First International Conferenceon Labyrinthulean Protists(ICoLP)
Program & Book of Abstract

August 28 (Wed) ‒ 30 (Fri), 2019

Koyu Hall in Konan University

KONAN GAKUEN 100th Anniversary
International Scientific Symposium Series
Sponsored by The Hirao Taro Foundation of
KONAN GAKUEN for Academic ResearchFirst International Conference on
Labyrinthulean Protists (ICoLP)
KONAN GAKUEN 100th Anniversary International Scientific Symposium Series
Sponsored by The Hirao Taro Foundation of KONAN GAKUEN for Academic Research

PeriodAugust 28 (Wed) – 30 (Fri), 2019

Venue

Koyu Hall in Konan University

Organizers
Dr. Daiske Honda (Konan University, Japan)
Dr. Makoto Ito (Kyusyu University, Japan)
Dr. Masahiro Hayashi (Miyazaki University, Japan)
Dr. Tsunehiro Aki (Hiroshima University, Japan)
Dr. Jackie L. Collier (Stony Brook University, USA)
Co-organized by
Department of Biology and Institute for Integrative Neurobiology,
Konan University

Sponsors
The Hirao Taro Foundation of KONAN GAKUEN for Academic Research
Tsutomu Nakauchi Foundation
Kobe Convention Bureau
Kobe Tourism Bureau
Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science

SupportSea Act Co., LTD (Tokyo, Japan)
Nepa Gene Co., Ltd. (Chiba, Japan)

Welcome to the First International Conference on
Labyrinthulean Protists (ICoLP)Labyrinthulean protists are used as industrial microorganisms to produce high-value
compounds such as polyunsaturated fatty acids and terpenes including carotenoids. They also
play important roles in food webs and elemental cycles in marine ecosystems, and are
involved in some diseases of marine plants and animals. Research on labyrinthulean protists
therefore extends over a wide variety of academic and industrial fields, and so opportunities
for information exchange among the diverse researchers working on these organisms arelimited. Small symposia have been held several times in Japan since 2000. To reach abroader audience, we decided to hold the first international conference so that labyrinthulid
researchers can gather together to promote future research and development on theseinteresting and useful microorganisms. This conference will also commemorate the 100th
anniversary of Konan Gakuen in 2019. We are hoping to gather 200 participants from many
countries to visit the land of Kobe beside the Seto Inland Sea, where many labyrinthulids live,
for active discussions at keynote lectures, oral and poster presentations.

Invited Speakers
Seshagiri Raghukumar (Ex-Scientist of CSIR-National Institute of Oceanography, India)
Chandralata Raghukumar (Ex-Scientist of CSIR-National Institute of Oceanography, India)
Guangyi Wang (Tianjin University, China)
Xiao Qiu (University of Saskatchewan, Canada)Jackie L. Collier (Stony Brook University, U.S.A.)
Varada Damare (Goa University, India)
Ning He (Xiemen University, China)
Javier del Campo (University of Miami, U.S.A.)
Brandon T. Hassett (University of Tromso - The Arctic University of Norway, Norway)
Yoko Hamamoto (Konan University, Japan)
Daiske Honda (Konan University, Japan)
Tsunehiro Aki (Hiroshima University, Japan)
Makoto Ito (Kyusyu University, Japan)2VenueKoyu Hall in Konan University

甲南大学 甲友会館

https://goo.gl/maps/oJvrsfUUbKTjBjUA69-chōme-5-5 Okamoto, Higashinada-ku, Kobe, Hyogo 658-0072
兵庫県神戸市東灘区岡本 9-5-5

Route
From Settsu-Motoyama Station (JR Kobe Line)
From Okamoto Station (Hankyu Kobe Line)

Free Wi-Fi in the venue
ID: guest001

PW: icolp-2019

ICoLP 2019 Secretariat
Konan University
8-9-1 Okamoto, Higashinada, Kobe, Hyogo 658-8501, Japan
TEL: +81-78-435-2607
FAX: +81-78-435-2548
E-mail: icolp@ml.konan-u.ac.jp

ICoLP 2019 Home Page
http://syst.bio.konan-u.ac.jp/icolp/index_en.html
http://syst.bio.konan-u.ac.jp/icolp/index_j.html

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Koyu Hall

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Koyu Hall

Infinity Commons(iCommons)

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Main gate

Campus Map

First International Conferenceon Labyrinthulean Protists

28-30 August, 2019
in Konan University

Please find this mascot sign5

Program of First International Conference on Labyrinthulean Protists(ICoLP)
Invited Papers & Contributed papers (Oral Presentations)
Wednesday, August 28
12:30 -

Registration at Koyu Hall

13:00 - 15:00

Tea Ceremony (Applicants only) at iCommons

15:15 - 15:30

Opening Remarks

15:30 - 16:10
Invited Paper 1

Seshagiri Raghukumar(Ex-Scientist of CSIR-National Institute of Oceanography, India)
Eight decades and more: Emergence of Labyrinthulomycetes as major
eukaryotic, marine osmotrophs

16:10 - 16:50
Invited Paper 2

Makoto Ito(Kyusyu University, Japan)
LipoQuality of thraustochytrids: Synthesis and accumulation of DHA in
thraustochytrids

17:00 - 19:00

Welcome Reception
Poster Session 1 (Odd number)

6

Thursday, August 29
09:20 - 10:00
Invited Paper 3

Guangyi Wang
(Tianjin University, China)
Significant role of Labyrinthulomycetes protists in the ecological andbiogeochemical processes of coastal and pelagic waters

10:00 - 10:40
Invited Paper 4

Yoko Hamamoto(Konan University, Japan)
Studies on ecological role and effect of labyrinthulids in marine
environment

10:40 - 11:00

Tea Break

11:00 - 11:40
Invited Paper 5

Varada Damare(Goa University, India)
Relation of aplanochytrids with the zooplankton in the marine environment

11:40 - 12:05
Chandralata Raghukumar
Contributed paper 1 (Ex Scientist: National Institute of Oceanography, India)
Occurrence of thraustochytrids in an extreme marine niche
12:05 - 12:30
Yousuke Taoka
Contributed paper 2 (University of Miyazaki, Japan)
In vitro analysis of gastrointestinal tolerance and isolation ofthraustochytrids from Japanese littleneck clam (Ruditapes philippinarum)
12:30 - 13:30

Lunch

13:30 - 14:10
Invited Paper 6

Jackie L. Collier(Stony Brook University, U. S. A.)
Insight into the ecology of the thraustochytrid QPX, an opportunistic
pathogen of hard clams

14:10 - 14:50
Invited Paper 7

Javier del Campo(University of Miami, U. S. A.)
Unveiling the ecological role of novel labyrinthulid diversity. A call toaction

14:50 - 15:30
Invited Paper 8

Brandon T. Hassett(University of Tromso - The Arctic University of Norway, Norway)
Arctic marine thraustochytrids: culturing and metabarcoding reveals novel
diversity

15:30 - 15:55
Ryosuke Nakai
Contributed paper 3 (National Institute of Advanced Industrial Science and Technology, Japan)
Unexpected distribution of labyrinthulomycetes in Antarctic freshwater
habitats
16:00 - 16:50

Poster Session 2 (Even number)

17:00 - 18:00

Transfer to the Banquet Hall

18:00 - 20:30

Conference Banquet at Sakabayashi

7Friday, August 30
09:20 - 10:00
Invited Paper 9

Xiao Qiu(University of Saskatchewan, Canada)
Biosynthesis and assembly of very long chain polyunsaturated fatty acids in
Thraustochytrium

