Paul R. Jensen, Ph.D. - US grants

Project Summary    This  renewal  application  builds  upon  a  productive  collaboration  between  the  Moore  (biosynthesis/natural  products  chemistry)  and  Jensen  (bioinformatics/microbiology)  labs.  It  seeks  to  capitalize  on  the  biosynthetic  potential  maintained  in  bacterial  genomes  by  developing  three  specific  areas  of  research:  1)  Transcriptome  guided  natural  product  discovery,  2)  Heterologous  expression,  production,  and  characterization  of  Salinispora  secondary  metabolites,  and  3)  Characterization  and  exploitation  of  secondary  metabolite  regulation.  The  research  will  be  implemented  using  the  model  marine  actinomycete  genus  Salinispora,  for  which  a  unique  culture  collection  and  a  large  number  of  genome  sequences  are  available.  Comparative  transcriptomics  will  be  used  to  distinguish  between  silent and expressed gene clusters, identify key regulatory elements associated with their silencing, and  prioritize  clusters  for  heterologous  expression  and  product  characterization.  The  resulting  compounds  will  be  isolated,  characterized,  and  subjected  to  biological  testing.  A  workflow  has  been  developed  to  target  gene  clusters  that  possess  the  highest  probability  to  yield  structurally  unique  and  biologically  active  compounds.  The  mechanistic  enzymology  associated  with  unique  structural  features  will  be  interrogated  and  used  to  inform  future  structure  predictions.  This  project  will  specifically  address  the  regulation  of  secondary  metabolism  by  testing  the  effects  of  salinipostin  as  an  A-­factor-­like  regulator,  identifying  the  genetic  basis  for  a  spontaneous,  pigment-­less  mutant  that  is  reduced  in  secondary  metabolite  production,  and  introducing  regulatory  elements  whose  disruption  has  been  linked  to  gene  cluster silencing in specific strains. The fundamental goals are to develop new tools and approaches to  identify  the  natural  products  encoded  in  bacterial  genomes,  obtain  new  information  about  the  mechanistic  biochemistry  responsible  for  their  assembly,  and  to  develop  more  efficient  approaches  to  mine bacterial genomes for the structurally unique and biologically active compounds they encode. The  research  effectively  combines  the  complimentary  expertise  of  two  research  groups  in  the  areas  of  marine microbiology, biosynthesis, and natural product discovery. 

DESCRIPTION (provided by applicant): Recent advances in DNA sequencing technologies and a better understanding of natural product biosynthesis provide newfound opportunities to improve the process by which microbial natural products are discovered. The objectives of this research are to establish a series of methodologies by which strains can be quickly assessed for natural product biosynthesis through the analysis of PCR-generated or genome sequence data. The methods will be developed using a model group of marine bacteria belonging to the genus Salinispora and then applied to a large and diverse collection of marine actinomycetes with the aim of discovering structurally diverse, new chemical entities, which will be provided to the NIH Molecular Libraries Small Molecule Repository (MLSMR). The methods include an initial, rapid molecular "fingerprinting" screen, from which the genetic potential of individual strains can be compared. Sequence-based approaches will then be applied to interpret the biosynthetic richness and novelty of strains with promising fingerprints. These methods will make it possible to predict if the metabolites produced by a strain will be new and how many different compounds in a particular structural class may be produced. Once strains with the greatest genetic potential are identified, detailed chemical studies will be performed. This approach represents a dramatic improvement over traditional paradigms in which large numbers of biosynthetically unknown strains are screened in a limited number of conditions. It will dramatically reduce the isolation of previously discovered compounds, a problem that has long plagued microbial natural product research. This approach provides a culture-independent, genome-level assessment of secondary metabolite biosynthesis as opposed to more traditional methods, which detect only those metabolites produced under a limited set of culture conditions. The methods developed will be broadly applicable to the scientific community and include the creation of a curated sequence database that can be readily downloaded and used to assess genome sequence data for pathways involved in secondary metabolite production. This research has the potential to dramatically increase the rates with which new chemical entities are discovered and made available for biomedical research. PUBLIC HEALTH RELEVANCE: The research presented in this proposal provides a method by which DNA sequence data can be used to dramatically improve the process by which natural products are discovered from microorganisms. It will generate considerable new chemical diversity that can be used for drug discovery research and to study basic biological processes. The methods developed will be made widely available to the research community and thereby have a broad impact on drug discovery and basic biomedical research.

