the finished genome of Drechmeria coniospora. Scientific Reports, 15(6), 23122. doi:10.1038/srep23122
• Molnar, I., & 4 co-authors, . (2016). Discrimination and quantification of true biological signals in LC-MS-based metabolomics analysis. Molecular Plant, 9, 1217-1220. doi:10.1016/j.molp.2016.05.009
• Molnar, I., & 6 coauthors, . (2016). A novel squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L. Nature Communications, 7, 11198. doi:10.1038/ncomms11198
• Molnar, I., Molnar, I., Gunatilaka, L., Gunatilaka, L., 7 co-authors, ., & 7 co-authors, C. (2016). Diversity-Oriented Combinatorial Biosynthesis of Hybrid Polyketide Scaffolds from Azaphilone and Benzenediol Lactone Biosynthons. Organic Letters, 18(6), 1262-1265. doi:10.1021/acs.orglett.6b00110
• Molnar, I., Stock, S. P., & 2 co-authors, . (2016). Bioprospecting for secondary metabolites in the entomopathogenic bacterium Photorhabdus luminescens subsp. sonorensis. Journal of Invertebrate Pathology, 141, 45-52. doi:10.1016/j.jip.2016.09.008
• Teng, C., Zhou, Z., Molnár, I., Li, X., Tang, R., Chen, M., Wang, L., Su, S., Zhang, W., & Lin, M. (2015). Whole-genome optical mapping and finished genome sequence of Sphingobacterium deserti sp. nov., a new species isolated from the Western Desert of China. PloS one, 10(4), e0122254.
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  A novel Gram-negative bacterium, designated ZWT, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5-42°C with an optimum of 30°C, and at pH 7.0-11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C16:1ω7c/C16:1ω6c or C16:1ω6c/C16:1ω7c) (39.22%), iso-C15:0 (27.91%), iso-C17:0 3OH (15.21%), C16:0 (4.98%), iso-C15:0 3OH (3.03%), C16:0 3OH (5.39%) and C14:0 (1.74%). The major polar lipid of strain ZWT is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZWT represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZWT (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZWT, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZWT features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZWT with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZWT to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.
• Zhou, Z., Zhang, W., Su, S., Chen, M., Lu, W., Lin, M., Molnár, I., & Xu, Y. (2015). CYP287A1 is a carotenoid 2-β-hydroxylase required for deinoxanthin biosynthesis in Deinococcus radiodurans R1. Applied microbiology and biotechnology, 99(24), 10539-46.
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  The carotenoid deinoxanthin is a crucial resistance factor against various stresses in the radiation-resistant bacterium Deinococcus radiodurans. Disruption of the gene dr2473 encoding the cytochrome P450 CYP287A1 led to the accumulation of 2-deoxydeinoxanthin in D. radiodurans, demonstrating that CYP287A1 is a novel β-carotene 2-hydroxylase. The dr2473 knockout mutant was shown to be more sensitive to UV radiation and oxidative stress than the wild-type strain D. radiodurans R1, indicating that the C2 alcohol of deinoxanthin is important for antioxidant activity.
• Xu, Y., Zhou, T., Espinosa-Artiles, P., Tang, Y., Zhan, J., & Molnár, I. (2014). Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae. ACS chemical biology, 9(5), 1119-27.
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  The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed "stuttering" by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions.
• Xu, Y., Zhou, T., Zhang, S., Espinosa-Artiles, P., Wang, L., Zhang, W., Lin, M., Gunatilaka, A. A., Zhan, J., & Molnár, I. (2014). Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases. Proceedings of the National Academy of Sciences of the United States of America, 111(34), 12354-9.
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  Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.
• Molnar, I., Xu, Y., Espinosa-Artiles, P., Schubert, V., Xu, Y., Zhang, W., Lin, M., Gunatilaka, A. A., Süssmuth, R., & Molnar, I. -. (2013). Characterization of the biosynthetic genes for 10,11-dehydrocurvularin, a heat shock response-modulating anticancer fungal polyketide from Aspergillus terreus. Applied and environmental microbiology, 79(6).
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  10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.
• Molnar, I., Xu, Y., Zhou, T., Zhang, S., Xuan, L., Zhan, J., & Molnar, I. -. (2013). Thioesterase domains of fungal nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. Journal of the American Chemical Society, 135(29).
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  A crucial step during the programmed biosynthesis of fungal polyketide natural products is the release of the final polyketide intermediate from the iterative polyketide synthases (iPKSs), most frequently by a thioesterase (TE) domain. Realization of combinatorial biosynthesis with iPKSs requires TE domains that can accept altered polyketide intermediates generated by hybrid synthase enzymes and successfully release "unnatural products" with the desired structure. Achieving precise control over product release is of paramount importance with O-C bond-forming TE domains capable of macrocyclization, hydrolysis, transesterification, and pyrone formation that channel reactive, pluripotent polyketide intermediates to defined structural classes of bioactive secondary metabolites. By exploiting chimeric iPKS enzymes to offer substrates with controlled structural variety to two orthologous O-C bond-forming TE domains in situ, we show that these enzymes act as nonequivalent decision gates, determining context-dependent release mechanisms and overall product flux. Inappropriate choice of a TE could eradicate product formation in an otherwise highly productive chassis. Conversely, a judicious choice of a TE may allow the production of a desired hybrid metabolite. Finally, a serendipitous choice of a TE may reveal the unexpected productivity of some chassis. The ultimate decision gating role of TE domains influences the observable outcome of combinatorial domain swaps, emphasizing that the deduced programming rules are context dependent. These factors may complicate engineering the biosynthesis of a desired "unnatural product" but may also open additional avenues to create biosynthetic novelty based on fungal nonreduced polyketides.
