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The Potential of Marine/Extremophilic Microbes to Explore Novel Biocatalysts Using Genomic Approach

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The Potential of Marine/Extremophilic Microbes to Explore Novel Biocatalysts Using Genomic Approach Global Forum On Biotechnology : Marine Biotechnology Session 1: Productivity and Sustainability of the Ocean on 30 May, 2012, Vancouver, Canada The potential of marine/extremophilic microbes to explore novel biocatalysts using genomic approach Sang-Jin Kim KORDI 김상진 Marine Biotechnology Research Centre Contents 1. Introduction: Feature of marine environment, Status of arts of Marine products, Development of genome sequencing tech. 2. Diversity of epoxide hydrolases identified by homology-driven screening approach 3. DNA polymerase mined from whole genome sequence of an hyperthermophile 4. Diversity of Lipase/Esterase explored from marine metagenome libraries 5. Conclusion and perspectives 김상진 Marine Biotechnology Research Centre Features of marine environment • Salinity: 3.5% Halophiles • Temp.: -2 ~ 400°C Psychrophiles and Hyperthermophiles • Average depth: 3,800 m Piezophilies • Photic zone: 0~200 m Oligotrophes 김상진 Marine Biotechnology Research Centre Cold-active, thermostable or any other biocatalysts representing unique characteristics could be preferentially obtained from the microorganisms or environmental DNA which were retrieved from the extreme marine habitats such as ocean trenches, deep- seas, polar seas, cold seeps, hydrothermal vents, etc (Bull et al., 2000, Microbiol. Mol. Biol. Rev. 64:573-606 ). 김상진 Marine Biotechnology Research Centre Products derived from marine organisms Products Application Source Protein, Peptide & Amino acids Deep-sea hydrothermalvent Vent TM DNA polymaerase Polymerase chain reaction (PCR) archaea Green Fluorescent Protein Reporter gene Aequora victoria (jellyfish) Aequorin Bioluminescent calcium indicator Aequora victoria (jellyfish) Ziconotide (Prialt) Analgesic Conus magnus (Mollusc) Antioxidant, immunostimulants nutraceutical Hormones, cyclic peptides Fish hydrolysates products Kainic acid Anthelmintic insecticide Red algae Saccharides Carragreenans Cosmetrics, thickener, Pharmacy, Agars mucoprotector, Red Algae Alginates Anti-coagulant, Antiviral Cosmetics, tissue replacement, Chondroitin sulfate Fish anticoagulant Cosmetics, colloids Pharmacy, Chitosan Crustacean shells, fungi icroencapsulation 김상진 Marine Biotechnology Research Centre <Continued> Fatty acids and miscellaneous Long chain PUFA Prevention of heart disease, mental development Microalgae, seaweed, fish (AA, EPA, DHA) is premature children; antimoural; lipid metabolism Fatty acids used as additive in infant formula Formulaid Marine microalgae nutritional supplement Pseudompterogorgia Resilience elisabethae (Caribbean Additive in skin creams gorgonia) Conjugated antibodies used in ELISAs and flow Phycoerythrin Red algae cytometry Manoalide Luffarella variabilis (sponge) Phospholipase- A inhibition Cephalosporins Marine fungi Antibiotic Spongoadenosine sponge Antiviral Herpes Cytarabine (Ara-C) sponge Antitumoral (cytostatic) 김상진 Marine Biotechnology Research Centre Development of Genome Sequencing 2003 Human Genome Project 13 years 14X $2.7B 2007 Craig Venter 4 years 7.5X $100M 2008 James Watson 2 years 7.4X $2 M 2009 Sung-Jin Kim 6 months 29X $0.17M 2009 Every genome 30X $48,000 2013 ??? 