Bradley S. Moore - US grants

A very important task in the ongoing search for new clinically useful drugs is the generation of large numbers of structurally diverse compounds. These molecules are required for screening by high-throughput bioassays in the discovery of new lead drug candidates. Combinatorial biosynthesis, in which nature's chemical capabilities are exploited in a combinatorial "mix-and-match" fashion, has generated libraries of novel molecules representing great structural diversity which are not available naturally or generated through combinatorial synthesis. The marine bacterium "Streptomyces maritimus" is uniquely capable of naturally producing a diverse series of structurally uncommon bactereostatic polyketides known as the enterocins and wailupemycins. Cloning, sequencing, and heterologous expression of the single biosynthesis gene cluster (enc) for these molecules revealed an unprecedented iterative type II polyketide synthase (PKS) system. Polyketide structural variability is probably achieved by the lack of a dedicated cyclase and the action of a rare enzyme-mediated Favorskii rearrangement. The novel architecture of this natural PKS gene set furnishes insight into engineering molecular diversity through genetic recombination and provides the major rationale for the proposed project. In this grant application, we propose to continue our biosynthetic analysis of the enterocin family at the chemical, biochemical and genetic levels. Our primary goal is to fully characterize this natural biosynthetically diverse pathway in order to engineer mutant organisms harboring and expressing altered gene clusters in which specific biosynthetic genes have been deleted, added or replaced with homologous genes from other biosynthetic pathways. The resulting recombinant will be chemically analyzed for the production of polyketide analogs and assayed for biological activities. Biosynthetic enzymes that will be exploited in recombinant systems include: EncM, a flavin-dependent oxygenase that is putatively involved in the derailment of the minimal enc PKS from generating aromatic endproducts, the enzymes involved in the synthesis and incorporation of the rare benzoyl-CoA PKS starter unit, the regiospecific P450 monooxygenase EncR, and the substrate tolerant methyltransferase EncK. Secondly, the "S. Maritimus" enc genes and their sequences will be used to analyze an enterocin-containing marine invertebrate and its associated microflora for the presence of homologous genes in order to model marine microbial-invertebrate symbiosis.

A new mechanism of polyketide assembly has emerged in bacteria for the biosynthesis of small aromatic residues that serve as important structural elements in a growing number of biologically active natural products. These small aromatic polyketides are synthesized by homodimeric (type III) polyketide synthases (PKSs) that are phylogenetically and biochemically related to ubiquitous plant PKSs such as chalcone synthase. Thus far, type III PKSs have been shown to be responsible for the biosynthesis of natural products such as 1,3,6,8- tetrahydroxynaphthalene (THN) and the formation of key components of more complex antimicrobial and antitumor natural products such as vancomycin, naphterpin, marinone, and kendomycin. While type III PKSs are architecturally simple, they arguably represent the most sophisticated PKSs mechanistically since embodied within their homodimeric architecture is the catalytic machinery necessary for starter molecule recognition and loading, malonyl- CoA decarboxylation and polyketide chain extension, and ultimately, multiple pathways for termination. Their simple gene and protein architecture makes them amendable for study using a variety of sophisticated approaches including heterologous biosynthesis, in vitro and in vivo biochemical analysis, directed and random approaches towards enzyme engineering, and atomic resolution protein x-ray crystallography. Although the analysis of related plant enzymes is fairly mature, research on the bacterial counterparts is only beginning and can be expected to yield novel, interesting, and potentially important information on these simple condensing enzymes. Moreover, the mechanistic and structural understanding of bacterial type III PKSs is likely to be relevant for the productive reengineering of modular type I and iterative type II bacterial PKSs. With the high resolution three-dimensional crystal structure of the first bacterial PKS, THN synthase from Streptomyces coelicolor A3(2), nearly in hand, the stage is set for a comprehensive structural and mechanistic analysis of this new subclass of bacterial PKS. Studies will extend to other bacterial type III PKSs, including those involved in the biosynthesis of the clinically important glycopeptide vancomycin, the broad spectrum antibiotic 2,4- diacetylphloroglucinol, and the antitumor antibiotic marinone.