• Molnar, I., Xu, Y., Zhou, T., Zhou, Z., Su, S., Roberts, S. A., Montfort, W. R., Zeng, J., Chen, M., Zhang, W., Lin, M., Zhan, J., & Molnar, I. -. (2013). Rational reprogramming of fungal polyketide first-ring cyclization. Proceedings of the National Academy of Sciences of the United States of America, 110(14).
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  Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.
• Yuquan, X. u., Zhou, T., Zhang, S., Xuan, L., Zhan, J., & Molnár, I. (2013). Thioesterase domains of fungal nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. Journal of the American Chemical Society, 135(29), 10783-10791.
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  PMID: 23822773;PMCID: PMC3780601;Abstract: A crucial step during the programmed biosynthesis of fungal polyketide natural products is the release of the final polyketide intermediate from the iterative polyketide synthases (iPKSs), most frequently by a thioesterase (TE) domain. Realization of combinatorial biosynthesis with iPKSs requires TE domains that can accept altered polyketide intermediates generated by hybrid synthase enzymes and successfully release "unnatural products" with the desired structure. Achieving precise control over product release is of paramount importance with O-C bond-forming TE domains capable of macrocyclization, hydrolysis, transesterification, and pyrone formation that channel reactive, pluripotent polyketide intermediates to defined structural classes of bioactive secondary metabolites. By exploiting chimeric iPKS enzymes to offer substrates with controlled structural variety to two orthologous O-C bond-forming TE domains in situ, we show that these enzymes act as nonequivalent decision gates, determining context-dependent release mechanisms and overall product flux. Inappropriate choice of a TE could eradicate product formation in an otherwise highly productive chassis. Conversely, a judicious choice of a TE may allow the production of a desired hybrid metabolite. Finally, a serendipitous choice of a TE may reveal the unexpected productivity of some chassis. The ultimate decision gating role of TE domains influences the observable outcome of combinatorial domain swaps, emphasizing that the deduced programming rules are context dependent. These factors may complicate engineering the biosynthesis of a desired "unnatural product" but may also open additional avenues to create biosynthetic novelty based on fungal nonreduced polyketides. © 2013 American Chemical Society.
• Gonzalez, D. J., Yuquan, X. u., Yang, Y., Esquenazi, E., Liu, W., Edlund, A., Duong, T., Liangcheng, D. u., Molnár, I., Gerwick, W. H., Jensen, P. R., Fischbach, M., Liaw, C., Straight, P., Nizet, V., & Dorrestein, P. C. (2012). Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes. Journal of Proteomics, 75(16), 5069-5076.
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  PMID: 22641157;PMCID: PMC3543690;Abstract: Many microbes can be cultured as single-species communities. Often, these colonies are controlled and maintained via the secretion of metabolites. Such metabolites have been an invaluable resource for the discovery of therapeutics (e.g. penicillin, taxol, rapamycin, epothilone). In this article, written for a special issue on imaging mass spectrometry, we show that MALDI-imaging mass spectrometry can be adapted to observe, in a spatial manner, the metabolic exchange patterns of a diverse array of microbes, including thermophilic and mesophilic fungi, cyanobacteria, marine and terrestrial actinobacteria, and pathogenic bacteria. Dependent on media conditions, on average and based on manual analysis, we observed 11.3 molecules associated with each microbial IMS experiment, which was split nearly 50:50 between secreted and colony-associated molecules. The spatial distributions of these metabolic exchange factors are related to the biological and ecological functions of the organisms. This work establishes that MALDI-based IMS can be used as a general tool to study a diverse array of microbes. Furthermore the article forwards the notion of the IMS platform as a window to discover previously unreported molecules by monitoring the metabolic exchange patterns of organisms when grown on agar substrates.This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User's Guide to a New Technique for Biological and Biomedical Research. © 2012.
• Gonzalez, J., Xu, Y., Yang, Y., Esquenazi, E., Liu, W., Edlund, A., Duaong, T., Du, L., Molnar, I., Gerwick, W., Jensen, P., Fischbach, M., Liaw, C., Straight, P., Nizet, V., & Dorrestein, P. (2012). Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes. Journal of Proteomics, 75, 5069-5076.
• Matthes, D., Richter, L., Muller, J., Denisiuk, A., Feifel, S. C., Xu, Y., Espinosa-Artiles, P., Suessmuth, R. D., & Molnar, I. -. (2012). In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues. Chemical Communications, 48(45), 5674-5676.
• Matthes, D., Richter, L., Müller, J., Denisiuk, A., Feifel, S. C., Yuquan, X. u., Espinosa-Artiles, P., Süssmuth, R. D., & Molnár, I. (2012). In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues. Chemical Communications, 48(45), 5674-5676.