There is overwhelming evidence that polybrominated diphenyl-ethers (PBDEs) and polybrominate pyrroles (PBPs) are bioaccumulating in the marine food chain and that these compounds are of biological origin. While the harmful effects of halogenated organic compounds (HOCs) on humans and the environment are only beginning to be understood, their structural similarities to anthropogenic pollutants suggest they represent an emerging and poorly characterized group of marine toxins. The overall goal of Project 1 is to identify the biological sources of PBDEs and PBPs in the Southern California Bight. This goal will be accomplished through a comprehensive sampling paradigm targeting bulk and size-fractionated plankton, sediments, benthic algae, and filter feeding invertebrates. Sampling efforts will capitalize on small boat and shipboard resources available through SIO and expertise provided by plankton expert Melissa Carter (SIO) and collaborators at the J. Craig Venter Institute (see letter of support from L. Zeigler). Project 1 activities will be tightly integrated with Project 3 and the Analytical Core, which will identify and quantify PBDEs and PBPs in the samples collected. This information will be used to link specific compounds with sample types and to guide subsequent microbial cultivation efforts and cultivation-independent diversity studies. Metagenomic data generated from Project 2 will, provide a complimentary analysis of microbial community composition and insight into the biosynthetic genes associated with HOC production. This information will be used in quantitative PCR and CARD-FISH studies designed to assess the numbers and location of HOC-producing bacteria and relevant biosynthetic genes in different sample types. HOCs that cannot be identified by Project 3 based on comparisons with reference data will be isolated, structurally characterized, and provided to Project 3 for toxicological evaluation and as GC-MS standards. Select PBPs previously identified from environmental samples will be synthesized to provide baseline toxicological data and GC-MS standards. A major effort will be placed on the cultivation of HOC-producing bacteria, as there is considerable preliminary evidence that they represent an important biological source of these compounds. This research represents the first major effort to identify the biological sources and sinks of an increasingly important group of marine pollutants in a major US coastal environment.

Intellectual Merit: This project explores the ecological functions of bacterial secondary metabolites as agents of chemical defense. It targets marine sediments, a major and poorly explored marine biome. The aims are to test three hypotheses related to the effects of bacterial secondary metabolites on co-occurring microorganisms and protistan grazers. The focus is on the bacterial genus Salinispora, which is well defined in terms of its diversity and distributions in marine sediments, and well characterized at the genomic level and in terms of secondary metabolite production. A genetic system recently developed for these bacteria will be employed to establish links between biological activities and specific secondary metabolites. By employing a variety of innovative methodologies including imaging mass spectrometry, it will be possible for the first time to gain insight into the potential roles of Salinispora secondary metabolites in structuring marine sediment microbial communities. The results will have broad implications for our understanding of the factors that regulate the diversity and distributions of bacteria in the marine environment. They will additionally address the supplemental hypothesis that secondary metabolites represent ecotype-defining traits that delineate Salinispora species.

The hypotheses to be tested are:
H1: Secondary metabolites inhibit microbial competitors,
H2: Secondary metabolites affect bacterial community composition, and
H3: Secondary metabolites function as invertebrate feeding deterrents.

A large collection of diverse, co-occurring microbes will be tested for sensitivity to Salinispora secondary metabolites using a direct challenge assay. These types of assays are highly informative in that they can detect behavioral and morphological responses in addition to toxicity. A recently developed imaging mass spectrometry technique will be used to visualize secondary metabolites associated with any observed biological activities. The results will be linked to existing genome sequences and used to aide in compound identification. The associated pathways will be knocked out to provide experimental support for the biological activities of specific compounds.

Given that most marine bacteria are not readily cultured, these experiments will additionally address the effects of secondary metabolites on the sediment bacterial community by employing culture independent techniques. In situ growth chambers and next generation sequencing technologies will be used to test extracts and pure compounds against a natural assemblages of sediment bacteria. The results will inform future cultivation efforts and provide a more comprehensive assessment of the organisms targeted by native chemical defenses. Finally, a robust feeding assay using two model protists will be developed and used to test the roles of bacterial secondary metabolites as invertebrate feeding deterrents. In situ experiments will provide insight into the natural assemblage of invertebrates affected by these defenses. The overall results of these studies have the potential to profoundly impact our understanding of the ecological functions of microbial secondary metabolites and the extent to which these compounds affect community composition.