DESCRIPTION (provided by applicant): Natural aromatic polyketides such as the antibiotic tetracycline and the anticancer agent daunorubicin represent an important class of Pharmaceuticals that, together with their semi-synthetic derivatives, command a vital role in human health. A basic understanding of aromatic polyketide assembly catalyzed by type II polyketide synthases (PKSs) at the biochemical and structural levels will undoubtedly increase our appreciation for these important biosynthetic processes and will aid in the rational engineering of new chemical entities. While the past decade has witnessed substantial growth in our basic knowledge on how aromatic polyketides are naturally synthesized, a number of fundamental gaps still persist today. We thus propose in this competitive renewal application to further our biosynthetic studies on the polyketide antibiotic enterocin, which has emerged as an important vehicle to address the early stages in aromatic polyketide assembly involving starter unit selection, timing of the ketoreduction reaction, cyclization potential as well as post-PKS modification reactions. Their simple gene and protein architecture makes them amendable for study using a variety of sophisticated approaches including heterologous biosynthesis, in vitro and in vivo biochemical analysis, directed and random approaches towards enzyme engineering, and atomic resolution protein x-ray crystallography. The specific aims of this proposal are thus: (1) to biochemically characterize the enterocin PKS priming and extension reactions using a recently developed in vitro process together with high-resolution protein mass spectrometry;(2) to biochemically characterize the flavoprotein EncM, which catalyzes an unprecedented series of biosynthetic reactions involving oxidative favorskii-like rearrangement, aldol condensation and heterocycle-forming reactions;and (3) to mechanistically and structurally characterize phenylalanine ammonia-lyase, a rare prokaryotic enzyme first discovered in the enterocin biosynthetic pathway and now in pre-clinical trials to treat the childhood disorder phenylketonuria. The outcome of this research plan will illuminate new biochemical reactions in natural product biosynthesis and will provide the opportunity to develop novel biocatalysts in bioorganic chemistry as well as therapeutic enzymes in the case of PAL.

DESCRIPTION (provided by applicant): Salinosporamide A is a potent irreversible proteasome inhibitor presently in phase Ib human clinical trials for the treatment of multiple myeloma and other cancers. This marine bacterial natural product has a distinctive mechanism of action based on its ?-lactam-ß-lactone pharmacophore that differs from the only FDA-approved proteasome inhibitor, the peptide boronate bortezomib. During the last period of support, we established the biosynthetic foundation of salinosporamide assembly and discovered a number of novel enzymatic reactions in halogenation, prephenate biochemistry, and polyketide precursor supply. Translation of this basic knowledge allowed us to rationally design through genetic engineering new salinosporamide analogues for biological evaluation. This work helped determine the structure-activity relationships within the salinosporamide family of anticancer agents. Despite our significant progress to date, we still only have a cursory understanding of how salinosporamide is biosynthesized due to its unprecedented assembly from novel molecular building blocks. Numerous questions remain, while new opportunities have surfaced in response to discoveries made in this ongoing research program. Opportunities in enzyme discovery, synthetic biology, chemoenzymatic synthesis, genome mining, and proteasome biochemistry are uniquely suited for this natural product biosynthetic program. To accomplish the broad goals outlined in this application, we propose a multidisciplinary project involving five specific aims. First, we plan to functionally and structurally characterize the SalC ketosynthase and its key biosynthetic role in the formation of the ?-lactam-ß-lactone core of salinosporamide. Second, we will apply the function of SalC to develop a streamlined chemoenzymatic synthesis of salinosporamide derivatives based on a focused library of ?-lactam-ß-lactones from synthetic acylamino acid thioesters with recombinant salinosporamide biosynthetic enzymes. Third, we aim to functionally characterize the biosynthetic enzymes responsible for the synthesis of salinosporamide's novel amino acid residue, cyclohexenylalanine, which is paramount to its potent proteasome binding affinity. Fourth, we will functionally characterize the dedicated proteasome ß-subunit SalI and its hypothesized role in S. tropica self-resistance against salinosporamide. And fifth, we plan to develop new crotonyl-CoA reductase-based expression systems for the engineered biosynthesis of new polyketide synthase extender units with halogenated (fluorine and chlorine) and branched side chains for the design of new polyketide molecules.