10:00 - 10:40
Invited Paper 10

Ning He(Xiemen University, China)
Studies on the polyketide synthase pathway and omega-3 polyunsaturated
fatty acids synthesis in Schizochytrium

10:40 - 11:00

Tea Break

11:00 - 11:25
Evgeniya Kiseleva
Contributed paper 4 (AJINOMOTO CO., INC., Japan)
Aurantiochytrium sp.1 AJ7869 strain, a novel production platform forfunctional lipids, others than DHA
11:25 - 11:50
Tonje M. B. Heggeset
Contributed paper 5 (SINTEF Industry, Norway)
Genome annotation and transcriptome analysis of the lipid- and DHAaccumulating Aurantiochytrium sp. T66
11:50 - 12:15
Inga Marie Aasen
Contributed paper 6 (SINTEF Industry, Norway)
Fatty acid synthesis in Aurantiochytrium sp., revealed by analyses ofgrowth and production kinetics and global proteomes
12:15 - 13:15

Lunch

13:15 - 13:40
Nozomu Okino
Contributed paper 7 (Kyushu University, Japan)
Visualization of endoplasmic reticulum and mitochondria in
Aurantiochytrium limacinum ATCC MYA-1381
13:40 - 14:05
Yohei Ishibashi
Contributed paper 8 (Kyushu University, Japan)
A novel thraustochytrid lipase/phospholipase with unique positional
specificity contributes to microbial competition and fatty acid acquisition
from the environment
14:05 - 14:45
Invited Paper 11

Tsunehiro Aki(Hiroshima University, Japan)
Gas-to-lipids bioprocessing by acetogens and thraustochytrids

14:45 - 15:00

Closing Ceremony

8

Contributed papers (Poster Presentations)
Odd number: Wednesday August 28, 17:00 –Even number: Thursday August 29, 16:00 –Contributed paper 9

Charose Marie Ting Perez(Hiroshima University, Japan)
Metabolite profile analysis of Aurantiochytrium limacinum SR21 grownon acetate-based medium for biotechnological applications

Contributed paper 10

Jackie L. Collier(University, Stony Brook, USA)
Characterization of carotenoid knockout mutants in Aurantiochytrium
limacinumContributed paper 11

Jiaqian Li(Tianjin University, China)
Labyrinthulomycetes protist communities in oxygen minimum zones(OMZs) of the bay of Bengal (the Indian Ocean)Contributed paper 12

Minato Kasari(Kyushu University, Japan)
Distribution of a novel sphingolipid, Ceramide Glyoxylic Ethanolamine(CGE), in thraustochytrids

Contributed paper 13

Toshiki Miyaoka(Konan University, Japan)
Search for nutrients required for growth of the strains of “difficult-toculture” aplanochytrids

Contributed paper 14

Ayako Matsuda(University of Miyazaki, Japan)
Metabolic profiling of xylose assimilating thraustochytrid
Aurantiochytrium sp.

Contributed paper 15

Yuki Kubo(Kyoto University, Japan)
Overexpression of the carotenoid synthesis gene, CarS, in
Aurantiochytrium limacinumContributed paper 16

Tomi Morimoto(Konan University, Japan)
Examination of quantitative PCR to quantify the abundance ofoblongichytrids in marine environment

Contributed paper 17

Yui Takeuchi(University of Miyazaki, Japan)
Characterization of novel eicosapentaenoic acid producing thraustochytrid

Contributed paper 18

Haruka Umazume(Konan University, Japan)
Examination of the condition in the gene transfer into Parietichytrium
cells by electroporation (Labyrinthulea, Stramenopiles)

9Contributed paper 19

Mai Shiroi(Kyoto University, Japan)
Research on a dehydratase gene which is responsible for DHA synthesis in
Aurantiochytrium limacinumContributed paper 20

Ryu Tsukasaki(University of Miyazaki, Japan)
Utilization of plant biomass wastes (soybean whey and molasses) for
cultivation of thraustochytrids

Contributed paper 21

Yoshitake Takao(Fukui Prefectural University, Japan)
Genomic analysis of Sicyoidochytrium minutum DNA virus (SmDNAV)

Contributed paper 22

Haruka Nakamura(Konan University, Japan)
Examination of the condition of CARD-FISH for specific detection of theaplanochytrid cells

10Invited Papers(Keynote Lecture)11

Invited Paper #1
Eight decades and more: Emergence of Labyrinthulomycetes as major
eukaryotic, marine osmotrophs
Seshagiri Raghukumar
Myko Tech Pvt. Ltd., 313 Vainguinnim Valley, Dona Paula, Goa – 403004, India
More than 150 years have elapsed since the first Labyrinthulomycete, Labyrinthula wasdescribed by Leon Cienkowski and over 80 years since F.K. Sparrow discovered the firstthraustochytrid, followed by those of Y. Kobayashi and M. Ookubo. My talk traces researchof early, eminent mycologists, particularly during the 1950’s to the 70’s that has led to ourpresent understanding on the ecology of Labyrinthulomycetes and the continuing relevance of
their work to the future. The discovery of many novel species by Solomon Goldstein
stimulated interest in their biodiversity. Nearly 50 species are currently known and many
novel species are being detected using metagenomics. Physiological studies carried out by
Goldstein and Helen S. Vishniac provided an insight to their habitat preference and role in thesea. Extending the studies of Willy Hoehnk, Alwin Gaertner not only described the life
cycles of many novel thraustochytrids, but also developed quantitative techniques that led to
the recognition that Labyrinthulomycetes are abundant in water column from the surface tothe deep sea, as well as in sediments. Novel methods to estimate their biomass, such as
epifluorescence microscopy, FISH, quantitative pCR and flow cytometry are presently being
developed and used. It is now clear that Labyrinthulomycetes often rival bacteria in terms ofbiomass in the oceans. While focus has been on their occurrence in the water column, theirimportance in coastal sediments, as well as deep sea waters and sediments has not beenadequately addressed. Annemarie Ulken reported the common occurrence of thraustochytrids
in mangrove sediments. Their important role in mangrove detrital dynamics has subsequently
been firmly established. It also now appears possible that thraustochytrids play a role inmicroaerophilic or anaerobic mangrove sediments, as well as in polluted waters.
Ultrastructure of ectoplasmic net elements and sagenetogenetosome by David Porter, Frank O.
Perkins and Steve Moss have been important in understanding the mechanism by which
Labyrinthulomycetes obtain their nutrition in nature. Guenther Bahnweg and F.K. Sparrowextended our knowledge of these organisms to the Antarctic and clearly showed thatpsychrophilic thraustochytrids occur in such cold waters. They also described Aplanochytrium,
species of which now seem to particularly play an important role in the water column. The
discovery of viruses in a thraustochytrid by F.Y. Kazama and K.L. Schornstein in has been
confirmed in recent years by Daiske Honda and his colleagues. An early report of athraustochytrid oyster parasite has been followed by the discovery of a number of invertebrateparasites, as well as possible commensals and mutualists. It now appears that theLabyrinthulomycetes are the most significant eukaryotic osmotrophs in the sea. In this, they
appear to play a role equivalent to that of the mycetaen fungi on land. The future challenge isto unravel their role in energy transfers in marine biogeochemical pathways and theirimportance in the food web.

