CONTENTS

1. Aim of this research

2. Emiliania huxleyi

3. Alkenone

4. Staff

5. Publication

6. Research Seeds

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About this site

This web-site is super-vised by Laboratory of Plant Physiology and Metablisim, University of Tsukuba (Shiraiwa-lab). Our project was financially supported by Core Research for Evolutional Science and Technology (CREST) organized by Japan Science and Technology Agency (JST). Research theme is "海洋ハプト藻類のアルケノン合成経路の解明と基盤技術の開発" (Investigation of biosynthetic pathway of alkenones in marine haptophytes and development of fundamental technology). This page was constructed for explanation and promotion of our study and progress report.

1. Aim of this research

Aim of this project

In this study, we manufacture strains that enhance the ability to synthesize alkenones (C₃₇-C₃₉, long-chain unsaturated ketones) occupying more than 20% in organic part of algal cells in coccolithophorids, while the enhanced stains need to maintain productivity of calcium carbonate and growth rate to a similar extent as native strains. For the purpose, we perform: (1) identification of limiting factors of photosynthetic carbon-fixation, (2) elucidation of synthetic pathways of alkenones and (3) establishment of a novel technique to modify metabolisms by transformation. Improvements of alkenone-productivity in marine algae, coccolithophorids, can contribute to development of techniques to produce biomass-based energy by using seawater. Elucidations of unknown synthetic pathways of alkenones can lead to explore novel biofuels derived from intermediary metabolites and crude materials of biorefinery. From these approaches, we construct a foundation to utilize various algal biomass-based metabolites that have not been ever discovered.
Achievements obtained in this study can lead to product crude materials of chemical industries by utilization of marine microalgae and seawater. The process includes improvement of productivity of alkenones that are crude materials of biomass-based energy, production of novel biofuels derived from intermediary metabolites of alkenones and crude materials of biorefinery, and production of end products that are alkenones modified by chemical methods. The attainment of our purposes contributes to supply of energy and chemical crude materials, independent of oil-consumption. When coccolithophorids synthesize a large amount of coccolith under nutritional deficient conditions, cells are quickly precipitated. Easy harvest of coccolithophorid cells gains an advantage in process of energy-consumption for harvest of algal cells. These innovations combined with innovations of large-scale culture bring us to on-site supply of energy and crude materials of chemical industry. The innovations largely contribute to changes in social structure to create low-carbon society that do not require long-distance transport of energy and crude chemical materials. The innovations to weaken consumption of water and oils can remove current problems in production of biomass-based energy. Therefore, the social impact on our study is very large.

Research background

Now, development of biofuel-resource is quickly required as one of innovations to create low-carbon society. Because oil-production by using microalgae is not competitive to food and amount of oil-production of per unit area is largest (Chisti 2007), studies in foreign countries including USA heat up and the importance of studies drastically gain predominant attention. By changes in social situation, competitions of research and development on collection of microalgae largely producing lipids and oils, secure reserve of useful algal strains and manufacture of algal cells provided with high ability to produce oils are heating up.
Since it is concerned that productions of terrestrial biomass derived from crops and algal biomass utilizing fresh water become serious problems against lack of food and water, one has a high request for technical development of production of aquatic biomass-based energy that is competitive to food and utilization of freshwater. Therefore, this study is proposed as a study that meets those social needs. Oceanic coccolithophorids (haptophyceae) have shells of calcium carbonate (coccolith) on the cell surface. It is thought that a part of limestone in the world is caused by huge amount of coccolith sedimentated in Mesozoic age, and that organic compounds derived from coccolithophorids might be degraded into present crude oil and natural gas. Since one of those coccolithophorids, Emiliania huxleyi causes huge bloom observed from a space satellite in the current ocean and produces a large biomass occupying approximately half of ocean primary production, the alga contributes to decrease in atmospheric CO₂. These results prove that a coccolithophorid, E. huxleyi has the potential to produce huge biomass, biofuels and crude materials for biorefinery.


Fig：SEM image of E. huxleyi cell（A）alkenones droplet in the cell stained by Nile-red（B）

The coccolithophorid synthesizes unsaturated long-chain alkylketons called alkenones (C₃₇-C₃₉) that have 2 to 4 trans-carbon double bonds (Boon 1978). The representative on this study identifies long-chain catabolites and component of crude oils and natural gas when dry cells of E. huxleyi are treated by pyrolysis in absence of oxygen, and show that it is possible to use coccolithophorids as a renewable energy resource (We et al. 1999a, b, c, d). Achievements obtained in these collaborative studies prove that idea of this study that coccolithophorids are utilized as algal biofuels and crude materials of biorefinery is justifiable. Further, it have been revealed by collaborative studies of the representative and the member in the study that changes in temperature of algal culture alter unsaturation ratios as well as amount of alkenones (Shiraiwa et al. 2005), and alkenones are located in organelles covered by membrane-like structure (Sawada and Shiraiwa 2004).