DESCRIPTION (provided by applicant): Bacteria belonging to the Order Actinomycetales, commonly called actinomycetes, account for approximately 75% of the microbial natural products used in human therapy. While major pharmaceutical companies have moved en masse away from natural products as a resource for small molecule drug discovery, it has recently become recognized that actinomycetes derived from marine sources represent an important new source of structurally diverse natural products. In particular, marine actinomycetes belonging to the genus Salinispora have proven to be a rich source of biologically active secondary metabolites including salinosporamide A, which recently completed phase I clinical trials for the treatment of cancer. The compounds produced by this chemically prolific genus span virtually all known biosynthetic classes and have led to the characterization of a growing number of unprecedented biosynthetic paradigms. After more than two decades of extensive marine sampling, we have amassed a collection of more than 5,000 diverse Salinispora strains. This collection provides unprecedented opportunities to further explore the biosynthetic potential of this extraordinary taxon and to address fundamental questions about mechanistic biochemistry and the evolutionary processes that generate new structural diversity. Here we propose the continuation of a genome-mining project that was initiated less than four years ago to analyze six Salinispora genome sequences. This renewal application builds upon the productive collaboration established between the Moore (biosynthesis/genome mining), Jensen (bioinformatics/microbiology) and William Fenical (natural product chemistry) laboratories. In this renewal, we specifically address five major aims: 1) the bioinformatic analysis of 101 Salinispora genome sequences, 2) the genome guided isolation and characterization of new, biologically active Salinispora natural products, 3) the expression of Salinispora biosynthetic gene clusters by transformation-associated recombination cloning, 4) the activation of 'silent' biosynthetic pathways by regulatory manipulation, and 5) the biosynthetic analysis of the Salinispora mTOR inhibitor lymphostin. These aims address fundamentally important questions related to the diversity and distributions of secondary metabolite biosynthetic pathways in a well-defined taxon and the evolutionary processes that generate new small molecule diversity.

Administrative Core Summary The Administrative Core for the Scripps Center for Oceans and Human Health will be managed out of Scripps Institution of Oceanography (SIO) at UC San Diego. The center will be under the leadership of Drs. Bradley Moore, Skaggs School of Pharmacy and Pharmaceutical Sciences and SIO, and Lihini Aluwihare, SIO. The two center directors will oversee the administrative requirements of the center, provide work direction for center staff, and ensure that frequent and substantive communication occurs across the research projects. Drs. Moore and Aluwihare will be supported by an internal steering committee comprised of the other program leaders. The center will also have a four-person External Advisory Committee (EAC) that will influence research directions and evaluate center progress. Twice in the course of the center's five years of funding, the annual EAC meeting will be broadened into a workshop allowing center investigators and the EAC to interact with basic research scientists, health professionals, policy makers, and the general public in a unique format to discuss halogenated organic compounds in the environment and how they relate to human health and public safety. This workshop will fill a critical gap currently existing in research conference offerings. The Scripps Center for Oceans and Human Health will employ two part-time employees - a business manager and data manager. These staff will be responsible for the day-to-day functioning of the center and be charged with fulfilling center outreach objectives by administering programs and maintaining the center's website. The center will also be supported by the administrative infrastructure already in place at SIO, including a business office well versed in NIH/NSF awards, and access to central departments such as communications, government relations, and foundation relations.