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  PMID: 22547105;Abstract: New beauvericins have been synthesized using the nonribosomal peptide synthetase BbBEAS from the entomopathogenic fungus Beauveria bassiana. Chemical diversity was generated by in vitro chemoenzymatic and in vivo whole cell biocatalytic syntheses using either a B. bassiana mutant or an E. coli strain expressing the bbBeas gene. © 2012 The Royal Society of Chemistry.
• Molnar, I., Lopez, D., Wisencaver, J., Devarenne, T., Weiss, T., Pellegrini, M., & Hackett, J. (2012). Bio-crude transcriptomics: Gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa). BMC Genomics, 13, 576.
• Molnar, I. -. (2011). A life with the bugs: In memoriam Istvan Ott. Society of Industrial Microbiology News, 68-69.
• Suessmuth, R., Muller, J., von, D. H., & Molnar, I. (2011). Fungal cyclooligomer depsipeptides: From classical biochemistry to combinatorial biosynthesis. Natural Products Reports, 28, 99-124.
• Süssmuth, R., Müller, J., Döhren, H. V., & Molnár, I. (2011). Fungal cyclooligomer depsipeptides: From classical biochemistry to combinatorial biosynthesis. Natural Product Reports, 28(1), 99-124.
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  PMID: 20959929;Abstract: This review surveys the biological activities and the iterative and recursive biosynthetic mechanisms of fungal cyclooligomer depsipeptides, and their structural diversification by various combinatorial biosynthetic methods. © The Royal Society of Chemistry 2011.
• Molnár, I., Gibson, D. M., & Krasnoff, S. B. (2010). Secondary metabolites from entomopathogenic Hypocrealean fungi. Natural Product Reports, 27(9), 1241-1275.
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  PMID: 20601982;Abstract: This review surveys the natural products described from entomopathogenic Hypocrealean fungi, including their structures, biological activities, potential utilities in medicine, roles in entomopathogenesis, and known or predicted biosynthetic pathways. © 2010 The Royal Society of Chemistry.
• Molnar, I., Xu, Y., Orozco, R., Kithsiri Wijeratne, E. M., Espinosa-Artiles, P., Leslie Gunatilaka, A. A., Patricia Stock, S., & Molnar, I. -. (2009). Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana. Fungal genetics and biology : FG & B, 46(5).
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  Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.
• Molnar, I., Xu, Y., Wijeratne, E. M., Espinosa-Artiles, P., Gunatilaka, A. A., & Molnar, I. -. (2009). Combinatorial mutasynthesis of scrambled beauvericins, cyclooligomer depsipeptide cell migration inhibitors from Beauveria bassiana. Chembiochem : a European journal of chemical biology, 10(2).
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  Fungal cyclooligomer depsipeptides such as beauvericin, bassianolide, and enniatins display antibiotic, antifungal, insecticidal, broad-spectrum cancer cell antiproliferative, and cell migration inhibitory activities. We have identified a gene encoding a novel enzyme, ketoisovalerate reductase (KIVR), which is the sole provider of D-hydroxyisovalerate (D-Hiv), a common precursor for cyclooligomer depsipeptide biosynthesis in Beauveria bassiana. KIVR and related hypothetical oxidoreductases encoded in fungal genomes are similar to ketopantoate reductases but not to D-hydroxycarboxylate dehydrogenases. We demonstrate that a KIVR knockout B. bassiana strain can be used for the efficient mutasynthesis of unnatural beauvericin congeners. Simultaneous feeding of precursor analogues enabled the combinatorial mutasynthesis of scrambled beauvericins, some assembled entirely from unnatural precursors. The effects of the introduced structural changes on the antiproliferative and cell migration inhibitory activities of these analogues were evaluated.
• Molnar, I., Wang, S., Xu, Y., Maine, E. A., Wijeratne, E. M., Espinosa-Artiles, P., Gunatilaka, A. A., & Molnar, I. -. (2008). Functional characterization of the biosynthesis of radicicol, an Hsp90 inhibitor resorcylic acid lactone from Chaetomium chiversii. Chemistry & biology, 15(12).
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  Fungal polyketides with the resorcylic acid lactone (RAL) scaffold are of interest for growth stimulation, the treatment of cancer, and neurodegenerative diseases. The RAL radicicol is a nanomolar inhibitor of the chaperone Hsp90, whose repression leads to a combinatorial blockade of cancer-causing pathways. Clustered genes for radicicol biosynthesis were identified and functionally characterized from the endophytic fungus Chaetomium chiversii, and compared to recently described RAL biosynthetic gene clusters. Radicicol production is abolished upon targeted inactivation of a putative cluster-specific regulator, or either of the two polyketide synthases that are predicted to collectively synthesize the radicicol polyketide core. Genomic evidence supports the existence of flavin-dependent halogenases in fungi: inactivation of such a putative halogenase from the C. chiversii radicicol locus yields dechloro-radicicol (monocillin I). Inactivation of a cytochrome P450 epoxidase furnishes pochonin D, a deepoxy-dihydro radicicol analog.
• Molnar, I., Xu, Y., Orozco, R., Wijeratne, E. M., Gunatilaka, A. A., Stock, S. P., & Molnar, I. -. (2008). Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus Beauveria bassiana. Chemistry & biology, 15(9).