Broader Impacts: This research presents the opportunity to fundamentally advance our understanding of the ecological roles of microbial secondary metabolites in a major marine biome. The activities are highly interdisciplinary and bring together aspects of microbiology, ecology, and marine natural products chemistry in unprecedented ways. It strengthens international collaborations with colleagues in Mexico and includes student and postdoctoral training and outreach to under-represented groups. The later includes participation in the anticipated UCSD/HBCU (Historically Black College and University) program and the UCSD Summer Training Academy for Research in the Sciences (STARS) program. Separate NIH funding will be leveraged to explore the medicinal potential of any new secondary metabolites discovered. These compounds will also be provided to the NIH Molecular Libraries program where they will be made broadly available to the scientific community. The project leverages existing NSF-funded ship time and has the potential to yield new assay models that will be broadly applicable to the chemical ecology research community.

AP1: Exploiting Microbial Diversity for Natural Product Discovery Project Summary/Abstract The primary objective of the proposed research is to discover new, small molecule drug candidates from marine microorganisms cultured from Fiji and the Solomon Islands. This objective will be facilitated through the continued development of a productive microbial drug discovery program at the University of the South Pacific. Bioassays targeting cancer, infectious disease, neurological disorders, and neglected tropical diseases will be used to guide the isolation of compounds relevant to these targets. The structures of new compounds will be solved using modern spectral analyses and produced in sufficient quantities for effective pre-clinical evaluation. The research will target chemically rich microbial taxa including the marine actinomycete genus Salinispora and explore the relationships between biotic diversity and natural product discovery. The research benefits from a wealth of genome sequence data that has been acquired through the Joint Genome Institutes Community Sequencing Program. Bioinformatic analyses will be used to prioritize strains for chemical evaluation and to establish relationships between secondary metabolite biosynthetic potential, taxonomy, and the habitats and locations from which the stains originate. This information will be used to develop more effective sampling strategies and to provide new insight into the extant biosynthetic potential of marine bacteria and the evolutionary processes that generate structural diversity. Genome mining approaches will be used to link molecules to the pathways responsible for their production and to facilitate discovery and de-replication. The web-based tool NaPDoS (Natural Product Domain Seeker), which simplifies the analysis of genes involved with secondary metabolite biosynthesis, will be further developed to include additional pathway types and reference sequences. New cultivation methods will be developed that mimic natural conditions and provide ecologically relevant stimuli in an effort to induce secondary metabolite production. These studies will be coupled with transcriptome analyses, which will be used to determine the effects of culture conditions on biosynthetic gene expression. Highly sensitive methods in mass spectrometry will be used to better visual the secondary metabolome and generate networks that can be used to recognize new molecules, de-replicate known compounds, and search for correlations between geographic origin, phylogeny, and secondary metabolite production. Extensive post-doctoral, graduate, and undergraduate training will be provided throughout the program including training for host country scientists. Ultimately, this program aims to develop improved methods for natural product discovery and apply these approaches to the microbial resources in Fiji and the Solomon Islands in an effort to discover new drug candidates to treat diseases relevant to the US and the host nations.

There is overwhelming evidence that polybrominated diphenyl-ethers (PBDEs) and polybrominate pyrroles (PBPs) are bioaccumulating in the marine food chain and that these compounds are of biological origin. While the harmful effects of halogenated organic compounds (HOCs) on humans and the environment are only beginning to be understood, their structural similarities to anthropogenic pollutants suggest they represent an emerging and poorly characterized group of marine toxins. The overall goal of Project 1 is to identify the biological sources of PBDEs and PBPs in the Southern California Bight. This goal will be accomplished through a comprehensive sampling paradigm targeting bulk and size-fractionated plankton, sediments, benthic algae, and filter feeding invertebrates. Sampling efforts will capitalize on small boat and shipboard resources available through SIO and expertise provided by plankton expert Melissa Carter (SIO) and collaborators at the J. Craig Venter Institute (see letter of support from L. Zeigler). Project 1 activities will be tightly integrated with Project 3 and the Analytical Core, which will identify and quantify PBDEs and PBPs in the samples collected. This information will be used to link specific compounds with sample types and to guide subsequent microbial cultivation efforts and cultivation-independent diversity studies. Metagenomic data generated from Project 2 will, provide a complimentary analysis of microbial community composition and insight into the biosynthetic genes associated with HOC production. This information will be used in quantitative PCR and CARD-FISH studies designed to assess the numbers and location of HOC-producing bacteria and relevant biosynthetic genes in different sample types. HOCs that cannot be identified by Project 3 based on comparisons with reference data will be isolated, structurally characterized, and provided to Project 3 for toxicological evaluation and as GC-MS standards. Select PBPs previously identified from environmental samples will be synthesized to provide baseline toxicological data and GC-MS standards. A major effort will be placed on the cultivation of HOC-producing bacteria, as there is considerable preliminary evidence that they represent an important biological source of these compounds. This research represents the first major effort to identify the biological sources and sinks of an increasingly important group of marine pollutants in a major US coastal environment.