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Invited Paper #2
LipoQuality of thraustochytrids: Synthesis and accumulation of DHA in
thraustochytrids
Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource andBioenvironmental Sciences, Kyushu University, Fukuoka, Japan
LipoQuality is the synthetic word indicating the Quality of Lipids of cells, tissues andorganisms, which may reflect the evolutional process including environments. In this
symposium, the author will report and discuss the LipoQuality of thraustochytrids, which aresingle-cell marine protists capable of accumulating a large amount of docosahexaenoic acid
(DHA, 22:6n-3) in their lipid droplets. We first developed a versatile transformation systemapplicable to thraustochytrids (1) and analyzed the global lipid metabolism of thraustochytrids.
The author will talk about how thraustochytrids synthesize DHA (2), and how the DHA is
incorporated into glycerolipids in thraustochytrids (3,4). They accumulate DHA in lipid
droplets, mainly as constituents(s) of triacylglycerol (TG). We identified the thraustochytridspecific lipid droplet protein (TLDP1) that regulates the TG accumulation and lipid droplet
morphology in thraustochytrids (5). Finally, the author will discuss about the biologicalsignificance of DHA synthesis in thraustochytrids using DHA-null mutants ofThraustochytrium aureum.
References
1. Sakaguchi K, et al. Versatile transformation system that is applicable to both multiple
transgene expression and gene targeting for thraustochytrids. Appl Environ Microbiol, 78,
3193-3202, 2012.
2. Matsuda T, et al. Analysis of delta12-fatty acid desaturase function revealed that two
distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304. J Lipid
Res, 53, 1210-1222, 2012.
3. Abe E, et al. Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium
limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine. PLOS ONE 9:e102377, 2014.
4. Nutahara E, et al. The glycerol-3-phosphate acyltransferase PLAT2 functions in thegeneration of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b. PLOS ONE
14:e0211164, 2019.
5. Watanabe T, et al. Regulation of TG accumulation and lipid droplet morphology by thenovel TLDP1 in Aurantiochytrium limacinum F26-b. J Lipid Res, 58, 2334-2346, 2017.

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Invited Paper #3
Significant roles of Labyrinthulomycetes protists in the ecological andbiogeochemical processes of coastal and pelagic waters
Guangyi Wang1,2
12School of Environmental Science & Engineering, Tianjin University, China
Center for Marine Environmental Ecology, Tianjin University, China

Heterotrophic microbes dominate the secondary production and biogeochemical cycles in
the world’s oceans. Although heterotrophic bacteria and archaea have been shown to play key
roles in these processes, the function of their eukaryotic counterparts (e.g., marine fungi andheterotrophic protists) remain largely unknown in the vast majority of ocean provinces. To
that end, this talk will discuss results of our recent studies on Labyrinthulomycetes protists inthe coastal waters of China and North Carolina (USA) as well as in the pelagic waters of theSouth China Sea and the Indian Ocean. In the coastal water, they can dominate the biomass ofheterotrophic microbial communities and display interesting complementary dynamic changes
in the biomass of heterotrophic microbes. Particularly, some groups of these protists display
interesting temporal patterns with complicated interactions with specific bacterial groups inthe coastal waters. Although the abundance of these protists in the pelagic waters is slightly
less than that in coastal ecosystems, their relative abundance can be more dominant in thesecondary production of heterotrophic communities in the deep waters. Clearly,
Labyrinthulomycetes protists can be a key component of heterotrophic microbialcommunities with unique ecological and biogeochemical roles in both coastal and pelagic
oceans.

14

Invited Paper #4
Studies on ecological role and effect of labyrinthulids in marine
environment (Labyrinthulea, Stramenopiles)
Yoko Hamamoto1,2, Takanori Shono1, Ryosuke Nakai3, Mayumi Ueda4,
Satoshi Nagai5, and Daiske Honda1,2,
1

Department of Biology, Faculty of Science and Engineering, Konan University, Kobe,
Hyogo, Japan.
2
Institute for Integrative Neurobiology, Konan University, Kobe, Hyogo, Japan
3
National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
4
Research Institute of Environment, Agriculture and Fisheries, Osaka, Japan
5
National Research Institute of Fisheries Science, Japan Fisheries Research and Education
Agency, Yokohama, Kanazawa, Japan
Thraustochytrids inhabit the ocean all over the world and they have been recognized asimportant eukaryotic decomposers in the marine ecosystem. Our recent studies revealed a
newly recognized pathway in the grazing food chain in the marine ecosystem forthraustochytrids, especially in Aplanochytrium. For example, Aplanochytrium strains obtainnutrients by consuming living diatoms (Hamamoto & Honda, 2019, PLoS ONE 14:
e0208941). Also, Aplanochytrium is detected in the gut of zooplankton copepods, suggesting
undetected predator–prey relationships in marine ecosystems (Hirai, Hamamoto et al., 2018,
Plankton & Benthos Research, 13: 75–82). Here, we investigate their biomass to understandthe ecological impact of Aplanochytrium and also thraustochytrids. First, we collected theenvironmental sample at Osaka Bay, Japan and cell number of Aplanochytrium in theseawater were estimated by quantitative PCR (qPCR) method. The averages of cell number
were 2,100 cells l-1and 13,000 cells l-1 in the inner and middle part of Osaka Bay respectively.
We next evaluated Illumina MiSeq-based metagenomic analyses using these samples. In thisresult, Aplanochytrium were particularly abundant among the taxa identified as
thraustochytrids, accounting at the inner and middle part of Osaka Bay, 77.1% (min. 0.0, max.
100.0%) and 67.8% (min. 0.0, max. 100.0%) respectively. Finally, we considered the carbon
flow among major components of food web in Osaka Bay to assess the trophic roles ofthraustochytrids. Based on the biomass of thraustochytrids, it was estimated thraustochytrids
graze 6.1% and 19.0% of the carbon produced by phytoplankton which is consumed by the
predator, on the other hand, they also supply 3.6% and 16.2% of the carbon consumed by the
microzooplankton (e.g., ciliates) and 1.8% and 6.3% of the carbon consumed by theherbivorous net-zooplankton (e.g., copepods) in the inner and middle part respectively. The
previous studies on copepod diets showed that the copepods consumed heterotrophic
dinoflagellates (41%), ciliates (36%), diatoms (15%), nanoflagellates (6%), other microalgae(3%) (Yang et al., 2009, Journal of Plankton Research, 31: 647–659). From these results, we
speculate that thraustochytrids may also supply almost equal amount of the carbon tocopepods which is known for nanoflagelates.

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Invited Paper #5
Relation of Labyrinthulomycetes protists, the aplanochytrids, withzooplankton in the marine environment
Varada S. Damare1,2
1

Biological Oceanography Division, CSIR-National Institute of Oceanography, Dona Paula,
Goa 403004, India
2
Current address: Department of Microbiology, Goa University, Taleigao Plateau, Goa
403206, India
Labyrinthulomycetes protists form an essential component of marine microbial biota. The
ectoplasmic net elements produced by these organisms help them not only to secrete various
degradative enzymes but also for anchoring to various substrates or particulate matter inpelagic waters. They, therefore, form a part of marine aggregates and attract attention of the
predators feeding on aggregates. Zooplankton are sloppy feeders in marine waters, and they
are known to consume phytoplankton and bacteria. The occurrence of Labyrinthulomycetes
protists in the guts of the mesozooplankton (2-20 mm size range) was revealed by in situ
hybridization studies using probes specific for the aplanochytrids. The aplanochytrids are thedominant Layrinthulomycetes in the oceanic water column, mainly associated with thezooplankton in coastal waters too. The protists were also found in faecal pellets of thezooplankton. Dilution experiments revealed microzooplankton (20-200 µm size range)
grazing on aplanochytrids. Labyrinthulomycetes protists produce polyunsaturated fatty acids(PUFAs) that provide nourishment to the zooplankton for their development and reproduction.
The Labyrinthulomycetes, therefore form an important part of the marine microbial loop,
forming the so-called ‘mycoloop’, transferring nutrients back to the classical food chain. In
addition to this, the protists-fed zooplankton such as rotifers increase the nutritional status of
the fish when fed to them in aquaculture. All these organisms harbor on marine aggregates,
which are also called hotspots of microbial activity. The aplanochytrids gain significance inmarine environment as the aggregates eventually settle to the bottom, thus transferring theparticulate organic matter there and serving as carbon sink.