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  Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence.
• Molnar, I., Xu, Y., Zhan, J., Wijeratne, E. M., Burns, A. M., Gunatilaka, A. A., & Molnar, I. -. (2007). Cytotoxic and Antihaptotactic beauvericin analogues from precursor-directed biosynthesis with the insect pathogen Beauveria bassiana ATCC 7159. Journal of natural products, 70(9).
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  Precursor-directed biosynthesis was used to produce analogues of the cyclic depsipeptide mycotoxin beauvericin (1) using the filamentous fungus Beauveria bassiana ATCC 7159. Feeding 30 analogues of D-2-hydroxyisovalerate and L-phenylalanine, the natural 2-hydroxycarboxylic acid and amino acid precursors of beauvericin, led to the biosynthesis of novel beauvericins. Six of these were isolated and characterized, and their cytotoxicity and directional cell migration (haptotaxis) inhibitory activity against the metastatic prostate cancer cell line PC-3M were evaluated. Replacement of one, two, or all three of the D-2-hydroxyisovalerate constituents in beauvericin (1) with 2-hydroxybutyrate moieties (beauvericins G(1-3), compounds 2-4) caused a parallel decline of cell migration inhibitory activity and cytotoxicity, suggesting a requirement for a branched side chain for both of these biological activities at the corresponding positions of beauvericins. Replacement of one, two, or all three N-methyl-L-phenylalanine residues of beauvericin with N-methyl-L-3-fluorophenylalanine moieties (beauvericins H(1-3), compounds 5-7) increased cytotoxicity without affecting antihaptotactic activity.
• Molnár, I., Zirkle, R., & Ligón, J. M. (2007). Biosynthesis of the antifungal polyketide antibiotic soraphen A in Sorangium cellulosum and Streptomyces lividans. ACS Symposium Series, 955, 217-230.
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  Abstract: The derivatization of soraphen by combinatorial biosynthesis has been a longstanding goal of the Natural Product Genetics group at Ciba Geigy - Novartis - Syngenta. We have cloned and characterized the soraphen A biosynthetic gene cluster of Sorangium cellulosum So ce26. To facilitate the genetic manipulation of So ce26, a central regulator of gliding motility was identified and its encoding gene disrupted to create a nonswarming strain. Despite this improvement, engineering of soraphen biosynthesis remained elusive in So ce26. Thus, the genes for the soraphen polyketide synthase, the post-polyketide tailoring steps, and methoxymalonate biosynthesis were introduced into Streptomyces lividans. Production of soraphen A in this genetically tractable strain also required provision of benzoyl-CoA by feeding benzoate or cinnamate. © 2007 American Chemical Society.
• Trefzer, A., Jungmann, V., Molnár, I., Botejue, A., Buckel, D., Frey, G., Hill, D. S., Jörg, M., Ligon, J. M., Mason, D., Moore, D., Pachlatko, J. P., Richardson, T. H., Spangenberg, P., Wall, M. A., Zirkle, R., & Stege, J. T. (2007). Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution. Applied and Environmental Microbiology, 73(13), 4317-4325.
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  PMID: 17483257;PMCID: PMC1932781;Abstract: Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
• Molnár, I., Jungmann, V., Stege, J., Trefzer, A., & Pachlatko, J. P. (2006). Biocatalytic conversion of avermectin into 4″-oxo-avermectin: Discovery, characterization, heterologous expression and specificity improvement of the cytochrome P450 enzyme. Biochemical Society Transactions, 34(6), 1236-1240.
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  PMID: 17073793;Abstract: 4″-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strainswith the ability to oxidize avermectin to 4″-oxoavermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP+ reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in coexpression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly™, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening. ©2006 Biochemical Society.
• Jungmann, V., Molnár, I., Hammer, P. E., Hill, D. S., Zirkle, R., Buckel, T. G., Buckel, D., Ligon, J. M., & Pachlatko, J. P. (2005). Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Characterization of biocatalytically active bacterial strains and of cytochrome p450 monooxygenase enzymes and their genes. Applied and Environmental Microbiology, 71(11), 6968-6976.
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  PMID: 16269732;PMCID: PMC1287622;Abstract: 4″-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4″-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Molnár, I., Hill, D. S., Zirkle, R., Hammer, P. E., Gross, F., Buckel, T. G., Jungmann, V., Pachlatko, J. P., & Ligon, J. M. (2005). Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Heterologous expression of the ema1 cytochrome p450 monooxygenase. Applied and Environmental Microbiology, 71(11), 6977-6985.
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  PMID: 16269733;PMCID: PMC1287623;Abstract: The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Zirkle, R., Black, T. A., Gorlach, J., Ligon, J. M., & Molnár, I. (2004). Analysis of a 108-kb region of the Saccharopolyspora spinosa genome covering the obscurin polyketide synthase locus. DNA Sequence - Journal of DNA Sequencing and Mapping, 15(2), 123-134.
