Bioprospecting Value for science and industry

1. BioprospectingValue for science and industry Tamas Torok Center for Environmental Biotechnology Lawrence Berkeley National Laboratory, Berkeley, CA Development of International Collaboration in Infectious Disease Research Sosnovka, Novosibirsk Region, Russia 8-10 September 2004

2. Acknowledgements • Work reported here was funded by federal tax dollars and private money • Progress was possible because of the active participation of scores of • dedicated, eager-to-learn students • outstanding US and foreign exchange faculty • industrial collaborators, and • my Center colleagues whom I have been privileged to work with over the years • They all deserve my sincere appreciation and heartfelt “Thank-you!” Tamas Torok, Ph.D.

3. Outline for today • Microbial diversity - what is it? • Bioprospecting • “…sharing in a fair and equitable way…” • Value for science and industry • research project examples • extremophiles from Kamchatka, Russia • crop protection story • radiation resistant fungi from the Chernobyl Exclusion Zone (CEZ), Ukraine • Stachybotrys story

4. Hypothesis • Bioprospecting is the continuous search for “old” and “new” microorganisms with novel genetic traits to • advance science • preserve a sustainable genetic pool • fairly and meaningfully commercialize microbial products by industry • benefit humankind

5. Rational for bioprospecting • “…Where there is life, there are microbes…” (Carl Woese, 2002)

6. Microbial diversity • “...Microbial diversity includes the genetic composition of microorganisms, their environment or habitat where they are found, and their ecological or functional role within the ecosystem…” (J. C. Hunter-Cevera, 2000)

7. Microbial diversity and societal concerns • “...Can a monetary value be placed on the annual worth of microbial diversity when the very existence of life is dependent on microorganisms?” (James Staley, University of Washington) • “...The true value of microbial diversity includes not only the diversity with respect to species richness but also the direct and indirect economic value of profits resulting from commercialization of microbial metabolic products and processes.” (Jennie Hunter-Cevera, UMBI) • “…20-50% of outpatient prescriptions and 25-45% of antibiotics prescriptions in hospitals are inappropriate” (Antibiotic Resistance Project) • Keyword for winning over skeptics is sustainability

8. Bioprospecting vs. biopiracy • “Earth Summit”, Rio de Janeiro, 1992 • “...peace, development, and environmental protection are interdependent and indivisible…” • Convention on Biodiversity, 1992 (Article 15) • “...facilitate access to genetic resources for environmentally sound uses…” • “...develop and carry out scientific research based on genetic resources…” • “...share in a fair and equitable way the results of research and development and the benefits arising from the commercial and other utilization of genetic resources...”

9. Value for science and industry • Research project examples • extremophiles from Kamchatka, Russia • crop protection story • radiation resistant fungi from the Chernobyl Exclusion Zone (CEZ), Ukraine • Stachybotrys story

10. Use of microbial diversity for novel biotechnology applications • Objective • develop a multi-year program for the collection of microbial diversity in the extreme environments of Kamchatka, Russia

11. Extreme environments • Kamchatka in the Russian Far-East uniquely provides a wide variety of pristine environments, including permafrost areas, volcanic and hydrothermal sites • Great diversity of thermophilic and acido- and alkaliphilic microorganisms • Uzon Caldera is a 7 x 10-km volcano-tectonic depression • elevation of about 400 m • underlying magma chamber with its top about 5 km down • hundreds of mud pools and hot springs • pH range 1-10 • high concentrations of Hg, As, Zn, Sb, and S • Valley of Geysers includes several hundred active hot springs and small geysers - temperatures from 35°C to 97°C