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Invited Paper #6
Insight into the ecology of the thraustochytrid Quahog Parasite Unknown(QPX), an opportunistic parasite of hard clams Mercenaria mercenaria
Sabrina Geraci-Yee, Bassem Allam, and Jackie L. Collier
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY,
USA
Only a few labyrinthulomycetes are known to be pathogens of marine plants or animals,
and the best studied of those infecting animals is Quahog Parasite Unknown (QPX), an
opportunistic pathogen of the hard clam, Mercenaria mercenaria. To better understand theecology of QPX, we surveyed the distribution and abundance of QPX and total
labyrinthulomycetes over 3 years throughout the marine district of New York, USA. To
assess total labyrinthulomycetes we used a quantitative PCR (qPCR) assay based on LABY-A
and LABY-Y. To assess QPX we used an existing specific qPCR assay for clam samples, andalso created a specific nested qPCR (nqPCR) assay because two rounds of PCR were required
to detect and quantify the single thraustochytrid genus in environmental samples. Sequencingof PCR products confirmed the specificity of all three assays. We examined surface seawater,bottom seawater, settleable aggregates, and sediment samples as well as clam tissue andpallial fluid samples. Labyrinthulomycetes were always detected, and sometimes we observed‘thraustochytrid spikes’ (Ueda et al. 2015) and QPX spikes. QPX was detected in about halfof the samples, of all types, and contributed a small fraction of the total labyrinthulomycete
community, usually less than 1%. The presence and abundance of QPX in the environment
were not related to the prevalence or intensity of QPX detected in clam tissues. QPX wasfrequently detected in clam pallial fluid, and we detected an intriguing inverse relationshipbetween the abundance of QPX in pallial fluid vs in tissue of individual clams.

17Invited Paper #7
Unveiling the ecological role of novel labyrinthulid diversity.
A call to action.
Javier del Campo
Rosenstiel School of Marine and Atmospheric Science – University of Miami, USA
Labyrinthulids have a diverse lifestyle in the range of heterotrophy. They can besaprophytic or phagotrophic, they can be free living or parasites, they can be gliders or they
can be free-swimmers, etc. This plasticity of life strategies allows them to occupy a wide
range of ecological niches. Recently, they have been revealed by high-throughput
environmental metabarcoding studies as very diverse, abundant, and ubiquitous.
Metabarcoding data are somehow limited in order to define their ecological functions, anddespite their potential environmental relevance little is known about the role played by mostof this newly described diversity. Thus, we need to increase our culturing and genome
sequencing efforts in order to have a better understanding of their biology. The labyrinthulidcommunity traditional expertise of using isolation and culturing methods to describe new
species stablishes the perfect foundation to succeed in this endeavor.

18

Invited Paper #8
Arctic marine thraustochytrids: culturing and metabarcoding reveals novel
diversity
Brandon T. Hassett and Marti A. Arumi
UiT – The Arctic University of Norway, Department of Arctic and Marine Biology, Tromsø,
NorwayHigh-latitude warming is modifying Arctic marine ecosystem structure and ushering in
novel biological interactions as southerly species migrant north and heterotrophic metabolic
rates increase. The Labyrinthulea are heterotrophic eukaryotic stramenopiles that have beencharacterized as important degraders of marine detritus and selectively as parasites ofphotosynthetic organisms. Throughout the Arctic Ocean, the Labyrinthulea have been
detected in general molecular surveys of eukaryotic diversity and found in association withalgae. However, this diversity has not been characterized and the corresponding ecology has
not been explored. In an effort to fill this gap of knowledge, we analyzed rDNA metabarcode
data from across the Arctic Ocean and conducted culturing surveys using pine pollen andaxenic diatom cultures. These culturing efforts have yielded several thraustochytrid isolateswith low molecular identity, including the recently described saprotroph Labyrinthulochytrium arktikum and a new diatom parasite with ~90% 18s rRNA identity to any organism
in GenBank. To supplement this culturing-based assessment of Labyrinthulea diversity,
metabarcoding was conducted across the Arctic Ocean in sea ice, open ocean, and under-ice
sediment. These efforts have revealed that the Labyrinthulea are widely distributed
throughout the Arctic marine realm and generally exist in low quantities in the open ocean,but are more abundant in coastal marine ecosystems. Warming temperatures and regionally
increasing export of permafrost suggest that more carbon might flow through saptrotrophic
thraustochytrids in the future Arctic Ocean, with unknown consequences on algal bloom
dynamics.

19

Invited Paper #9
Biosynthesis and assembly of very long chain polyunsaturated fatty acids in
Thraustochytrium
Xiao Qiu
Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon,
Saskatchewan, S7N 5A8, Canada
Thraustochytrium is a marine protist that can accumulate a large amount of very long
chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n3), a nutritionally important omega-3 fatty acid for humans and animals. Our interest in thisprotist went back about 20 years ago when little was known about the biosynthesis of DHA.
We discovered the first delta-4 desaturase in the aerobic pathway for the biosynthesis of DHA
in Thraustochytrium, which opened the opportunity to implement the aerobic pathway to
produce DHA in oilseed plants. Recently, we found that the aerobic pathway for thebiosynthesis of DHA in Thraustochytrium was actually partial with missing a few
desaturation steps, and the anaerobic pathway catalyzed by a PUFA synthase, a mega-enzyme
with three subunits each with multiple catalytic domains, was solely responsible for thebiosynthesis of DHA. Dissecting the embedded catalytic domains of the PUFA synthase andexpressing them as standalone enzymes in E. coli and Arabidopsis thaliana showed they
could functionally replace the corresponding discrete enzymes of Type II fatty acid synthases
for the synthesis of long chain fatty acids. Very recently, we demonstrated that freshly
synthesized DHA was initially incorporated into phosphatidylcholine (PC), and thenmobilized to triacylglycerol (TAG) for storage via diacylglycerol (DAG) as the intermediatein Thraustochytrium. Elucidating the biosynthesis and assembly of VLCPUFAs would be
instrumental in improving the production of VLCPUFAs in native microorganisms anddesigning new strategies for effective metabolic engineering of these fatty acids in heterologous systems, particularly oilseed crops.

20Invited Paper #10
Studies on the polyketide synthase pathway and omega-3 polyunsaturated
fatty acids synthesis in Schizochytrium
Zhipeng Li1,2, Yanyan Shi1,2, Xingyu Cao1,2, and Ning He1,2
1

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen 361005, People’s Republic of China
2
The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen 361005, People’s
Republic of China
Polyunsaturated fatty acids (PUFAs), especially ω-3 docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA), have been widely used in food, medical service, health care andother fields. However, the mechanism of PUFAs synthesis by polyketide synthase (PKS)
pathway is still unclear, which limited further development of PUFAs production. MalonylCoA: ACP transacylase (MAT) was overexpressed in Schizochytrium ATCC1381.Lipids and
PUFAs yields in MAT overexpressing strain were increased by 10.1 and 24.5%,respectively.
The total lipids yield was further increased to 110.5 g/L with fed-batch fermentation, whichwas 39.6% higher than the wild strain. DHA and EPA yields were enhanced to 47.39 g/L and1.65 g/L, respectively. Dehydrogenase (DH)-disrupted and chain length factor (CLF)disrupted strains had significant decreases (65.85 and 84.24%) in PUFAs yield, while thesaturated fatty acids proportion in lipids was slightly increased. Meanwhile, the disruption of
CLF decreased C-22 PUFA proportion by 57.51% without positive effect on C-20 PUFAs
accumulation while DH-disrupted mutant decreased the production of each PUFA. The
knockdown of ketoacyl reductase (KR) and ketoacyl synthase (KS) decreased the ratio of
DPA and DHA and increased the content of saturated fatty acids. MAT, ER and DH genes
were speculated to play a role in the synthesis of lipidsin both PKS and FAS pathways while
KR, KS and CLF are essential in PKS pathway.