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  PMID: 15346767;Abstract: A 108-kb genomic DNA region of Saccharopolyspora spinosa NRRL 18395, producer of the agriculturally important insecticidal antibiotics spinosyns, has been cloned, sequenced and analyzed to reveal clustered genes encoding a type I polyketide synthase (PKS) complex. The genes for the PKS are flanked by genes encoding homologs of enzymes that are involved in the urea cycle, valine, leucine and isoleucine biosynthesis and energy metabolism. While the disruption of the PKS genes by insertional inactivation was not expected to abolish the production of spinosyns, no differences were found in the antibacterial, antifungal, or insecticidal activities either of the parental and the knockout mutant strains under the growth conditions tested. Deduction of the most likely structure of the polyketide core of the cryptic metabolite, termed obscurin, from the predicted modules and domains of the PKS suggests the formation of a highly unsaturated substituted C22 carboxylic acid that might undergo further processing after its release from the PKS.
• Zirkle, R., Ligon, J. M., & Molnár, I. (2004). Cloning, sequence analysis and disruption of the mglA gene involved in swarming motility of Sorangium cellulosum So ce26, a producer of the antifungal polyketide antibiotic soraphen A. Journal of Bioscience and Bioengineering, 97(4), 267-274.
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  PMID: 16233626;Abstract: The myxobacterium Sorangium cellulosum So ce26, the producer of the agriculturally important fungicide antibiotic soraphen A, displays coordinated gliding motility (swarming) on agar surfaces. The consequent failure to form detached colonies represents a major obstacle for microbiological and genetic studies, since single cells representing discrete genetic events cannot be reliably separated and propagated as clones. The MglA protein, the product of the mglA gene, has been shown to be a central regulator of gliding motility and swarming in the related myxobacterium Myxococcus xanthus. We have cloned and sequenced a chromosomal locus from S. cellulosum So ce26 that shows similarity to the M. xanthus mglA locus. Insertional inactivation of the chromosomal copy of the S. cellulosum So ce26 mglA homolog resulted in a strain with a non-swarming colony phenotype. This strain is able to form distinct colonies presumably derived from single cells. This is the first report on the characterization of a genetic element of the gliding motility system in the myxobacterial suborder Sorangineae.
• Zirkle, R., Ligon, J. M., & Molnár, I. (2004). Heterologous production of the antifugal polyketide antibiotic soraphen A of Sorangium cellulosum So ce26 in Streptomyces lividans. Microbiology, 150(8), 2761-2774.
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  PMID: 15289572;Abstract: The antifungal polyketide soraphen A is produced by the myxobacterium Sorangium cellulosum So ce26. The slow growth, swarming motility and general intransigence of the strain for genetic manipulations make industrial strain development, large-scale fermentation and combinatorial biosynthetic manipulation of the soraphen producer very challenging. To provide a better host for soraphen A production and molecular engineering, the biosynthetic gene cluster for this secondary metabolite was integrated into the chromosome of Streptomyces lividans ZX7. The upstream border of the gene cluster in Sor. cellulosum was defined by disrupting sorC, which is proposed to take part in the biosynthesis of methoxymalonyl-coenzyme A, to yield a Sor. cellulosum strain with abolished soraphen A production. Insertional inactivation of orf2 further upstream of sorC had no effect on soraphen A production. The genes sorR, C, D, F and E thus implicated in soraphen biosynthesis were then introduced into an engineered Str. lividans strain that carried the polyketide synthase genes sorA and sorB, and the methyltransferase gene sorM integrated into its chromosome. A benzoate-coenzyme A ligase from Rhodopseudomonas palustris was also included in some constructs. Fermentations with the engineered Str. lividans strains in the presence of benzoate and/or cinnamate yielded soraphen A. Further feeding experiments were used to delineate the biosynthesis of the benzoyl-coenzyme A starter unit of soraphen A in the heterologous host. © 2004 SGM.
• Nowak-Thompson, B., Hammer, P. E., Hill, D. S., Stafford, J., Torkewitz, N., Gaffney, T. D., Lam, S. T., Molnár, I., & Ligon, J. M. (2003). 2,5-Dialkylresorcinol biosynthesis in Pseudomonas aurantiaca: Novel head-to-head condensation of two fatty acid-derived precursors. Journal of Bacteriology, 185(3), 860-869.
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  PMID: 12533461;PMCID: PMC142816;Abstract: 2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a β-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of β-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.
• Ligon, J., Hill, S., Beck, J., Zirkle, R., Molnár, I., Zawodny, J., Money, S., & Schupp, T. (2002). Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26. Gene, 285(1-2), 257-267.
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  PMID: 12039053;Abstract: A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT. © 2002 Elsevier Science B.V. All rights reserved.
• Molnár, I., Schupp, T., Ono, M., Zirkle, R. E., Milnamow, M., Nowak-Thompson, B., Engel, N., Toupet, C., Stratmann, A., Cyr, D. D., Gorlach, J., Mayo, J. M., Hu, A., Goff, S., Schmid, J., & Ligon, J. M. (2000). The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. Chemistry and Biology, 7(2), 97-109.