The bioaccumulation of halogenated organic compounds (HOCs) in the marine food web provides a direct route for human exposure to several classes of persistent organic pollutants. Natural polybrominated organic compounds such as polybrominated diphenyl ethers, polybrominated dibenzodioxins and polybrominated bipyrroles are collectively proposed to be synthesized by marine organisms such as cyanobacteria and red algae involving unknown metabolic pathways harboring yet to be discovered halogenating enzymes. The goals of this project are to provide a genetic and biochemical foundation for the microbial biosynthesis of HOCs in the marine environment. Our research strategy includes a comprehensive genetic, biochemical, and enzyme structure-based analysis of polybrominated metabolite biosynthesis in two model marine bacterial groups, Pseudoalteromonas and Streptomyces, as well as other HOC producing strains discovered in the course of the research. We will provide a direct interrogation of natural maririe samples enriched in HOCs to identify and characterize the prevalence of these biosynthetic pathways in the marine environment. The proposed work will be undertaken jointly by the laboratories of Allen and Moore at Scripps who have a proven track record of collaboration and joint student mentorship. The success of this Project is based on biochemists, microbiologists, structural biologists and genome scientists working together; thus we have enlisted the help of Moore's long-standing collaborator Prof. Joseph Noel (Salk Institute for Biological Studies, La Jolla) to join the Project through a sub-contract to assist in the protein crystallography of brominating enzymes in order to provide a detailed understanding of the structural basis behind enzymatic bromination. Collectively, this project will deliver new molecular-based insight into organohalogen biosynthesis that will be united with other Center investigations to explore the diversity and ecology of these compounds and their impacts on oceans and human health. 1

Project Summary / Abstract    The  emergence  of  antibiotic  resistance  has  created  a  global  dilemma  for  the  need  to  discover  new antibacterial lead agents. Realizing this critical need for new antibacterial agents with new  structure types and targets, we focus here on the biosynthetic interrogation and development of  structurally  distinct  marine  bacterial  natural  products.  An  underlying  theme  associated  with  many marine microbial antibiotics involves the use of aromatic polyketide frameworks that have  undergone  extensive  oxidative  tailoring  reactions  catalyzed  by  halogenase  and  oxygenase  biosynthetic  enzymes.  In  this  application,  we  propose  a  multidisciplinary  project  involving  heterologous  biosynthesis,  mechanistic  enzymology,  atomic  resolution  protein  X-­ray  crystallography, chemoenzymatic synthesis, and genetic engineering to understand and control  the molecular basis of polyketide diversification in a series of marine bacterial compounds with  promising  antimicrobial  properties.  To  accomplish  the  broad  goals  outlined  in  this  application,  we propose four specific aims. First, we plan to functionally and structurally characterize diverse  meroterpenoid  V-­dependent  chloroperoxidases  and  their  catalytic  properties  in  promoting  antimicrobial  chemical  diversity.  Second,  we  will  discover,  characterize,  and  engineer  biosynthetic  pathways  for  structural  diversification  of  halogenated  pyrrole  containing  bioactive  natural  products.  Third,  we  aim  to  functionally  characterize  the  unprecedented  biosynthesis  of  thiotetronic  acid  polyketide  antibiotics  and  apply  new  biosynthetic  reactions  to  extend  the  synthesis  and  bioengineering  of  novel  molecules.  And  fourth,  we  will  interrogate  the  antimicrobial  activity  and  mechanism  of  new  meroterpenoid,  bipyrrole,  and  thiotetronate  compounds. 

DESCRIPTION (provided by applicant): Ribosomally encoded natural products were once thought to be of limited structural diversity and uncommon amongst microbes. Over the past few years, however, this viewpoint has changed due to the increased discovery rate of RNPs possessing newly described structural motifs previously ascribed to their nonribosomal counterparts. Nearly every sequenced genome, including invertebrates, contains the genetic capacity to biosynthesize ribosomally- encoded, post-translationally modified natural products such as lantibiotics, bacteriocins, microcins, cyanobactins, thiopeptides, and lasso peptides, thereby making this class of underappreciated natural products perhaps the most dominant in all of nature. What is lacking, however, is a systematic approach to harvest this ubiquitous class of natural products and assess their unique biosynthetic capacity. The difficulty associated with characterizing RNPs in a systematic fashion can be attributed to their falling outside the scope of not only most therapeutic screening programs but also metabolomic or proteomic approaches due to their larger size, structural diversity and extraordinary number of post-translational modifications. This proposal outlines the developmental strategies to create a set of tools for harnessing the biosynthetic potential of ribosomally encoded natural products through mass spectrometry based genome mining. The techniques and methodologies created as a result of the proposed work will not only be important for the detection of therapeutic lead compounds, but also for the efficient characterization of ribosomally encoded toxins secreted by pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus and Clostridium difficile as well as defensins produced by higher eukaryotes such as marine snails, primates and humans.