21

Invited Paper #11
Gas-to-lipids bioprocessing by acetogens and thraustochytrids
Charose M. T. Perez1, Ran Hirotani1, Motomu Ishigaki1, Kenshi Watanabe1,
Yoshiko Okamura1, Takahisa Tajima1, Yukihiko Matsumura1, Yutaka
Nakashimada1, Yusuke Sumita2, Shinzo Mayuzumi3, and Tsunehiro Aki1
1
2

Hiroshima University, Hiroshima, Japan
Chugoku Electric Power, Hiroshima, Japan, 3Idemitsu Kosan, Chiba, Japan

Research and development is being promoted as a pressing issue worldwide on the
reduction and reuse of greenhouse gases. We have previously established composite
fermentation systems for producing useful lipids from various unutilized biomass using two
kinds of microorganisms having different metabolic systems. These outcomes led us to findthe condition where the oleaginous microorganism, Aurantiochytrium sp., shows highassimilability of organic acids, especially acetic acid. We therefore focused on acetogens,which release acetic acid as the main product of their metabolism, to examine its combinationin lipid fermentation. Acetic acid fermentation with Acetobacterium woodii was performed
using carbon dioxide as a substrate, and the culture broth was successfully utilized by
Aurantiochytrium sp. for efficient production of triacylglycerols. Furthermore, the metabolic
profile of lipid fermentation was subjected to the metabolome analysis to extract improvement
factors for optimization of this new gas-to-lipids bioprocess.

22

Contributed Papers(Oral Presentation)

23

Contributed Paper (Oral Presentation) #1
Occurrence of thraustochytrids in an extreme marine niche
Chandralata Raghukumar
Ex Scientist: National Institute of Oceanography, Dona Paula, Goa 403 004, India.
Stramenopiles have been reported from hydrothermal vents. We isolated thraustochytrids from the shallow waterhydrothermal vent of the D. João de Castro Sea mount, Azores in the Atlantic Ocean. The 1000 m high D. João deCastro seamount lies in the middle of Azores Archipelago (Portugal) on the hyperslow-spreading Terceira rift in
Atlantic. Hydrothermal vents are found on top of this seamount, at about 25 m below surface. The vent sites are
surrounded by two distinct color zonation, white and yellow. The white zone is characterized by high content of H2S,
CH4, H2, Pb and Co and temperature of ca. 37ºC. The yellow zone is characterized by high concentrations of Fe, Ba
and Mn and a temperature of ca. 60ºC . Species diversity of benthic diatoms was marginally higher in the white zone. A
higher numbers of heterotrophic bacteria were isolated from the yellow zone. Almost equal numbers of thraustochytrids
were isolated from both these zones. One of the thraustochytrid isolates, Ulkenia visurgensis from this site showed
better growth and protease production in the presence of Fe, Mn and Pb suggesting its adaptation to survive in metalenriched waters of hydrothermal vent. The protease enzyme was halophilic and showed maximum activity at alkaline
pH and 40 to 50 ºC temperature. The shallow water vent (23 m at the deepest) was rich in algal growth and thus algal
detritus. Thraustochytrids in such vent sites may play a role in degradation of algal detritus and enrichment of waters
with dissolved organic matter.

Contributed Paper (Oral Presentation) #2
In vitro analysis of gastrointestinal tolerance and isolation of thraustochytrids from Japanese littleneck clam
(Ruditapes philippinarum)
Kazuaki Hidaka1 and Yousuke Taoka2
1

Laboratory of Marine Environmental Microbiology , Graduate School of Agriculture, University of Miyazaki,
Miyazaki, Japan; 2Laboratory of Marine Environmental Microbiology, Department of Marine Biology andEnvironmental Sciences, University of Miyazaki, Miyazaki, Japan.
Thrausotochytrids are widely distributed in marine environment, and isolated from various samples, such asseawater, sediments, dead leaves, coral and seaweed but there is very little information about isolation from digestive
tract in aquatic animals. Previously, we observed that the tolerance of thraustochytrids against gastric juices isdepending on genus based on in vitro assay, in short, genera Aurantiochytrium and Ulkenia had higher tolerance andgenus Oblongichytrium had lower tolerance. In this study, we evaluated the tolerance of thraustochytrids isolated frommarine environment to artificial gastrointestinal juices in vitro. Ulkenia sp. strain Nob9 and Oblongichytrium sp. Strains
TakIII and Aos14 isolated from seashore area in Miyazaki, Japan were exposed to artificial acidic buffer, gastric juices
and intestinal juices. Results showed that Oblongichytrium sp. was sensitive to lower pH (less than pH 3.0) as comparedwith Ulkenia sp. We reared Japanese littleneck clam R. philippinarum in artificial seawater for two weeks, anddissected the digestive tract out. The digestive tract samples were homogenized in a 0.01M phosphate buffer saline anddirectly inoculated on B1 agar plate medium containing antibiotics, penicillin, streptomycin and amphotericin. Afterincubation, thraustochytrids-like colonies on agar plates were randomly picked up, and phylogenetic analysis using 18S
rDNA gene sequence was performed. Results showed that 4 strains and 7 strains were identified to genus
Aurantiochytrium and Siyoidochytrium, respectively. In this study, thraustochytrids were firstly isolated from digestive
tracts in R. philippinarum.

24

Contributed Paper (Oral Presentation) #3
Unexpected distribution of labyrinthulomycetes in Antarctic freshwater habitats
Ryosuke Nakai1, Yuiki Takahashi2,3, Masaki Yoshida4, Megumu Tsujimoto5, Atsushi C. Suzuki6, Sakae Kudoh7,8,
and Satoshi Imura7,8
1

National Institute of Advanced Industrial Science and Technology, Sapporo, Hokkaido, Japan; 2Graduate School ofLife and Environmental Sciences, University of Tsukuba, Tsukuba, ibaraki, Japan; 3Toho Junior and Senior HighSchool Affiliated to Toho University, Narashino, Chiba, Japan; 4Faculty of Life and Environmental Sciences,
University of Tsukuba, Tsukuba, ibaraki, Japan; 5Keio University SFC (Shonan Fujisawa Campus), Fujisawa,
Kanagawa, Japan; 6Research and education center for natural sciences, Keio University, Yokohama, Kanagawa, Japan;
7
National Institute of Polar Research, Tachikawa, Tokyo, Japan; 8SOKENDAI (The Graduate University for Advanced
Studies), Tachikawa, Tokyo, Japan.
Labyrinthulomycetes are heterotrophic straminipilan protists and occur in detectable amounts mostly in the marine
habitats. They possess the unique ability to produce long-chain polyunsaturated fatty acids such as docosahexaenoic
acid (DHA) and docosapentaenoic acid (DPA) and to degrade a wide variety of organic substrates by means of
extracellular enzymes. While the phylogenetic diversity and distribution of labyrinthulomycetes in marine environments
has been well documented, information pertaining to their freshwater lineage remains limited. In addition to the several
lineages already documented from the freshwater environments (e.g. species of the genera Diplophrys and Fibrophrys),
our previous clone library surveys reported a baseline existence of unseen freshwater lineages inhabiting aquatic mosses
in an Antarctic lake (Nakai et al., Polar Biol., 2012; Nakai et al., Chapter 2 in The Ecological Role of Micro-organisms
in the Antarctic Environment, 2019). In the present study, we examined moss specimens and lake water samples
collected from the freshwater lakes in the vicinity of Syowa Station during the 56th Japanese Antarctic Research
Expedition (JARE56). Using the 18S rRNA gene amplicon sequencing, we detected several phylotypes belonging tolabyrinthulomycete groups. These phylotypes showed very low sequence similarity with known cultured
labyrinthulomycetes, and they formed an independent cluster on the phylogenetic tree. This implies that uncultured
labyrinthulomycetes with high phylogenetic novelty are distributed in the studied freshwater habitats.