30X $1,000 김상진 Marine Biotechnology Research Centre 836 (5%) Marine genome project Genome Project (2012) Eukarya Metagenome Total 17874 project 29 (3%) 72 (9%) Complete Incomplete 12 (41%) Complete 17 (59%) 26 (36%) Archea Incomplete 46 (64%) 107 (13%) Incomplete 19 (18%) Complete 88 (82%) Bacteria 628 (75%) Incomplete Complete 326 (52%) 302 (48%) 836 Marine Genome Project 김상진 Marine Biotechnology Research Centre Phylogenetic tree of putative Epoxide Hydrolases 3c 1a 3b Group 3 Group 1 3a 1b Symbols :Blue line is isolated from 2f marine source Red circle : Enzymes identified in this study 2a 2e Green circle: Enzymes with known 2b 2d EHase activity 2c Gene containing GXSXG, DXG, HGXP motif of Epoxide hydrolase Group 2 김상진 Marine Biotechnology Research Centre Acquisition of genome sequenced marine microorganisms (15 st. from collaborator, 2 st. from ATCC ) Oceanospirillum sp. MED92 Marinomonas sp. MED121 Roseobacter sp. MED193 Leeuwenhoekiella blandensis MED217 Vibrio sp. MED222 Reinekea sp. MED297 Limnobacter sp. MED105 Roseovarius sp. HTCC2601 Oceanicaulis alexandrii HTCC2633 Janibacter sp. HTCC2649 Oceanobacter sp. RED65 Rhodobacterials sp. HTCC2654 Erythrobacter litoralis HTCC2594 Oceanicola batsensis HTCC2597 HTCC2143 Sphingopyxis alaskensis Novosphingobium aromaticivorans 김상진 Marine Biotechnology Research Centre 11 putative Epoxide hydrolase genes were expressed M EEH2,3 M NEH M SEH M REH EEH1 EEH3 NEH SEH EEH2 REH kDa M MH M 1H M MH M 121 193 MH 1H 2H MED121 MED193 김상진 Marine Biotechnology Research Centre Enantioselective activity of Ehases toward various epoxide substrates SO GPE EX EB ECH TSO CH3 CH3 Hydrolysis rate (x 10-2) mg/min Enzyme SO GPE EX EB ECH TSO (S) (R) (S) (R) (S) (R) (S) (R) (S) (R) (S) (R) EEH1 9.00 28.00 20.00 11.00 8.00 7.00 15.00 15.00 1.05 7.79 NA NA EEH2 0.06 0.05 0.07 0.06 0.07 0.07 0.08 0.08 0.23 0.23 ND ND EEH3 0.14 0.10 0.10 0.10 0.09 0.17 0.11 0.08 0.28 0.26 ND ND SEH 1.16 1.12 7.15 7.59 1.90 1.70 0.09 0.06 9.10 9.24 ND ND NEH 4.13 6.22 15.34 12.26 13.35 13.82 8.77 12.11 6.68 10.95 NA NA REH 1.15 7.29 32.70 1.39 2.86 6.02 0.86 0.60 2.71 2.37 0.01 0.07 MH 0.29 0.34 0.13 0.12 0.43 0.49 ND ND 0.64 0.64 ND ND 1H 0.45 0.85 0.67 0.32 0.65 2.31 ND ND 0.88 0.75 ND ND 2H 0.24 0.22 0.23 0.24 0.45 0.40 ND ND 0.52 0.52 ND ND MED121 0.06 0.06 ND ND ND ND ND ND ND ND ND ND MED193 0.08 0.07 ND ND ND ND ND ND ND ND ND ND •ND: not determined, •NA: no activity (activity too low for determination) 김상진 Marine Biotechnology Research Centre REH (20 ug) + SO 5 mM REH toward various epoxide substrates 3.0 100 2.5 ) % 80 , e 2.0 e ( s ) s e M 60 c x m ( 1.5 e . c c i (R)-Styrene oxide r n e 1, styrene oxide (SO) o (S)-Styrene oxide C 1.0 40 m o ee (%) i t 2, ortho-chlorostyrene oxide (2CSO) n a 0.5 n 3, meta-chlorostyrene oxide (3CSO) 20 E 0.0 4, para-chlorostyrene oxide (4CSO) 0 0 2 4 6 8 10 Time (min) Epoxides Time (min) ee (%) c E Yield (%) Config. 