DESCRIPTION (provided by applicant) The specific aims of the Administrative Core are to: 1) Provide the administrative infrastructure for the Center, including the initial point of contact and information clearinghouse for all stakeholders, including collaborators, funding agencies, and the community. 2) Facilitate communication and collaboration between the three R0Is and the Analytical Facility Core. 3) Provide a platform for Center activities, including evaluation. 4) Manage all conference and workshop logistics. 5) Maintain the website and engage in community outreach by updating content with Center research and related activities. 6) Facilitate communication and collaboration across the currently funded Centers for Ocean and Human Health and applicable R0Is funded under the companion FOA RFA-ES-11-013. 7) Ensure all NIH reporting requirements are met.

This award will provide NSF support for the establishment of the Scripps Center for Oceans and Human Health at the Scripps Institute of Oceanography of the University of California - San Diego. The Scripps COHH research team will apply a multidisciplinary approach to elucidate the marine cycling of small, natural, brominated, aromatic compounds that share chemical characteristics with some anthropogenic contaminants. The study will be focused in the Southern California Bight where we have applied a new non-targeted analytical approach to demonstrate the presence of >300 halogenated organic compounds in dolphins - an apex marine predator - feeding either offshore or inshore. As many as 30% of these compounds contain bromine and have no known anthropogenic source. In some cases, similar compounds have been previously hypothesized to be of natural origin in other marine environments. Given that these compounds bioaccumulate in apex marine predators they must be available to enter human populations through seafood consumption.

These brominated, likely natural organic compounds will be the focus od Center activity for two main reasons. First, although several studies have documented the presence of these purported natural compounds in top predators, most have been unable to delineate trophic transfer, and no study has definitively identified source organisms. the team has recently identified a biosynthetic cluster in a marine bacterium that is capable of producing most of the carbon skeletons and bromination patterns of interest. To establish spatial patterns and ubiquity of source organisms they will continue to characterize this biosynthetic pathway through culture studies and examine environmental distributions through metagenomics. Furthermore, by using our non-targeted analytical approach to comprehensively survey all trophic levels in benthic and pelagic habitats they will directly demonstrate how these compounds enter apex predators. This will further enable us to delineate potential pathways by which these compounds enter human populations.

Secondly, the presence of these compounds in apex predators indicates that they must enter the human population via seafood consumption, but this has not been documented. To test this the team will apply a non-targeted method to analyze breast milk from local mothers who have been surveyed to document their seafood consumption habits. Since these compounds resemble anthropogenic contaminants such as PBDEs, PCBs and PCDDs, they are expected to have similar toxic effects in both humans and wildlife. Brominated, natural compounds that are most abundant in dolphins have pyrrole backbones and their toxic impacts are poorly documented, and so, they will examine the potential toxicity of these compounds in the zebra fish model. Together, these efforts seek to identify source organisms and biosynthetic mechanisms of production, and also delineate modes of transfer to human populations.

Broader Impacts. A more complete picture of the marine cycling of these compounds will enable us to assess how global change may impact sources -- something that up to now has been impossible. Furthermore, we will be in a position to assess the role that the burgeoning aquaculture industry and general seafood consumption plays in transferring these compounds to human populations. The Center organizational structure and ongoing collaborations with NOAA and local water resources management agencies will insure that the research approach and findings benefit from the input of individuals who dictate public health policy decisions, carry out environmental monitoring, and manage resources. The Center web portal (www.scohh.ucsd.edu) and personal outreach efforts will also keep the public informed and engaged in our research activities through interaction with K-12 classrooms and local communities. A special effort will be made to engage URM undergraduates in the Center's research through participation in the UCSD STARS program, the SIO NSF-sponsored SURF program and the UCSD-Howard University Pathways program.