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  PMID: 10662695;Abstract: Background: Epothilones are produced by the myxobacterium Sorangium cellulosum So ce90, and, like paclitaxel (Taxol®), they inhibit microtubule depolymerisation and arrest the cell cycle at the G2-M phase. They are effective against P-glycoprotein-expressing multiple-drug-resistant tumor cell lines and are more water soluble than paclitaxel. The total synthesis of epothilones has been achieved, but has not provided an economically viable alternative to fermentation. We set out to clone, sequence and analyze the gene cluster responsible for the biosynthesis of the epothilones in S. cellulosum So ce90. Results: A cluster of 22 open reading frames spanning 68,750 base pairs of the S. cellulosum So ce90 genome has been sequenced and found to encode nine modules of a polyketide synthase (PKS), one module of a nonribosomal peptide synthetase (NRPS), a cytochrome P450, and two putative antibiotic transport proteins. Disruptions in the genes encoding the PKS abolished epothilone production. The first PKS module and the NRPS module are proposed to co-operate in forming the thiazole heterocycle of epothilone from an acetate and a cysteine by condensation, cyclodehydration and subsequent dehydrogenation. The remaining eight PKS modules are responsible for the elaboration of the rest of the epothilone carbon skeleton. Conclusions: The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.
• König, A., Schwecke, T., Molnár, I., Böhm, G. A., A., P., Staunton, J., & Leadlay, P. F. (1997). The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin - Nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus. European Journal of Biochemistry, 247(2), 526-534.
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  PMID: 9266694;Abstract: An open reading frame (rapP) encoding the putative pipecolate-incorporating enzyme (PIE) has been identified in the gene cluster for the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rnpP by phage insertion abolished rapamycin production in S. hygroscopicus, and the production of the antibiotic was specifically restored upon loss of the inserted phage by a second recombination event. rapP was expressed in both Escherichia coli and Streptomyces coelicolor, and recombinant PIE was purified to homogeneity from both hosts. Although low-level incorporation of [14C]β-alanine into recombinant PIE isolated from E. coli was detected, formation of the covalent acylenzyme intermediate could only be shown with the PIE from S. coelicolor, suggesting that while the recombinant PIE from S. coelicolor was phosphopantetheinylated, only a minor proportion of the recombinant enzyme from E. coli was post-translationally modified.
• Aparicio, J. F., Molnár, I., Schwecke, T., König, A., Haydock, S. F., Khaw, L. E., Staunton, J., & Leadlay, P. F. (1996). Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: Analysis of the enzymatic domains in the modular polyketide synthase. Gene, 169(1), 9-16.
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  PMID: 8635756;Abstract: The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowed further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), β-ketoacyl-ACP synthase (KS) and acyltransferase (AT) domains (the core domains) have an extremely high degree of mutual sequence homology. The KS domains in particular are almost perfect repeats over their entire length. Module 14 shows the least homology and is unique in possessing only core domains, The enoyl reductase (ER), β-ketoacyl-ACP reductase (KR) and dehydratase (DH) domains are present even in certain modules where they are not apparently required. Four DH domains can be recognised as inactive by characteristic deletions in active site sequences, but for two others, and for KR and ER in module 3, the sequence is not distinguishable from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity that may modify the starter unit after attachment, The sequence comparison has revealed the surprisingly high sequence similarity between inter-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisation into active species.
• Molnár, I., Aparicio, J. F., Haydock, S. F., Khaw, L. E., Schwecke, T., König, A., Staunton, J., & Leadlay, P. F. (1996). Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: Analysis of genes flanking the polyketide synthase. Gene, 169(1), 1-7.
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  PMID: 8635730;Abstract: Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosypthesis of the polyketide immunosuppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in 'late' modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster.
• Choi, K. -., Molnar, I., & Murooka, Y. (1995). Secretory overproduction of Arthrobacter simplex 3-ketosteroid Δ¹-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector. Applied Microbiology and Biotechnology, 43(6), 1044-1049.
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  PMID: 8590655;Abstract: The gene for 3-ketosteroid Δ1-dehydrogenase (ksdD) of Arthrobacter simplex was expressed in Streptomyces lividans and the secreted enzyme was overproduced by using a multi-copy shuttle vector composed of pIJ702 and pUC19. Deletional analysis of the recombinant plasmid showed that the entire coding sequence of the ksdD gene was located within a 7-kb segment of the chromosomal DNA obtained from the enzyme-producing strain of A. simplex. When S. lividans carrying the recombinant plasmid was grown in an appropriate medium, the cells produced about 100-fold more 3-ketosteroid Δ1-dehydrogenase than the original strain. Although the percentage of enzyme secreted was changed during cultivation, a maximum 55% of the enzyme was secreted into the cultured medium of S. lividans, while A. simplex did not produce the enzyme extracellularly. Secretory overproduction of 3-ketosteroid Δ1-dehydrogenasein S. lividans was also identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and on native gel, and the enzyme reaction was confirmed by reverse-phase HPLC using 4-androstene-3,17-dione as a substrate.
• Choi, K. -., Molnar, I., Yamashita, M., & Murooka, Y. (1995). Purification and characterization of the 3-ketosteroid-Δ¹-dehydrogenase of arthrobacter simplex produced in Streptomyces lividans. Journal of Biochemistry, 117(5), 1043-1049.
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  PMID: 8586617;Abstract: The 3-ketosteroid-Δ1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyopearl HFV55S from the supernatant of culture broth and cell-free extracts of S. Lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence, The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (M(r) 54,329), The optimum conditions for its activity were pH 10.0 and 40°C, The enzyme catalyzed the conversion of several 3-ketosteroids, but those containing 11α- or 11β-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.