5 mM SO 3 99.99 0.525 192.8 47.5 S 5 mM 3CSO 5 99.99 0.583 55.7 41.7 S 5 mM 4CSO 1 99.99 0.522 226.5 47.8 S 5 mM 4NSO 15 99.99 0.612 39.8 38.8 S * The extent of conversion (c) [c={1−(ERs+ESs/ERso+ESso)}], where the initial epoxide of (R) and (S) was denoted as Eso, and the remaining epoxide of (R) and (S) was as Es * The enantiomeric ratio (E) is derived from the extent of conversion (c) and the enantiomeric excess of the remaining enantiomer of the substrate (ees) [E= In{(1−c) (1−ees)}/In{(1−c) (1+ees)}] obesity care, diabetes care neuroprotective property b3-adrenergic receptor agonist 김상진 Marine Biotechnology Research Centre DNA polymerase Microorganism Name of polymerase Reference Thermus aquaticus Taq polymerase Chien et al., (1976) T. litoralis Vent polymerase Cariello et al., 1991 Mattila et al., 1991) P. furiosus Pfu polymerase Lundberg et al., (1991) P. woesi Pwo polymerase Frey and Suppmann, (1995) Pyrococcus strain GB-D Deep Vent polymerase Perler et al., (1996) Thermococcus sp. strain polymerase Southworth et al., 1996) 9N-7 Thermococcus KOD polymerase Takagi et al., (1997) kodakaraensis KOD1 The DNA polymerase world market is currently more than 350 million$ (282 million Euro) and growing. (2012) (www.in-pharmatechnologist.com) 김상진 Marine Biotechnology Research Centre Isolation, classification and genome sequencing of Thermococcus onnurineus NA1 PACMANUS Basin 김상진 Marine Biotechnology Research Centre Isolation, classification and genome sequencing of Thermococcus onnurineus NA1 95 Thermococcus kodakaraensis KOD1T (D38650) Thermococcus peptonophilus JCM 9653T (AB055125) Thermococcus stetteri DSM 5474T (Z75240) Thermococcus profundus DT5342T (Z75233) Thermococcus acidaminovorans DSM 11906T (AB055120) Thermococcus onnurineus NA1 Thermococcus gorgonarius JCM 10552T (AB055123) Thermococcus fumicolans JCM 10128T (AB055128) Thermococcus guaymasensis DSMZ11113T (Y08385) Thermococcus gammatolerans EJ3T (AF479014) ‘Thermococcus barossii’ (U76535) Thermococcus celer DSM 2476T (M21529) Thermococcus hydrothermalis AL662T (Z70244) Thermococcus pacificus JCM 10553T (AB055124) Thermococcus zilligii JCM 10554T (U76534) Thermococcus siculi DSMZ 12349T (AJ298870) Thermococcus atlanticus MA898T (AJ310754) Thermococcus sibiricus DSM Z12597T (AJ238992) Thermococcus alcaliphilus DSM 10322T (AB055121) 89 97 Thermococcus litoralis JCM 8560T (Z70252) 97 Thermococcus aegaeus DSMZ 12767T (AJ012643) Thermococcus aggregans DSM 10597T (Y08384) 97 Thermococcus barophilus DSM 11836T (U82237) 80 Pyrococcus sp. NA2 90 Pyrococcus abyssi GE 5T (L19921) 100 Pyrococcus horikoshii JCM 9974T (D87344) 100 Pyrococcus furiosus JCM 8422T (U20163) Pyrococcus glycovornas CNCMI-2120T (Z70247) Palaeococcus ferrophilus JCM 10246T (AB019239) Methanocaldococcus jannaschii JAL-1 DSM2661T (M59126) Methanopyrus kandleri av19 DSM 6324T (M59932) 0.01 김상진 Marine Biotechnology Research Centre In silico analysis of novel biocatalysts and characterization of them 2-D Gel Electrophroesis of Thermostable Proteins of NA1 Identification of a novel dITPase (Kim et al. AMB. 79, 571-) Identification of a novel aminopeptidase (Lee et al. AEM. 72, 1886-) Characterization of Amylase (Lim et al. JMBiotechnol. 17, 1242-) Study on Deblocking aminopeptidase. (Lee et al. JBB. 104,188-) Identification of a dUTPase (Lee et al., Mar.Biotechnol. 9, 450-) Study on a Prolyl Oligopeptidase. (Lee et la., JBB. 103, 221-) Study on a DNA Polymerase (Kim et al. JMBiotechnol. 17, 1090-) Study on a Methionylaminopeptidase (Lee et al. Mar.Biotechnol. 8, 425-) Study on a carboxypeptidase (Lee et al. BBB 70, 1140-) Study on a DNA ligase (Kim et al. Biotechnol. Lett. 28, 401-) 김상진 Marine Biotechnology Research Centre TNA1 DNA polymerase PCR using TNA1 DNA polymerase Purified enzyme M 2 5 8 10 12 15 M 2 4 8 (kb) λ DNA Genomic DNA Source Expressed and purified from E.
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