Poster Session 1 Natural Product Discovery and Development in The

Session Poster Session 1 Natural Product Discovery and Development in the Genomic Era Paper Sonogashira diversification of unprotected halotryptophans, halotryptohan containing tripeptides; and generation of a new to nature bromo-natural product and its diversification in water Chris Cartmell, University of St. Andrews, Strathkinness, United Kingdom Natural Product Discovery and Development in the Genomic Era The blending together of synthetic chemistry and natural product biosynthesis provides a potentially powerful route to new natural product analogues2. Cystargamide is a structurally interesting lipo-depsi peptide containing a 5-hydroxy tryptophan as well as a 2, 3-epoxydecanoyl fatty acid chain3. We envisaged that by installing a sufficiently reactive handle (e.g. a C-Br bond) and developing compatible mild aqueous chemistries, biosynthesis of the halo-cystargamides and its subsequent chemical modification can be achieved. Precursor directed biosynthesis (PDB) provides a great potential for the production of new natural product analogues by exploiting the natural promiscuity of the enzymes involved in the biosynthesis of natural product. Using PDB method, we achieved the incorporation of various chloro/bromo-tryptophans and generated a series of halogenated analogues of cystargamide. 6-Br-cystargamide was subsequently diversified using aqueous cross coupling chemistries such as the Sonogashira reaction to obtain new derivatives of cystargamide. The installation of bromo/chloro handle also provided an excellent analytical handle for investigation of all metabolites by LC-MS/MS. With knowledge gained from the fragmentation of the natural cystargamide, we analysed the MSn data to identify and characterise the new natural products analogues produced via the Sonogashira reaction1. References 1. J. Corr, S. V. Sharma, C. Pubill-Ulldemolins, et al. Chem Sci., 8, 2039-2046, (2017) Paper Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus Dr. Matthew Lebar1, Dr. Jeffrey Cary1, Rajtilak Majumdar1, Carol Carter-Wientjes1, Brian Mack1, Qijian Wei1, Valdet Uka2, Prof. Sarah DeSaeger3 and Dr. Jose Di Mavungu3, (1)USDA, New Orleans, LA, USA, (2)University of Ghent, Ghent, Belgium, (3)University of Ghent, Gent, Belgium Natural Product Discovery and Development in the Genomic Era Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. In silico analysis of the A. flavus genome has revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 was shown to be composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus previously identified as ferriaspergillin. We observed significantly less fungal growth on maize kernels infected with A. flavus ΔasaC compared to A. flavus control and are currently investigating the role aspergillic acid plays in maize kernel infection, possibly through iron sequestration. Paper Photoprotection of the potent polyene antibiotic Marinomycin Christopher Bailey1, Joseph Zarins-Tutt1, Matthias Agbo1, Alberto Diego Taboada2, Maoluo Gan1, Emily Abraham1, Refaat Hamed1, Grahame Mackenzie2, Andrew Evans3 and Rebecca Goss1, (1)University of St. Andrews, St Andrews, United Kingdom, (2)Sporomex Ltd, Bridlington, United Kingdom, (3)Queen's University, Kingston, ON, Canada Natural Product Discovery and Development in the Genomic Era Bioactive polyenes are abundant in nature showing a broad range of chemical diversity and biological activities. Despite the promising bioactivity of many isolated polyenes, the inherent instability of polyene systems towards photoisomerization continues to deter their development towards potential lead drug candidates. Marinomycin is a particularly noteworthy, but photolabile, polyene antibiotic with excellent activity against MRSA and VRSA.1 We envisaged that microencapsulating polyenes using a chemically inert, biocompatible, non-toxic natural polymers could be utilised to improve photostability and as a drug carrier. We have demonstrated a striking level of photoprotection (t1/2 extended from 8 seconds – 34 minutes, when exposed directly to 312 nm UV radiation at 48 W) using a natural biopolymer. Our results show promise for the clinical development and administration of polyenes, such as marinomycin, that would otherwise be too unstable to consider. Additionally a serendipitous discovery revealed that our biopolymer could be used to selectively extract culture broths containing marinomycin providing higher recovery than extraction with conventional XAD resins. References H. C. Kwon, C. A. Kauffman, P. R. Jensen and W. Fenical, J. Am. Chem. Soc., 2006, 128, 1622–1632. Paper Biochemical role of multicore precursor peptides in a RiPP pathway Wenjia Gu, Debosmita Sardar and Eric Schmidt, University of Utah, Salt Lake City, UT, USA Natural Product Discovery and Development in the Genomic Era Cyanobactin pathways are diversity-generating biosynthetic pathways that can process hypervariable substrates. Each cyanobactin precursor peptide contains multiple core sequences encoding different natural products, which is not common in RiPPs. The role of these multicore precursor peptides remains unclear. Preliminary in vivo data suggests that the cassette position influences the production yield. Here, we perform biochemical experiments that define the role of multiple cassettes in cyanobactin RiPP biosynthesis. Investigation of the mechanisms behind this recognition will help us better control cyanobactin production for potential therapeutic applications. Paper Biosynthetic and genome mining approaches to identify triazine containing natural products Khaled Almabruk1, Michael Fenwick2, Steven Ealick2 and Benjamin Philmus1, (1)Oregon State University, Corvallis, OR, USA, (2)Cornell University, Ithaca, NY, USA Natural Product Discovery and Development in the Genomic Era Triazine containing natural products are of interest because of their attractive biological activities and the unique chemistry involved in their biosynthesis. One of the most studied examples is toxoflavin, which has been isolated from plant pathogenic strains of Burkholderia, and plays a key role in virulence. Genome mining has revealed that triazine natural product gene clusters are found in pseudomonads and actinobacteria suggesting that triazine-containing natural products are more prevalent than previously realized. In this study, we present our results from precursor labeling studies in an effort to shed light on the complex biosynthetic steps involved in forming the N-N-bond of the triazine moiety during toxoflavin biosynthesis. We also discuss the crystallographic and the in vitro characterization of two N- methyltransferases that use 1,6-didesmethyltoxoflavin (1,6-DDMT) as a substrate. Paper Metagenomics-guided natural product discovery and engineering in bacteria Teresa Shock, Xiao Yang, Deepak Chandran, Jennifer Shock, Oliver Liu and Jeffrey Kim, Radiant Genomics, Emeryville, CA, USA Natural Product Discovery and Development in the Genomic Era Metagenomics continues to play an increasingly important role in the discovery of natural products. With the combination of next-generation sequencing, bioinformatics, genetic engineering and natural product chemistry, previously-inaccessible chemical diversity in our environment are being gradually uncovered. These technologies also circumvent the constraints of traditional culture-based natural product discovery, allowing for targeted approaches in heterologous hosts. This work focuses on recent progress in pipeline development and process engineering in a startup-scale industrial setting. Paper Autometa: Automated extraction of microbial genomes from shotgun metagenomes Ian Miller, Evan Rees, Jennifer Ross, Izaak Miller, Jared Baxa, Dr. Juan Lopera, Robert Kerby, Dr. Federico Rey and Dr. Jason Kwan, University of Wisconsin - Madison, Madison, WI, USA Natural Product Discovery and Development in the Genomic Era Culture-independent sequencing (metagenomics) is a powerful, high resolution technique enabling the study of microbial communities in situ. With modern sequencing technology and bioinformatics, individual genomes can be assembled and extracted directly from environmental samples containing complex microbial communities by a process known as metagenomic "binning." However, available binning programs suffer from methodological and practical shortcomings, such as the

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