Fig：Molecular structure of a general fatty acid, {alpha}-linoleic acid (upper) and alkenones (lower)

Since cultural technique of coccolithophorid cells possessing alkenones have not prevailed and studies on alkenones were extremely biased toward restitution of paleotemperature, analyses of molecular property and biosynthetic system of alkenones and application of alkenones to industry have not performed. In this study, we aim to develop techniques to achieve ‘carbon-zero emission’ by research and development to make best use of alkenones that coccolithophorids produce.

2. Haptophyta, Emiliania huxleyi

Emiliania huxleyi

Plants acquiring chloroplasts (the ability of photosynthesis) by symbiosis are a group of some phylogenetically different organisms. Studies on photosynthesis in one taxon as well as many different taxa are necessary to understand plant photosynthesis. They are actively performed in green plants, but they are still poor in other plants and algae. There is an important group of algae that are phylogenetically different from higher plants and the algae have large impact on global environments. A coccolithophorid such as E. huxleyi is one of them.


Fig：A scanning electron microscope (SEM) image of a coccolithophorid, E. huxleyi.
(The partially modified picture provided by Prof. Isao Inouye, University of Tsukuba)

Coccolithophorids possess CaCO₃ shells namely coccolith

Coccolithophorids are a group of marine haptophyte algae that form calcium carbonate crystals in the cell. As shown in an above picture, E. huxleyi also possesses specific shells on the cell surface. The shell morphorogy depends on species. Shells composed of calcium carbonate are named ‘coccolith’. It is still unknown why coccolith is produced in coccolithophorids and how coccolith is produced in coccolithophorids.

Coccolithophorids often cause 'bloom'

Coccolithopholid is known to sometimes cause so-called "bloom", large-scale mass generation in ocean. In the coast area, we often observed similar generation named "Akashiwo" which is blown-red color and is caused by Raphidophyta or Dinophyta. In contrast to Akawhiwo, a bloom by coccolithiphorid is colored brilliant emerald green as you can see the satellite image below. It is thought that coccolithophorid is an important microalgae for the big contribution on global carbon cycle.


Fig：Satellite image of coccolithophorid bloom taken by National Oceanography Centre, Southampton
（ http://www.noc.soton.ac.uk/soes/staff/tt/eh/cwall99.html ）

Coccolithophorids require selenium for their growth

In previous studies, we revealed that selenium (Se) is essential for growth of E. huxleyi. While mammals including human, a part of algae and bacteria require Se for their growth, terrestrial plants do not require Se. It is still unknown why Se requirement for the growth is different between E. huxleyi and terrestrial plants. However, we recently found unique characteristics of Se-utilization and novel selenoproteins containing Se in place of S. Therefore, it is expected to elucidate species-specific strategy for Se-utilization by the analyses of Se-metabolic mechanism of E. huxleyi.

Ocean acidification

Since the Industrial Revolution, the green house effect thought to be caused by increase of atmospheric CO₂ concentration have been a crucial problem. Besides increase of air temperature, higher CO₂ concentration may cause another effect on ocean. CO₂ released into air will be dissolved into ocean and decrease pH, so-called 'ocean acidification'. Nobody expects the effect of ocean acidification on marine organisms. As blooming by coccolithophorid indicating the great contribution on marine ecosystem, we are studying the effect of ocean acidification on E. huxleyi focusing on growth, photosynthesis and coccolith formation.

3. Alkenone

What is alkenone?

Only four species in haptophytes are known to produce long-chain unsaturated ketone, named "alkenones". Carbon length of alkenones is C₃₇-C₃₉, and in carbon backbone, there are 2 to 4 trans-type double bonds. In contrast to well-known membrane lipids which carbon length are normally C₁₈ to C₂₂, alkenones are composed of more number of carbons. Nobody knows the physiological function and the biosynthetic pathway of alkneones in haptophytes. Alkenones are known to be produced in five species of haptophytes: Emiliania, Gephyrocapsa, Isochrysis, Pseudoisochrysis and Chrysotila.


Fig：Molecular structures of alkenone species

Are alkenones the material of crude oil?

Alkenones are found in sediment of ocean over the world. According to the pyrolysis experiment of E. huxleyi, Gephyrocapsa cells at 100 to 500 degree, there appeared several hydrocarbons at C₁₀ to C₃₀. Alkenones produced anciently by haptophytes are thought to be material of crude oil or natural gas by a long-term degradation by microoraganism or naturally.

Membrane lipids or storage compounds?

There are several functional lipids found in organisms. In the cell, lipids can be divided into two by their functions. One is storage lipid for energy storage of carbons, e.g. tryacylglyceride. The other is membrane lipids which are component of cell/thylakoid/ER membranes. In the case of E. huxleyi, they highly accumulate alkenones under the daylight but, the amount of alkenones in the cell is decreased under dark condition indicating the existence of metabolic pathway of alkenones and that alkenones fucntion as storage lipid.

Do alkenones synthesized via fatty acid synthesis cycle?

Whole synthetic pathway for alkenones is still unknown. However there are some knowledges about alkenones physiology. In E. huxleyi Inhibition of a key enzyme of fatty acid synthesis, β-ketoacyl ACP synthase, causes the decrease of the amount of alkenones. It was indicated that the alkenone are sinthesized through fatty acid synthesis pathway. It was thought that, using fatty acid (C₁₆-C₂₂) as a precursor, alkenones are synthesized via elongation of carbon length, introduction of trans-type double bond and keto-group.