12. Uzon Caldera

13. Use of microbial diversity for novel biotechnology applications • Kamchatka in the Russian Far East uniquely provides a wide variety of pristine environments, including permafrost areas, volcanic and hydrothermal sites • Great diversity of thermophilic and acidophilic microorganisms • Uzon Caldera is a 7 x 10-km volcano-tectonic depression • elevation of about 400 m • underlying magma chamber with its top about 5 km down • hundreds of mud pools and hot springs • pH range 1-10 • high concentrations of Hg, As, Zn, Sb, and S • Valley of Geysers includes several hundred active hot springs, mud pools, and small and large geysers • temperatures from 35°C to 100°C • acidic and alkaline pH

14. Valley of Geysers

15. Course of events • Environmental samples collected • Pure cultures isolated and grown under proprietary secondary metabolites producing conditions  

16. HTP screening • Proprietary screening for novel small molecules and natural products to be used in the agriculture

17. Value for industry • Experimental approach • primary hits verified in secondary screens • actives identified, chemically characterized • reverse engineering to find microbial genomic coding sequences • potential for generation of transgenic plants • After three years of research, about 100 novel environmental microorganisms and their active biomolecules are considered for IP protection

18. Value for science • Valuable research progress • unconventional isolation and non-trivial fermentation techniques used • scale-up protocols designed • molecular-level techniques applied to filamentous fungi identification

19. Total genomic DNA extraction 

20. PCR amplification  

21. Sequence analysis 

22. Exciting results • New filamentous fungal isolates • express multiple bioactive molecules under proprietary secondary metabolite producing growth conditions • strains are identified by • classical fungal taxonomy • comparative sequencing of 18S rDNA and the D1/D2 domain (~ 600 variable nucleotides of 28 S rDNA) to establish phylogenetic relatedness

23. Screening of botanical and microbial species for pharmaceutical and agrochemical activities • Overall objective • screen plant and microbial samples collected in and around the failed Chernobyl nuclear power plant over a period of 18 years for lead molecules and natural products of biomedical and agricultural importance • Berkeley Lab’s assignments • screen for a new class of antibiotics that inhibits DNA polymerase III in Gram (+) microorganisms • detect novel traits in fungi from the Exclusion Zone

24. Expectations and findings • Biological material exposed long-term to tremendous radiation in and around the failed nuclear plant might have experienced a “speeded up evolution” • Expression of a wide variety of biological activities found

25. Stachybotrys story • Stachybotrys chartarum implicated in “sick building” syndrome and infant pulmonary hemosiderosis • capability to produce mycotoxins and other harmful, often volatile chemical compounds • allergic reactions upon inhalation of conidia • Traditionally, fungal identification is based on colony and cell morphology • mycology skills and a functional culture collection required • limited training available worldwide • Molecular-level techniques need to be applied to fungal identification • routine detection and identification of S. chartarum and related species assists health care providers and building developers

26. Our contribution • Biodiversity of the species studied • clinical and building isolates from the Houston, TX area compared to wild-type organisms isolated in and around CEZ • Comparative analysis of conserved rRNA sequences • ITS regions 1 and 2 • complete sequence of 5.8S subunit • partial sequences of 18S and 28S subunits

27. Sequence analysis results • Three strains identified as S. chartarum • One strain as S. albipes (strong sequence similarity to S. chartarum) • Three strains determined earlier as S. chartarum, S. elegans, and S. bisbyi were identified as Penicillium spp.

28. Conclusions • Use of molecular-level methods enables more laboratories to attempt fungal identification • Databases are limited • “next best guess” • Multi-gene amplification leads to correct ID • conserved sequences • genus or species specific sequences • Genus Stachybotrys can be resolved using multiplex PCR amplifying conserved rRNA genes and the Tri5 gene that codes for trichodiene synthase

29. Concluding remarks • Microorganisms, especially extremophiles, produce unique biocatalysts and natural products that function under extreme conditions comparable to those prevailing in various industrial processes • Bioprospecting has elevated the search for microorganisms with novel capabilities to a safe, ethical, and meaningful conduct that equally benefits science and industry • Bioprospecting provides science with the opportunity to understand microbial diversity for it is the microbial world that is the foundation of the biosphere on our planet

30. Berkeley Lab at sunset