Bioprospecting Value for science and industry

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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

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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.

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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

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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

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Rational for bioprospecting

• Where there is life, there are microbes
• (Carl Woese, 2002)

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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)

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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

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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...

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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

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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

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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 35C to 97C

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Uzon Caldera
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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 35C to 100C
• acidic and alkaline pH

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Valley of Geysers
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Course of events
?
?

• Environmental samples collected
• Pure cultures isolated and grown under
  proprietary secondary metabolites producing
  conditions

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HTP screening

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

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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

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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

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Total genomic DNA extraction
?
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PCR amplification
?
?
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Sequence analysis
?
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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

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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 Labs 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

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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

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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

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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

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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.

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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

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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

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Berkeley Lab at sunset