JOINT FUNDING BY NSF AND NIEHS: The original proposal on which this project is based (P01 ES021921-01) was submitted to the National Institutes of Environmental Health Sciences (NIH/NIEHS) in response to Funding Opportunity Announcement RFA-ES-11-012 , "Centers for Oceans Human Health (P01)?" an opportunity jointly sponsored by NSF. This project is cooperatively funded through separate awards from NSF and NIEHS.

The bioaccumulation of halogenated organic compounds (HOCs) in the marine food web provides a direct route for human exposure to several classes of persistent organic pollutants. Natural polybrominated organic compounds such as polybrominated diphenyl ethers, polybrominated dibenzodioxins and polybrominated bipyrroles are collectively proposed to be synthesized by marine organisms such as cyanobacteria and red algae involving unknown metabolic pathways harboring yet to be discovered halogenating enzymes. The goals of this project are to provide a genetic and biochemical foundation for the microbial biosynthesis of HOCs in the marine environment. Our research strategy includes a comprehensive genetic, biochemical, and enzyme structure-based analysis of polybrominated metabolite biosynthesis in two model marine bacterial groups, Pseudoalteromonas and Streptomyces, as well as other HOC producing strains discovered in the course of the research. We will provide a direct interrogation of natural maririe samples enriched in HOCs to identify and characterize the prevalence of these biosynthetic pathways in the marine environment. The proposed work will be undertaken jointly by the laboratories of Allen and Moore at Scripps who have a proven track record of collaboration and joint student mentorship. The success of this Project is based on biochemists, microbiologists, structural biologists and genome scientists working together; thus we have enlisted the help of Moore's long-standing collaborator Prof. Joseph Noel (Salk Institute for Biological Studies, La Jolla) to join the Project through a sub-contract to assist in the protein crystallography of brominating enzymes in order to provide a detailed understanding of the structural basis behind enzymatic bromination. Collectively, this project will deliver new molecular-based insight into organohalogen biosynthesis that will be united with other Center investigations to explore the diversity and ecology of these compounds and their impacts on oceans and human health.

? DESCRIPTION (provided by applicant): The increasing prevalence of bacterial pathogens that are resistant to most of the clinically approved antibiotics is an alarming situation that has spurred renewed interest in antibiotic discovery programs. Since most antibiotics are derived from natural products produced by microorganisms, there is now intense interest in using new methods to screen genetically and chemically diverse collections of bacteria. However, identifying new molecules from bacterial extracts is confounded by the overwhelming presence of previously identified molecules as well as the fact that most of the biosynthetic potential of a organism is typically not expressed under laboratory growth conditions. We have characterized a unique collection of microbes obtained from deep within four different caves of New Mexico. Since these bacteria were isolated from remote, underexplored locations that are only just beginning to be mined for antibiotics, there is an increased probability of identifying molecules with unique chemical structures and new modes of action. The goal of this project is to use two new powerful platforms to identify and purify molecules active against multidrug resistant (MDR) bacteria from this diverse collection of cave bacteria. First, we will use our recently developed bacterial cytological profiling (BCP) approach to identify natural products with antibacterial activities in crude organic extracts or directly on plates. BCP uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely cellular target of newly isolated compounds in a few hours. BCP works in complex crude extracts and subsequent fractions, allowing it to be used to guide natural product purification. We will sequence strains producing antibiotics and then use target directed genome mining (TDGM) and heterologous biosynthetic gene cluster (BGC) overproduction to identify novel antibacterial producing BGCs. Heterologous overexpression of normally silent BGCs will allow us to identify molecules missed by traditional screening.

