Novel Bioactive Molecules with BiopharmaceuticalNovel Bioactive Molecules with BiopharmaceuticalPotential from the Marine Environment Potential from the Marine Environment
(ERI Open Day October 10(ERI Open Day October 10thth, 2012), 2012)
Professor Alan Dobson,Professor Alan Dobson,Environmental Research Institute and Microbiology Department,Environmental Research Institute and Microbiology Department,
University College Cork University College Cork
• Mapping and genetic characterisation of Ireland’s marine biodiversity
• Sampling, extraction and identification of biochemical components
• Screening and culturing process for isolation of bioactive compounds
• Application of research results into generation of new biomaterials, compounds and agents
• Integrated data management system
• Educational, outreach and technology transfer
Beaufort Marine Biodiscovery Work Programme
Initial Capacity Building
• Beaufort Marine Research Award:
– funding of €7.2m over 7 years
– to NUIG, UCC and QUB.
• MI IRCSET funding
– to DCU and UCD
Implementation of Marine Biodiscovery Programme
UCC
DCU
QUB
NUIG
UCD
PI, 2 PD, 4 PhD
2 PD, 4 PhD
2 PD, 4 PhD
1 PhD
2 PhD
Novel Marine Bio-actives for Health Novel Marine Bio-actives for Health
Marine-origin Bioactives in the Market
Pharmaceuticals• UpJohn: Anti-cancer drug Cytosar-U® $1.36 billion in 2009
- sponge• PharmaMar: Anti-cancer drug Yondelis® €45 million in
2009 - sea squirt• Parkedale: Anti- herpes and anti- shingles drug Vidarabine
€27 million in 2008 - sponge• Azurpharma: Anti-neuropathic pain drug Prialt® (IE) $16.5
million for 2008/9 – snail
Marine bioactives market = $1.9bn by 2012 Pharmaceuticals, nutraceuticals, cosmetics, agri products
Driven by life sciences research – turning to marine for novelty
Marine Natural ProductsMarine Natural Products• Distribution of new natural products by phyla
From Blunt et al., NPR 2009
Marine SpongesMarine SpongesPoriferaPorifera
– Simple animals• No nervous system• No internal organs
– Sessile filter feeders• 1000 L/kg/hr
– Chemical defence system• Important source of new bioactive metabolites
Suberites carnosus
Axinella dissimilis Cliona celataSuberites ficus
• Galway Bay Haliclona simulans
• Lough Hyne Marine Nature Reserve 16 sponge species (33 samples)
Amphilectus fucorum, Axinella dissimilis, Axinella damicornisCliona celata, Dysidea fragilis, Eurypon major, Leucosolenia sp.
Pachymatisma johnstonia, Polymastia boletiformis, Raspailia hispida, Raspailia ramosa, Rhaphidostyla kitchingi, Suberites carnosus,
Suberites ficus, Stelligera rigida, Tethya citrina
Sponge samples for antimicrobial screens
Sources of SpongesSources of Sponges
Deep Sea2010 and 2011
Biodiscovery Cruises on Celtic Explorer
Kilkieran Bay, Galway
Haliclona simulans
Lough Hyne, West Cork
Marine Nature ReserveUCC Research
Laboratories
Culture dependent approach
Sponges
CulturingMarine isolates
Bioactive compounds
Pseudovibrio sp.W19
Example 1
Pseudovibrio species
• Axinella dissimilis
- 16S RNA gene analysis- Pseudovibrio species
• Genetic diverse determined• Novel Pseudovibrio species characterised
• Majority of strains anti-microbial activity- Gram negative bacteria (e.g. E. coli, S. typhimurium, Y. enterocolitica)
- Gram positive bacteria (e.g. S. aureus, C. difficile, L. monocytogenes)
(other)
Pseudovibrio species
Extraction
Antimicrobial activity of crude extracts of W19 vs
S. aureus, MRSA, and VISA
region extracted; Rf ≈ 0.72
Purification of Ad2 bioactive compound by preparative TLC
Preliminary NMR
scaling up
MALDI-TOF mass spec analysis
Ad2 bioactive compound (MW 234)
• Characterisation of bioactive compound
Culture dependent approach
Sponges
CulturingMarine isolates
Bioactive compounds
SporeformersBacillus sp.
Example 2
Bacillus species
• Majority of strains antimicrobial activity
– Range of Gram positive bacteria– Gram negative; only Enterobacter species
Characterisation of B. subtilis MMA7 • Broad spectrum of antimicrobial activity
– Listeria monocytogenes– Clostridium difficile
L. monocytogenes overlay
HPLC purified compound
1 2 3 1 2 3
De novo sequencingALPHALYSE, Denmark
Bacillus species
• Lytic effect of putative lantibiotic
• Whole genome sequencing– Gene cluster identified– Further characterisation
Example 3 Streptomyces Streptomyces SM2SM2
Strong bioactivity:
Bacillus subtilis
Staphylococcus aureus
MRSA
VISA
VRE
Clostridium difficile
Kennedy, et al., Mar.Biotech. 2009,
Fermentation• 14 days
• 1.6 L grown
Extraction• Amberlite XAD-16 resin
• Eluted with acetone, methanol, and ethyl acetate
• Concentrated by evaporation
Purification• silica gel flash chromatography
– Active fractions pooled– Crude extract MIC (B. subtilis)
• ~0.45mg/ml– Most active fraction MIC
• ~0.015mg/ml
Extraction of antibioticExtraction of antibiotic
Qualitative Assay – Disk Diffusion
Quantitative Assay
Analysis of active fractionAnalysis of active fractionHPLC
Single major UV active peak
UV Spectrum
Mass Spectrum of major peak
MS and NMR data indicates major product is a novel compound.Structure currently being evaluated.
Functional MetagenomicsFunctional Metagenomics
Screen for activity
Isolate sponge DNA
Construct library
Transform into E. coli
Functional Screens
Anti-bacterialAnti-fungal
Quorum sensing inhibitors
Calcineurin inhibitionPHA production
Antibiotic resistanceBiosurfactant
ProteaseLipase
EsterasePhosphatase
Metagenomic HitsMetagenomic Hits
Lipase
(tributyrin)Protease
(skimmed milk)Phosphatase
(X-phos)Biosurfactant(oil spray and emulsification)
Olive oil
Mineral oil
Clones analysed by activity profileEnd-sequenced to determine phylogenetic origin
Selected clones fully sequenced
New funding opportunities
EU FP7• Excellent international partners• Large consortium
Granted• MicroB3• PharmaSEA• MaCuMBA
National postdoctoral fellowship granted• IRCSET EMPOWER
Marine Biotechnology Centre (Dobson)Biomerit Research Centre (O’Gara)
SummarySummary
O
HN
OO
OH
O
OO
O
O
HO
H
• Culturable microbes• 1,500 bacterial and 85 fungal isolates• >700 bacterial strains from deep sea
sponges
• Diverse collection of sponges (33)
Microbial Ecology
16s rRNA analysis of>1,400 bacteria from 9 coastal >350 bacteria from 5 deep sea
sponges
Bioactivities
3 lead antimicrobials SM2, SM8, MMA7All have novel structures and good anti-
bacterial, anti-fungal activities (g.ml range)
Genomics
Sequenced genomes of Streptomyces strains SM2, SM8,
Bacillus subtilis MMA7
Biocatalysts
LipasesProteases
Phosphatases
1617