Contributed Paper (Oral Presentation) #4
Aurantiochytrium sp.1 AJ7869 strain, a novel production platform for functional lipids, others than DHA
Evgeniya Kiseleva1, Shigeo Suzuki1, Yuki Nishida2, Yohei Ishibashi2, Nozomu Okino2 and Makoto Ito2
1

Research Institute for Bioscience Products and Fine Chemicals, AJINOMOTO CO., INC., Kawasaki, Japan;
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences,
Kyushu University, Fukuoka, Japan.

2

Thraustochytrids are well known as a w3-fatty acids producer, accumulating large amount of DHA in lipid
droplets. To clarify whether Thraustochytrids can be used as a platform for sterol production, the following study was
conducted. Aurantiochytrium sp.1 sterol-rich AJ7869 strain, accumulating 4.1% of total sterols/DCW, was isolated
from the nature. Modified versatile transformation system for Thraustochytrids was developed for this strain. We foundout, that disruption of 24-dehydrocholesterol reductase gene led to 24-methyl-sterols (stigmasterol and brassicasterol)
accumulation and animal sterols disappearance. Otherwise, disruption of sterol 24-C-methyltransferase gene led toanimal sterols (cholesterol) accumulation and terminated plant and fungal sterols biosynthesis. Thus, we showed, that
AJ7869 strain can be used as a potential producer of both, plant and fungal sterols, or animal sterols. To confirm
whether accumulation of a target sterol can be increased by breeding technologies, we constructed a cholesterol
producer strain from AJ7869 parent strain, accumulating 1.3% cholesterol/DCW. Expression of 7-dehydrocholesterol
reductase and 24-dehydrocholesterol reductase genes, disruption of 24-C-methytransferase and C-22 desaturase genes
led to cholesterol/DCW increase from 1.3% to 3.1%. Also we showed, that disruption of 7-dehydrocholesterol reductase
gene led to pro-vitamin D3 accumulation. These results allow to conclude, the AJ7869 strain might be used as aplatform for various sterols production.

25

Contributed Paper (Oral Presentation) #5
Genome annotation and transcriptome analysis of the lipid- and DHA-accumulating Aurantiochytrium sp. T66
Tonje M. B. Heggeset1, Helga Ertesvåg2, Bin Liu2 and Inga Marie Aasen1
1

SINTEF Industry, Department of Biotechnology and Nanomedicine, Trondheim, Norway; 2NTNU, The Norwegian
University of Science and Technology, Department of Biotechnology and Food Science, Trondheim, Norway.
Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine
n3 fatty acid docosahexaenoic acid (DHA). As part of studies of the DHA-production in Aurantiochytrium sp. T66(ATCC® PRA-276™), the 43 Mbp draft genome was annotated based on ab-initio prediction combined with RNA-Seq
mapping. The genome encodes 11683 putative genes, of which functional information could be assigned to 9090. T66
show high sequence similarity to Aurantiochytrium sp. S-3 (ATCC® 26185™), despite the publicly available genome
record of S-3 covering only 18.1 Mbp.
A global transcriptome analysis was carried out with the aim to provide more knowledge about factors that affectthe DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis inthraustochytrids, fatty acid synthetase (FAS) and Polyunsaturated fatty acid (PUFA) synthase. Batch cultivation withnitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared tocultivation with nitrogen in excess, where lipid accumulation was obtained by oxygen limitation. A significant upregulation of FAS under N-deficient conditions was found, while the PUFA-synthase genes were only marginally
upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that Nstarvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also
revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.

Contributed Paper (Oral Presentation) #6
Fatty acid synthesis in Aurantiochytrium sp., revealed by analyses of growth and production kinetics and global
proteomes
Inga Marie Aasen1, Hanne Haslene-Hox1, Helga Ertesvåg2, Tonje M. B. Heggeset1, Per Bruheim2
1
Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway; 2Department of
Biotechnology and Food Science, The Norwegian University of Science and Technology, Trondheim, Norway.

Thraustochytrids have become important industrial production organisms for the essential, n3 fatty aciddocosahexaenoic acid (DHA). They are the only microorganisms that produce long-chain polyunsaturated fatty acids(PUFA) as part of a triacylglycerol oil. Yet, the biosynthesis of DHA in thraustochytrids is not fully understood, inparticular which factors that control the distribution between the two pathways used for fatty acids synthesis, fatty acidsynthase (FAS) and PUFA-synthase, and how to obtain high DHA productivities.
When Aurantiochytrium sp. T66 was cultivated on a defined medium using glycerol or glucose as carbon source
and glutamic acid or ammonium as nitrogen source, the fatty acid production rates decreased some hours after nitrogen(N) exhaustion. Proteome analyses showed that the amounts of the majority of the enzymes related to the coremetabolism (glycolysis and TCA-cycle) were not affected by N-exhaustion, while the amounts of FAS and PUFAsynthase dropped rapidly after N-exhaustion. By feeding N in limiting concentrations, the enzyme concentrations weremaintained at higher levels for a longer period, and the overall fatty acid productivities increased. Complete fatty acidprofiles were acquired as a function of time at the different cultivation conditions. DHA and palmitic acid (C16:0) were
the dominating fatty acids. Myristic acid (C14:0) and the C16 and C18 monounsaturated fatty acids increased rapidly
immediately after N-exhaustion in batch fermentations. When N was supplied continuously, the content of these threefatty acids was even higher, confirming that the availability of N affects both the enzyme concentrations and the fatty
acid profiles.