Natural polybrominated organic compounds such as hydroxylated polybrominated diphenyl ethers (OH-BDEs) and polybrominated pyrroles (PBPs) have recently emerged as chemicals of human health concern. These natural product relatives of man-made halogenated persistent organic pollutants (POPs) are widely distributed throughout the marine food web and accumulate in seafood sources consumed by humans. Researchers have demonstrated that OH-BDEs such as 6-OH-BDE-47 (thyroid hormone receptor) and PBPs such as tetrabromopyrrole (ryanodine receptor) are potent toxins and thus pose a potential risk to humans. Many fundamental questions however remain about the extent of sources for these natural organobromine molecules, how these chemicals enter and move through the marine food web, whether changes in the climate will impact their production and accumulation, and whether humans are more or less impacted by natural halogenated POPs versus their man-made counterparts. This project seeks to advance the biology and chemistry of marine contaminants of emerging concern. A better understanding of the sources and sinks of these compounds will improve public understanding about links between oceans and human health and help improve guidelines and evidence-based policies regarding fish consumption and health. The project will support a graduate student and a postdoctoral researcher. The project is jointly supported by NSF and by the National Institute for Environmental Health Sciences (NIEHS).

Recent discoveries by these investigators have rigorously established the genetic and biochemical basis for the microbial synthesis of natural OH-BDE molecules in diverse lineages of marine bacteria. However, the global distribution and ubiquity of these polybrominated POPs in marine biota cannot be fully explained by the sources discovered thus far, suggesting additional biogenic sources exist and are actively contributing to OH-BDE and MeO-BDE accumulation in the marine food web. This information is critical to more accurately identify trophic connections and interconversions that lead to natural PBDE accumulation in marine fish and ultimately, human dietary exposure risks. In this project, new genetic and biochemical evidence for the biosynthesis and biotransformation of PBDE molecules will be established for marine macroalgae, a conspicuous but uncharacterized source of PBDE molecules in marine habitats, using transcriptome analysis coupled with biochemical enzyme characterization. Additional microbial sources for PBDE synthesis/transformation will be characterized by the comprehensive analysis of fish and marine-mammal associated microbiomes using integrated genomic and metabolomic approaches combined with experimental microbiome enrichment reactors amended with PBDE molecules or biosynthetic substrates.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary / Abstract Freshwater harmful algal blooms of cyanobacteria or cyanoHABs are increasing in frequency and impact worldwide due to natural and anthropogenic flooding events. In the USA, all 50 states now regularly experience major cyanoHAB incidents, including Florida and Ohio that have declared states of emergency in recent years to address increased cyanotoxin loads in public waterways posing human health, environmental, and economic threats. While harmful cyanobacteria and their toxins are actively monitored, one of the most neurotoxic cyanotoxins, the organophosphate anatoxin-a(s), is not monitored. This water-soluble, UV-insensitive, and zwitterionic toxin is notoriously reactive, which has led to the ongoing challenges in its environmental monitoring. We recently discovered the genes encoding anatoxin-a(s) biosynthesis from the planktonic cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through a series of genomic, chemical, and biochemical experiments, we have nearly reconstituted the entire biosynthetic pathway from arginine to anatoxin-a(s) with recombinant enzymes. Moreover, we identified nearly complete anatoxin-a(s) transcripts from the nearshore Western Basin of Lake Erie at Toledo, Ohio, suggesting that residents in Toledo as well as other communities across the USA may experience anatoxin-a(s) exposure without their knowledge and that of regional and national monitoring agencies. Our discovery sets the stage for this 2-year, R21 application to apply our biosynthetic expertise to fill the gap in knowledge about the prevalence, significance, and impact of this critical toxin in the environment. We propose four specific aims to address our broad goals. First, we plan to complete the functional assignment of all anatoxin-a(s) biosynthesis enzymes. Second, we will broadly analyze metagenomic and metatranscriptomic fresh water supplies for anatoxin-a(s) genes and to identify the producing cyanobacteria. Third, we aim to develop a rapid, PCR screen specific for environmental anatoxin- a(s) gene detection. And fourth, we will evaluate the native phosphatase AnsH and commercial phosphatases as anatoxin-a(s) degradation enzymes with potential bioremediation applications.