Marine Biological Research and Possible Application

Kazuhiro Kitazawa Advisor Marine Technology and Engineering Center (MARITEC) Japan Agency for-Marine Earth Science and Technology (JAMSTEC)

2 -3 May 2013, New York Intersessional Workshop on Marine Genetic Resources

ExploringExploring MarineMarine LifeLife

Studies on the origin of life and the Studies of the structures and functions of the mechanisms of its evolution Earth’s biosphere, particularly in the ocean • From the origin of life to • Biogeochemical cycles and their early ecosystem evolution history • Evolution of eukaryotes • Monitoring of the marine environment • Progress from monocellular • Adaptive ecology of organisms in to multicellular organisms extreme environments

4.0

3.0

2.0 Trophic level Trophic

1.0

Rocky shore ecosystem

Applied research on microorganisms and functional molecules such as biological materials and enzymes ・Development of reagents and materials using useful enzymes and membrane substances 1 Vessels Vessels GT: Gross tonnage R/V NATSUSIMA R/V KAIYO R/V HAKUHOMARU GT 1,739 t GT 3,350 t GT 3,991 t

SWATH-type vessel Multipurpose research vessel Various observations with ROV with large workspace with long-term cruise R/V YOKOSUKA R/V MIRAI R/V KAIREI GT 4,439 t GT 8,687 t GT 4,517 t

Support vessel for Large vessel able to perform Surveys the structure of sub- “SHINKAI6500” observation over wide areas bottoms mainly with MCS D/V CHIKYU R/V SHINSEIMARU R/V (under planning) GT 56,752 t GT 1,600 t GT 5,000 t (approx.) (approx.)

Operation from 2013 2 Drilling vessel with world-class Multipurpose R/V with an emphasis Operation from 2015 scientific drilling capacity on survey off the coast of TOHOKU Survey for submarine resources

Manned/UnmannedManned/Unmanned UnderwaterUnderwater VehiclesVehicles

Deep Submergence Vehicle Shinkai6500 Shinkai2000 URASHIMA （Retired） AUV

World-class manned submersible Japan's first full-fledged manned submersible for deep-sea research Large AUV capable of long-distance dives ROV ABISMO AUV/ROVPICASSO

Automatic Bottom Inspection and Plankton Investigatory Collaborating Sampling Mobile Autonomous Survey System Operon ROVHYPER-DOLPHIN ROVKAIKO 7000 II ROV under construction

Operation with high-sensitivity ROV for heavy-duty work Capable of diving up to a 3 camera and manipulators maximum depth of 7,000 m To be launched in 2013 ExamplesExamples ofof SamplingSampling

Manned Research Submersible ROV

ExamplesExamples ofof SamplingSampling

Multiple Corer

Bait Trap Piston Corer 5 SampleSample AnalysisAnalysis LaboratoryLaboratory

The state-of-the-art facilities for Life and Earth Planetary Sciences Toward systematic understanding of Life, Ocean and our planet Earth through core sample research, Kochi Institute for Core Sample Research will promote the overarching approaches using ultra-high resolution mass and molecular analyses.

Secondary ion mass spectrometry (NanoSIMS 50L and IMS1280HR)

Single Cell Analysis Lab. in ultra-clean room 6

DevelopmentDevelopment ofof findingsfindings （（ NucleicNucleic acidacid extractionextraction fromfrom AgaroseAgarose gelgel ））

Relatively large nucleotide fragments can be recovered without damage so that quick DNA and RNA purification became possible. (1)Screening of agarase-producing microorganisms from deep-sea sediments

•AGAR-DIGESTING ENZYME AND UTILIZATION Screening of agarase-producing THEREOF (Japanese Patent No. 4441486) microorganisms from deep-sea sediments

Enrichment culture Licensed

Screened from sediments obtained by Shinkai 6500 at a Deep-sea Halo formations depth of 2,406 m in Suruga Bay sediments on agar medium

(2)Hyper-production of target proteins Hyper-production of target proteins Target protein gene •METHOD FOR STABLY RETAINING FOREIGN Strong GENES IN CELLS promoter Plasmid DNA Thermostable β-Agarase

(Japanese Unexamined Patent Application Transfor- Publication Number 2009-17840) mation No need to add •NOVEL DNA FRAGMENT,RECOMBINANT antibiotics to VECTOR COMPRISING SAME, the medium TRANSFORMANT TRANSFORMED THEREWITH, produce AND USE THEREOF (Japanese patent No. 5126879) Target protein 7 WhatWhat isis HirondelleaHirondellea gigasgigas??

• They live in the Challenger deep (the deepest point in the world). • They belong to Amphipoda. • They were captured during a R.V. Yokosuka cruise by a bait trap with a cut of fish (1998 & 2009). 8

MysteriesMysteries ofof HirondelleaHirondellea gigasgigas

¾ What does Hirondellea gigas eat? 9 Very low population of fish/plankton in the deep sea ¾ Microorganisms? 9 Very rare in deep-sea sediment ¾ Digestive enzymes? 9 DigestiveDigestive enzymesenzymes

Enzyme detection using mashed H. gigas

Detected enzyme activities Substrate Source • Protease Protein • Lipase Lipid Animals • Amylase Starch, glycogen • Cellulase Cellulose Plants • Glucomannanase Glucomannnan • Maltase Maltose

CellulaseCellulase degradesdegrades cellulosecellulose What is cellulose? • Cellulose is mainly contained in the green plants, algae and oomycetes. • Cellulose is the most common organic carbon on Earth. • Cellulose is a polymer of glucose (ß1-4).

CH2OH CH2OH ß1-4 O O OH O OH O

OH OH Cellulase n 11 TLCTLC analysisanalysis ofof productsproducts

Glucose

Cellobiose

01246824 Reaction time (h) 30°C, 10 mM Sodium acetate (pH 5.6) Substrate; 1% Carboxymethyl-cellulose (CMC) 12

ReactionReaction withwith SawdustSawdust andand RecycledRecycled PaperPaper

Sawdust (Oak) Enzyme reaction at 35°C CMC

µg/ml) Sawdust （ Glucose

Recycled Paper Reaction Time（hour）

Enzyme reaction at room temperature Glucose in a red colour

Before enzyme reaction 15 hours later 13 WhatWhat drivesdrives scientists?scientists?

Scientific Interest

Sampling Basic Research Lab Work Application

Industry Commercial Benefit?

Acknowledgement: Special thanks to Dr Hideki Kobayashi, JAMSTEC Dr Yuji Hatada, JAMSTEC

Contact Details: Dr Kazuhiro Kitazawa [email protected]