• Haydock, S. F., Aparicio, J. F., Molnár, I., Schwecke, T., Khaw, L. E., König, A., Marsden, A. F., Galloway, I. S., Staunton, J., & Leadlay, P. F. (1995). Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Letters, 374(2), 246-248.
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  PMID: 7589545;Abstract: The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate. © 1995.
• Molnár, I., Choi, K., Yamashita, M., & Murooka, Y. (1995). Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-AΔ¹-dehydrogenase, 3-ketosteroid-Δ⁵-isomerase and a hypothetical regulatory protein. Molecular Microbiology, 15(5), 895-905.
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  PMID: 7596291;Abstract: The Arthrobacter simplex gene coding for 3-ketosteroid-Δ1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-Δ1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-Δ5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-Δ1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-Δ1-dehydrogenase. The expression of 3-ketosteroid-Δ5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.
• Schwecke, T., Aparicio, J. F., Molnár, I., König, A., Khaw, L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cortés, J., Lester, J. B., Böhm, G. A., Staunton, J., & Leadlay, P. F. (1995). The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proceedings of the National Academy of Sciences of the United States of America, 92(17), 7839-7843.
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  PMID: 7644502;PMCID: PMC41241;Abstract: The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identified so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.
• Molnár, I. (1994). Secretory production of homologous and heterologous proteins by recombinant Streptomyces: what has been accomplished?. Bioprocess technology, 19, 81-104.
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  PMID: 7764787;
• Molnar, I., & Murooka, Y. (1993). Helix-turn-helix DNA-binding motifs of Streptomyces - A cautionary note [1]. Molecular Microbiology, 8(4), 783-784.
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  PMID: 8332068;
• Molnar, I., & Murooka, Y. (1993). Nucleotide sequence analysis of a region upstream of the cholesterol oxidase-cytochrome P450 operon of Streptomyces sp. SA-COO revealing repeating units coding for putative transmembrane and DNA-binding proteins. Journal of Fermentation and Bioengineering, 76(4), 257-264.
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  Abstract: A 5.8-kb segment from the upstream region of the cholesterol oxidase (choA)-cytochrome P450 (choP) operon of Streptomyces sp. SA-COO was sequenced. Computer assisted analysis of the sequence revealed four open reading frames (ORFs), whose deduced gene products could be classified into two groups. Cho-Orf1, the C-terminal segment of Cho-Orf3, and Cho-Orf4 were homologous to each other and showed similarities to the DNA-binding domains of bacterial response regulators of the UhpA subfamily, while a putative transmembrane protein, Cho-Orf2, and the N-terminal segment of Cho-Orf3 were homologous to each other but no homologies to known proteins were found. The genes coding for these putative proteins appeared to be organized as repeating units. Structural features of the nucleotide sequence and the homologies of the predicted gen products are discussed.
• Molnár, I., Hayashi, N., Choi, K. -., Yamamoto, H., Yamashita, M., & Murooka, Y. (1993). Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one. Molecular Microbiology, 7(3), 419-428.
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  PMID: 8459768;Abstract: A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-cholesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.
• Molnar, I., Choi, K. -., Hayashi, N., & Murooka, Y. (1991). Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector. Journal of Fermentation and Bioengineering, 72(5), 368-372.
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  Abstract: Deletion of a 1.2-kb fragment adjoined upstream of the choP-choA operon and subcloning of the operon into a multi-copy shuttle vector composed of pIJ702 and pUC19 in Streptomyces lividans resulted in the overproduction of Streptomyces cholesterol oxidase extracellularly about 70-fold more than that of the original producer, Streptomyces sp. SA-COO. When the cells of S. lividans carrying the plasmid were grown in an appropriate medium, about 90 to 99% of the enzyme produced was secreted into the medium. Increased concentration of glucose in the culture medium resulted in a significant decrease in the production of the enzyme, while elevated peptone concentrations led to a further increase in the production. The level of the overproduction of cholesterol oxidase was dependent on the copy numbers of the plasmids as well as the presence of the sequences derived from pUC19.

Presentations

• Molnar, I. (2018, Fall). Perfecting fungal leads: Combinatorial biosynthetic approaches towards novel benzenediol lactones. International Symposium: From Mutases to Megasynthases. Cambridge, U.K.: University of Cambridge.
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  Invited plenary lecture
• Molnar, I. (2016, August). Combinatorial biosynthesis of fungal bioactive polyketides. Invited plenary lecture at the 2016 symposium “Environment•Microorganism•Health”. Wuhan, China: Huazhong Agricultural University.
• Molnar, I. (2016, August). Use, discovery, manufacture and biosynthesis of secondary metabolites for agricultural use: a 4-lecture mini course. Invited colloquium series at the Huazhong Agricultural University, Wuhang, China. Wuhang, China: Huazhong Agricultural University.
• Molnar, I. (2016, May). Combinatorial biosynthesis of bioactive benzenediol lactones.. Invited lecture at Dalian University China. Dalian, China: Dalian University.
• Molnar, I. (2016, May). Combinatorial biosynthesis of bioactive benzenediol lactones. Invited lecture at the College of Biological Sciences, China Agricultural University. Beijing, China: College of Biological Sciences, China Agricultural University.