26

Contributed Paper (Oral Presentation) #7
Visualization of endoplasmic reticulum and mitochondria in Aurantiochytrium limacinum ATCC MYA-1381
Nozomu Okino, Hiroyoshi Wakisaka, Yohei Ishibashi and Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu
University, Fukuoka, Japan.
Thraustochytrids, single cell marine eukaryotes, produce large amounts of polyunsaturated fatty acids such as
docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) andmitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using theenhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We first expressed the egfp genein A. limacinum. The fluorescent signals of free EGFP were mainly detected in the cytosol and nucleus, indicating that
free EGFP was similarly expressed in mammalian cell lines. We next expressed the egfp gene with ER
targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the
distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker Red, an authentic fluorescentprobe for the visualization of ER in mammalian cells, also stained the same region. Expression of the egfp gene withthe mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the
inner leaflet of the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker Red
CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum werevisualized for the first time by EGFP with thraustochytrid organelle-specific targeting/retaining signals. These results
will contribute to investigations of the intracellular localization of proteins expressed in ER and mitochondria as well as
analyses of these organelles in thraustochytrids.
Ref: Okino, N. et al, Mar. Biotechnol. 20(2):182-192 (2018)

Contributed Paper (Oral Presentation) #8
A novel thraustochytrid lipase/phospholipase with unique positional specificity contributes to microbial
competition and fatty acid acquisition from the environment
Yohei Ishibashi1, Keisuke Aoki1, Nozomu Okino1, Masahiro Hayashi2, Makoto Ito1
1

Department of Bioscience and Biotechnology, Kyushu University, Fukuoka, Japan; 2Faculty of Agriculture, Universityof Miyazaki, Miyazaki, Japan.
Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine
ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic
clustering analysis using thraustochytrid draft genome databases revealed that novel putative proteins with a Lipase_3
domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous
expression in Escherichia coli and His tag-based purification, protein ID: 145138 was identified as alipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted
into the culture medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular TG
and PC. Fatty acids generated by the action of 145138 were reused for the biosynthesis of PC and TG, and A. limacinum
was able to survive even in medium containing TG as a sole source of carbon. 145138 hydrolyzed all the acyl-ester
linkages of TG; however, the enzyme showed strict positional specificity toward PC and phosphatidylethanolamine(PE), generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed very strong antimicrobial activity
compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes
to the acquisition of fatty acids from the environment after the digestion of TG and phospholipids, as well as to generate
antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine
environment.

27

28

Contributed Papers(Poster Presentation)

29

Contributed Paper (Poster Presentation) #9
Metabolite profile analysis of Aurantiochytrium limacinum SR21 grown on acetate-based medium forbiotechnological applications
Charose Marie Ting Perez, Kenshi Watanabe, Yoshiko Okamura, Yutaka Nakashimada, and Tsunehiro Aki
Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
Thraustochytrids, genus Aurantiochytrium, are excellent microbial catalyst to produce lipid products for various
biotechnological applications. In order to expand their application range, it is desirable to be able to convert diverse
resources to lipids. Therefore, lipid production system using Aurantiochytrium species from unutilized biomass such as
food waste and marine macroalgae has been constructed. In this study, acetate, which can be easily generated fromvarious resources by acetogenic microorganisms, proved to have a potential as a substrate of Aurantiochytrium
limacinum SR21. Specific growth rates (µ) of the strain SR21 in 3% acetate (APY)-or glucose(GPY)-based medium
were 0.55 and 0.98 h-1, respectively, while maximum yield of total fatty acid were 4.8 g/L at 48 h and 6.8 g/L at 30 h,
respectively. Metabolome analysis was performed to comprehensively elucidate metabolic fluctuations caused byacetate assimilation to understand the difference in growth profile between two substrates. Results indicated that cells
grown in GPY showed significant activity in glycolysis, pentose phosphate pathway and amino acids metabolism whilecells grown in APY showed significant activity in glyoxylate cycle, and mevalonate pathway. The phenotype of thestrain SR21 was interpreted based on results of metabolome analysis and possible targets for optimization of lipid
productivity were theorized.

Contributed Paper (Poster Presentation) #10
Characterization of carotenoid knockout mutants in Aurantiochytrium limacinum
Jackie L. Collier1, Mariana Rius1, Joshua S. Rest2
1

School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA; 2Department of
Ecology and Evolution, Stony Brook University, Stony Brook, NY, USA,

Some labyrinthulomycetes have the ability to synthesize carotenoids, which is uncommon among heterotrophic
eukaryotes. We have found that the trifunctional gene crtIBY, which encodes the first three carotenogenesis-specificreactions (phytoene synthase, phytoene desaturase, lycopene cyclase) in thraustochytrids, was acquired by horizontal
gene transfer from bacterial sources. However, the selective forces that favor the acquisition and maintenance of
carotenoid biosynthesis in some, but not all, labyrinthulomycetes are unknown. One hypothesis is that carotenoids may
protect the large amounts of essential omega-3 polyunsaturated fatty acids stored in lipid droplets from oxidative
damage. To evaluate this hypothesis and understand carotenoid function in thraustochytrid physiological ecology, we
have produced mutants of Aurantiochytrium limacinum ATCC MYA-1381 in which crtIBY has been interrupted bydouble homologous recombination with a construct containing a zeocin resistance (shble) expression cassette. As
predicted, the crtIBY knockout mutants lack the carotenoid pigmentation found in the wild-type. While the carotenoid
knockout mutants don’t exhibit any clear growth defects, we have detected differences between the wild-type andknockout strains in lipid accumulation in late growth and stationary phase, and in sensitivity to oxidative stress causedby treatment with hydrogen peroxide. Complementation with the wild-type gene to confirm that these phenotypes are
due to the knockout is in progress, as are studies of gene expression related to redox stress response under a variety ofgrowth conditions.

30

Contributed Paper (Poster Presentation) #11
Labyrinthulomycetes protist communities in oxygen minimum zones (OMZs) of the bay of Bengal (the Indian
Ocean)
Jiaqian Li1,2, Lu Liu1,2, Mohan Bai1,2, and Guangyi Wang1,2,
1School of Environmental Science & Engineering, Tianjin University, China; 2Center for Marine Environmental
Ecology, Tianjin University, China.
Dissolved oxygen (DO) plays a key role in shaping the structure and function of marine ecosystems. Below the
sunlight-zone (>200 m), heterotrophic microbial communities can deplete DO much more quickly through respiration
than it is replenished by phytoplankton and other physical processes. Oxygen minimum zones (OMZs) have beencommonly defined as those oceanic regions containing oxygen level below low nanomolar concentrations. The OMZ
communities are considered to be featured with the dominant microbial anaerobic activities. However, the structure anddiversity of heterotrophic microbial communities in OMZs remain largely unknown.
In this study, we investigate the diversity of bacterioplankton and Labyrinthulomycetes (fungus-like protists) in
the water columns of OMZs in the Bay of Bengal (the Indian Ocean). Nutrient analyses revealed interesting verticalprofiles of nitrate, nitrite, total nitrogen (TN), total phosphate (TP) and Silicate with their much lower concentrations
detected in the water samples of the OMZ stations than those of normal oxygen zone (NOZ). The abundance ofbacterioplankton and Labyrinthulomycetes is generally lower in OMZs than NOZs. High-through put sequencing
analyses revealed intriguing community structures of bacterioplankton and Labyrinthulomycetes with considerable
fraction of novel OTUs detected in OMZs. This study represents the first report of eukaryotic heterotrophic microbialcommunities in OMZs.