Project Summary/Abstract Natural products research is advancing at a remarkable pace. Driven by advances in analytical techniques, new methods to identify molecular targets, and omics-based technologies, major gains are being made in the approaches used to identify drugs derived from natural sources. In particular, natural products from marine organisms continue to deliver unique molecular scaffolds that interact with cellular macromolecules and pathways in ways not seen with molecules from other sources. Efforts to isolate and identify marine natural products have led to at least five compounds in clinical use and many more undergoing clinical trails for diseases such as cancer and pain relief. In addition, marine derived products continue to play prominent roles as nutraceuticals and marine natural products researchers are being funded by the NCCIH to develop important new technologies in natural products research. With this proposal, the PIs request support for the 2018 Marine Natural Products Gordon Research Seminar (GRS) and Gordon Research Conference (GRC) to be held in Ventura, California on March 3-8, 2018. The GRS is a unique forum for graduate students and postdoctoral scholars to present and exchange new data and cutting edge ideas encompassing the field of marine natural products with applications to human health. The GRC will bring together early career and established researchers from academia, industry, and government (including all of the GRS participants) to address the discovery and development of marine natural products. Presentations and the following discussions will focus on new methods to isolate and characterize compounds from complex mixtures, mechanisms of action and pharmacology, drug development, and the applications of ?omic? techniques in natural products research. As such, these topics will transcend the field of marine natural products and encourage the application of new techniques and synergistic activities. The three overarching aims of these two meetings are 1) to provide education in the form of cutting edge science, 2) catalyze collaborations by providing opportunities for individuals from disparate fields to interact, and 3) provide training and guidance for early career scientists. The format of the meeting is designed to foster interaction and discussion both in the formal sessions and during the meals and free time. An emphasis has been placed on the inclusion of women, under-represented groups, and handicapped individuals in the GRC program. NIH support will help offset the registration and travel fees of these individuals along with students attending the GRS and ensure that these meetings achieve their stated goals of excellence.

1 Microbial natural products represent one of our most important sources of medicines. Traditionally, these 2 compounds have been discovered from microbes cultured in the laboratory using conditions that bear little 3 resemblance to the environments from which they were derived. The failure of traditional discovery approaches 4 to keep pace with the need for new drug leads, coupled with our improved understanding of the molecular 5 genetics of natural product biosynthesis, spurred the development of new approaches for natural product 6 discovery. These efforts have largely focused on genome mining and the genetic manipulation of orphan 7 biosynthetic gene clusters with the aim of bypassing the regulatory mechanisms that control compound 8 production. While important progress has been made, these approaches have yet to yield a wealth of new 9 chemical scaffolds. Here we propose an alternative discovery paradigm that takes a compound first approach. 10 We developed a Small Molecule In situ Resin Capture (SMIRC) technique to recover microbial natural products 11 directly from environmental samples thus by-passing the need for cultivation. We have applied this approach to 12 ocean sediments, where complex microbial communities occur and interstitial water flow facilitates compound 13 capture. Coupled with mass-spectrometry based metabolomics, we demonstrate that SMIRC can capture known 14 microbial metabolites and thousands of compounds that cannot be readily identified, thus hinting at the discovery 15 potential afforded by this technique. Here we propose to test the applications of a new capture and analysis 16 pipeline to discover microbial natural products directly from marine sediments. By introducing an antibiotic screen 17 into the workflow, we target biomedically relevant compounds for isolation and structural characterization. A 18 micro-fractionation technique will be used to separate complex mixtures and guide the isolation of active, low 19 abundance compounds. We capitalize on innovations in 'nanomole-scale' natural product characterization and 20 the integration of microcryoprobe NMR and MS analyses for sub-micromole level structure determination 21 (including stereostructures). Our ability to prioritize leads based on MS de-replication and biological activity 22 provide opportunities to tease out deep chemical diversity from complex extracts. We further propose to use 23 metagenomics to link compounds to their biogenic sources and thus gain taxonomic perspective on unrealized 24 biosynthetic potential. This approach eliminates the need to establish laboratory conditions suitable for cultivation 25 and gene cluster expression (two of the major bottlenecks hampering current natural product discovery efforts) 26 and instead allows the environment to act as a natural bioreactor. Ultimately, lead compounds discovered using 27 this approach can be produced via chemical synthesis, bulk environmental extraction, synthetic biology, or 28 metagenomics informed cultivation.