Alkenone as biomarker

Because of its persistent of alkenones, alkenones are used as a biomarker for specific strains of haptophytes. For instance, alkenone detected in Cretaceous sediment indicates the existence of haptophytes in Cretaceous period. Besides, because number of carbon double bond in alkenone structure is known to reflect the culturing temperature, we can estimate paleotemperature by measuring unsaturation index of alkenones found in sediment. Alkenones have been frequently used by organic geochemistry researchers as biomarker of haptophytes and paleotemperature meter.

4. Staff

Responsible Researcher

白岩善博（Prof. Dr. Yoshihiro SHIRAIWA）

Affiliation：Faculty of Life & Environmental Sciences, University of Tsukuba
Research Field：Plant physiology and metabolism

Subdelegate

沢田健（Prof. Dr. Ken SAWADA）

Affiliation：Faculty of Science, Hokkaido University
Research Field : Organic Geochemistry

5. Publication

Peer-Reviewed Paper

[9]
Quantitative analysis of carbon flow into photosynthetic products functioning as carbon storage in the marine coccolithophore, Emiliania huxleyi.
Tsuji Y, Yamazaki M, Suzuki I, Shiraiwa Y
Marine Biotechnology, in press

[8]
n-Nonacosadienes from the marine haptophytes Emiliania huxleyi and Gephyrocapsa oceanica.
Nakamura H, Sawada K, Araie H, Suzuki I, Shiraiwa Y
Phytochemistry, in press, PMID:25595675

[7]
Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi.
Kotajima T, Shiraiwa Y, Suzuki I
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1842: 1451-1458, PMID:25046625

[6]
Difference in physiological responses of growth, photosynthesis and calcification of the coccolithophore Emiliania huxleyi to acidification by acid and CO₂.
Fukuda S, Suzuki Y, Shiraiwa Y
Photosynthesis Research, 121: 299-309, PMID:24500605

[5]
Pan genome of the phytoplankton Emiliania underpins its global distribution.
Read BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, Young J, Aguilar M, Claverie JM, Frickenhaus S, Gonzalez K, Herman EK, Lin YC, Napier J, Ogata H, Sarno AF, Shmutz J, Schroeder D, de Vargas C, Verret F, von Dassow P, Valentin K, Van de Peer Y, Wheeler G; Emiliania huxleyi Annotation Consortium, Allen AE, Bidle K, Borodovsky M, Bowler C, Brownlee C, Mark Cock J, Elias M, Gladyshev VN, Groth M, Guda C, Hadaegh A, Debora Iglesias-Rodriguez M, Jenkins J, Jones BM, Lawson T, Leese F, Lindquist E, Lobanov A, Lomsadze A, Malik SB, Marsh ME, Mackinder L, Mock T, Mueller-Roeber B, Pagarete A, Parker M, Probert I, Quesneville H, Raines C, Rensing SA, Riano-Pachon DM, Richier S, Rokitta S, Shiraiwa Y, Soanes DM, van der Giezen M, Wahlund TM, Williams B, Wilson W, Wolfe G, Wurch LL, Dacks JB, Delwiche CF, Dyhrman ST, Glockner G, John U, Richards T, Worden AZ, Zhang X, Grigoriev IV
Nature, 499: 209-213, PMID:23760476

[4]
Long chain alkenes, alkenones and alkenoates produced by the haptophyte alga Chrysotila lamellosa CCMP1307 isolated from a salt marsh.
Nakamura H, Sawada K, Araie H, Suzuki I, Shiraiwa Y
Organic Geochemistry, 66: 90-97, Abstract

[3]
Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68^oC enhances generation of n-alkane-rich biofuels after pyrolysis.
Yamane K, Matsuyama S, Igarashi K, Utsumi M, Shiraiwa Y, Kuwabara T
Applied and Environmental Microbiology, 79: 924-930, PMID:23183975

[2]
Changes in alkenone and alkenoate distributions during acclimatization to salinity change in Isochrysis galbana: Implication for alkenone-based paleosalinity and paleothermometry.
Ono M, Sawada K, Shiraiwa Y, Kubota M
Geochemical Journal, 46: 235-247, Abstract

[1]
Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C₄ compounds.
Tsuji Y, Suzuki I, Shiraiwa Y
Plant and Cell Physiology, 53: 1043-1052, PMID:22492231

6. Research Seeds

1. Metabolome/Lipidome system

Comprehensive detection of more than 1000 metabolites by using combination of capillary electrophoresis (CE), liquid chromatography (LC), gas chromatography (GC) equipped with mass spectrometer (MS/MS) to analyze primary products and lipid biosynthesis pathway.

2. GC & GC-MS

Gas chromatograph (GC) and gas chromatograph equipped with mass spectrometry (GC-MS) are used for detection and quantification of alkenones, alkenes and fatty acid species. In contrast to metabolome as a complehensive analysis of all kinds of metabolites, GC and GC-MS methods are used focusing specifically on a certain lipid class.