• Molnar, I. (2016, May). Combinatorial biosynthesis of bioactive benzenediol lactones. Invited lecture at the College of Food Sciences, China Agricultural University. Beijing, China: College of Food Sciences, China Agricultural University.
• Molnar, I. (2016, May). Genetic manipulation of microbial natural product biosynthesis: Agricultural perspectives. Invited lecture at the College of Agronomy, China Agricultural University. Beijing, China: College of Agronomy, China Agricultural University.
• Molnar, I. (2015, July). Invited plenary lecture: Towards “Unnatural Products”: Combinatorial Biosynthesis of Bioactive Benzenediol Lactone Polyketides. 17th International Congress of the Hungarian Society for Microbiology. Budapest, Hungary.
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  Invited plenary lecture
• Molnar, I. (2015, March). Combinatorial biosynthesis of bioactive benzenediol lactones. Invited lecture at the Helmholtz-Institute for Pharmaceutical Research, University of Saarland, Saarbruecken Germany. Saarbruecken, Germany.
• Molnar, I. (2014, 06-26-2014). Nematicidal peptaibols from Drechmeria coniospora. Invited lecture at the China Pharmaceutical University. Nanjing, China.
• Molnar, I. (2014, 06-30-2014). Molecular genetics of benzenediol lactone polyketide biosynthesis. Invited lecture at the Zhejiang Academy of Agricultural Sciences. Hangzhou, China.
• Molnar, I. (2014, 07-04-2014). Genomics of Drechmeria coniospora, a near-obligate endoparasitoid of nematodes. Invited lecture at the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences. Beijing, China.
• Molnar, I. (2014, 2014-06-24). Heterologous biosynthesis of paecilomycins in Saccharomyces cerevisiae. Invited lecture at the South China Institute of Botany, Chinese Academy of Sciences. Guangzhou, China.
• Molnar, I. -. (2013, January). Bio-crude transcriptomics. Annual NAABB meeting. Tempe, AZ: National Alliance of Biofuels and Bioproducts.
• Anderssen, E., Hackett, J., & Molnar, I. (2012, August). Sequencing and De Novo Annotation of the Transcriptomes of Botryococcus Braunii Races A and L for Creating Biofuels. McNair Scholars Symposium at UC Berkeley. Berkely CA.
• Anderssen, E., Hackett, J., & Molnar, I. (2012, August). Sequencing and De Novo Annotation of the Transcriptomes of Botryococcus Braunii Races A and L for Creating Biofuels. UA Ronald E. McNair Summer Research Colloquia at the UA. Tucson, AZ.
• Anderssen, E., Hackett, J., & Molnar, I. (2012, November). Sequencing and De Novo Annotation of the Transcriptomes of Botryococcus Braunii Races A and L for Creating Biofuels. Annual Biomedical Research Conference for Minority Students. San Jose, CA.
• Molnar, I. -., Molnar, ., & et, a. l. (2012, February). Exploring modularity. Guest seminar for the students and faculty of PLS. Tucson, AZ: School of Plant Sciences.
• Molnar, I. -., Molnar, ., & et, a. l. (2012, January). Bio-crude transcriptomics in Botryococcus. NAABB project conference. San Diego: NAABB algal biofuels consortium.
• Xu, Y., & Molnar, I. (2012, May). Biosynthetic reprogramming of heat shock response modulatory metabolites of filamentous fungi. Beijing, China: Chinese Academy of Agricultural Sciences.
• Xu, Y., & Molnar, I. (2012, May). Collaborating polyketide synthases from filamentous fungi. Beijing, China: Chinese Academy of Sciences, Dept. Microbiology.
• Xu, Y., & Molnar, I. (2012, May). Combinatorial biosynthesis of insecticidal secondary metabolites of filamentous fungi. Hefei, China: Chinese Academy of Agricultural Sciences, Hefei branch.
• Molnar, I. -. (2011, February). Botryococcus molecular genetics for biofuels production.. Guest seminar for the UA members of the NAABB consortium and students of CEE. Tucson, AZ: Dept Chemical and Environmental Engineering.
• Molnar, I. -. (2011, September). Cooperating fungal iterative polyketide synthases for resorcylic acid lactone production. 2nd Annual CIPKEBIP conference. Ljubljana, Slovenia: CIPKEBIP.
• Molnar, I. -. (2011, September). Exploring modularity - Cooperating fungal iterative polyketide synthases for resorcylic acid lactone production. 12th Intl. Conf. on the Chemistry of Antibiotics and Other Bioactive Compounds. Berlin, Germany.

Poster Presentations

• Pocsi, I., Miskei, M., Xu, Y., Lin, M., & Molnar, I. (2015, July). Sequencing and annotation of the genome of the nematophagous fungus Drechmeria coniospora reveals the arsenal of an endoparasitoid fungus for the invasion of its host organisms. 17th International Congress of the Hungarian Society for Microbiology. Budapest, Hungary.
• Molnar, I. -. (2011, March). Characterization of the biosynthesis of radicicol from Chaetomium chiversii. 16th Annual Institute of Biological Engineering Conference. Atlanta, GA.