Contributed Paper (Poster Presentation) #12
Distribution of a novel sphingolipid, ceramide glyoxylic ethanolamine (CGE), in thraustochytrids
Minato Kasari1, Natsumi Hasegawa1, Yohei Ishibashi1, Tomofumi Miyamoto2, Masahiro Hayashi3, Daiske
Honda4, Nozomu Okino1, and Makoto Ito1
1
Department of Bioscience and Biotechnology, Kyushu University, Fukuoka, Japan; 2Faculty of Pharmaceutical
Sciences, Kyushu University, Fukuoka, Japan; 3Faculty of Agriculture, Miyazaki University, Miyazaki, Japan; 4Facultyof Science and Engineering, Konan University, Kobe, Japan

Sphingolipids, a class of lipids containing a sphingoid base, are ubiquitous cellular membrane components of all
eukaryotes and some prokaryotes. Since thraustochytrids have the characteristic of producing large quantities of DHAcontaining glycerolipids, special attention has been paid to glycerolipids and their metabolism so far. However, thecomposition and metabolism of sphingolipids are largely unknown in thraustochytrids. In this study, we analyzed thesphingolipid profile of different genera of thraustochytrids, Aurantiochytrium, Thraustochytrium, Schizochytrium,
Aplanochytrium and Parietichytrium with LC-ESI MS/MS. Sphingolipid profile of Thraustochytrium is completely
different from that of Aurantiochytrium; that is, the former possesses ceramide phosphoethanolamine (CPE), a
phosphosphingolipid composed of a ceramide and phosphoethanolamine, as a major complex sphingolipid, while thelatter does not contain known sphingolipids such as CPE or glycosphingolipids. Instead, Aurantiochytrium possesses anovel sphingolipid showing unkown fragmentation pattern in MS/MS analysis. NMR and high-resolution mass
spectrometry analyses revealed that the novel sphingolipid, tentatively designated as Ceramide Glyoxylic Ethanolamine(CGE), has a novel head group containing amino and carboxyl groups, which is bound to ceramide via acetal structure.
CGE is distributed in not only Aurantiochytrium, but also in Thraustochytrium, Schizochytrium, and Aplanochytrium,
although the content is quite different depending on the genera. At present, CGE has not been found in mammals, fishes,
algae, and other stramenopiles except thraustochytrids, suggesting that CGE is the thraustochytrid-specific sphingolipid.

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Contributed Paper (Poster Presentation) #13
Search for nutrients required for growth of the strains of “difficult-to-culture” aplanochytrids
Toshiki Miyaoka1, Ryosuke Nakai2, and Daiske Honda3,4
1

Graduate School of Natural Science, Konan University, Kobe, Hyogo, Japan; 2National Institute of Advanced
Industrial Science and Technology, Sapporo, Hokkaido, Japan; 3Institute for Integrative Neurobiology, Konan
University, Kobe, Hyogo, Japan; 4Faculty of Science and Engineering, Konan University, Kobe, Hyogo, Japan.
Many of the thraustochytrids, especially Aurantiochytrium strains, grow well in standard media for heterotrophs
including glucose and yeast extract. However, the most strains of genus Aplanochytrium are hard to grow in these
media. Recently, it was reported that Aplanochytrium cells grew well by obtaining nutrients from living diatoms in twomembered culture examination. This result suggests that Aplanochytrium cells potentially have the ability of wellgrowth but they cannot grow in the standard media because the unknown materials obtaining from living diatoms is thelimiting factor. In this study, we searched for essential nutrients required for growth of the strains of Aplanochytrium.
First, the proliferation test was examined using PM plates (BIOLOG, Hayward, USA) containing nutrients such as
sugars, amino acids, nucleoside, and vitamins in 96-well plates. The Aplanochytrium cells appeared to be proliferating,for example, in the wells of PM plates containing nicotinic acid and quinolinic acid, but re-examination on a larger
culture scale did not show particularly greater growth. While, it was investigated whether there was anything in the foodthat could enhance the growth of the cells. The highest cell growth was observed in the culture with the medium
including the glucose, yeast extract, minerals and a liquid prepared by soaking the dried shrimps in Milli-Q water. It has
been reported that the aplanochytrids detected in the guts of the copepods and the other zooplankton. Enhancing the cell
growth by adding crustacean extracts may be related to the association of aplanochytrids with crustaceans found inmarine environments.

Contributed Paper (Poster Presentation) #14
Metabolic profiling of xylose assimilating thraustochytrid Aurantiochytrium sp.
Ayako Matsuda¹, Aya Nagaoka2 and Masahiro Hayashi¹¹University of Miyazaki, Miyazaki, Miyazaki, Japan; ²Hayashikane Sangyo, Co., Ltd., Shimonoseki, Yamaguchi, Japan.
Thraustochytrids remarkably accumulate lipids in the cells. Their lipids rich in fatty acids are expected utilization
for biodiesel fuel. As a cheap carbon source for cultivation of thraustochytrids, lignocellulose containing 30-40% of
pentose (mainly xylose) is promising recently. For microbial production of the biodiesel fuel from lignocellulosic
biomass by thraustochytrids, xylose assimilating thraustochytrids have been isolated.In the present study, to make clear
a mechanism of lipid synthesis of the isolates, the major metabolites of central metabolism in the cells of the isolate
cultivated in the medium containing glucose or xylose as a sole carbon source were analyzed by GC-MS and LCMS/MS. Moreover, the cells cultivated in the nitrogen sufficient medium or nitrogen deficient medium were compared,
respectively. As a result, the metabolites of TCA cycle were decreased in the cells cultivated in the medium containing
glucose and deficient nitrogen. It was suggested that the carbon incorporated in the cells were used for lipid synthesis.
The contents of total lipids and fatty acids in the cells were remarkably increased. However, the metabolites of TCA
cycle were not decreased in the cells cultivated in the medium containing xylose. And the cellular lipids were notchanged drastically in the cells. Therefore, it was suggested that the differences of the carbon source and nitrogen
deficiency affected on the metabolic carbon flow in the isolate. These results showed that the optimization of carbon
flow and coenzyme balance made the biofuel production from lignocellulosic biomass much more efficient in the future.

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Contributed Paper (Poster Presentation) #15
Overexpression of the carotenoid synthesis gene, CarS, in Aurantiochytrium limacinum
Yuki Kubo, Mai Shiroi, Saki Tokunaga, Takahisa Koyama, Daichi Morimoto, Satoshi Nakagawa, and Shigeki
Sawayama
Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Labyrinthuleans are heterotrophic and eukaryotic microorganisms that inhabit marine and brackish water
environments, and are known to accumulate large amounts of ω3 fatty acids such as docosahexaenoic acid (DHA) thathave anti-inflammatory effect. Aurantiochytrium limacinum, which is a kind of labyrinthuleans, can accumulate not
only ω3 fatty acids but also a small amount of antioxidant carotenoids such as astaxanthin. Therefore, A. limacinum isthought to be the potential source of both DHA and carotenoids for health food and feed. In this study, by using genetic
engineering, we aim to improve the carotenoid synthesis ability of A. limacinum, and to create a strain which canaccumulate large amounts of both DHA and carotenoids. To achieve this goal, we first screened an endogenous
carotenoid synthesis gene CarS homolog, which is identified in closely related species Schizochytrium sp. and encodesa multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase, in
the A. limacinum genome. After that, we amplified the corresponding region of CarS from the A. limacinum genome by
PCR, ligated it to a protein expression vector for A. limacinum, and then transformed A. limacinum. After screening
with antibiotics G418, carotenoids were extracted from the transformants and quantified by HPLC. As compared withthe wild strain, carotenoids accumulation in the transformants was enriched, suggesting that putative CarS had anability to synthesize carotenoids, and overexpressing it resulted in increasing carotenoid accumulation in the cell.

Contributed Paper (Poster Presentation) #16
Examination of quantitative PCR to quantify the abundance of oblongichytrids in marine environment
Tomi Morimoto1, Yoko Hamamoto3, Mayumi Ueda2 and Daiske Honda1,3
1

Graduate School of Natural Sciences, Konan University, Kobe, Japan; 2 Research Institute of Environment, Agriculture
and Fisheries, Osaka Prefecture, Osaka, Japan; 3Faculty of Science and Engineering, Konan University, Kobe, Hyogo,
Japan.
The labyrinthuleans have been recognized as ubiquitous and abundant heterotrophs in the marine and brackish
water. Recent studies suggested that each phylogenetic group has different ecological roles such as decomposers andpredators, so that the quantitative PCR (qPCR) method is suitable for quantifying the abundance of each phylogenetic
group of labyrinthuleans. In this study, we focused on the phylogenetic group including the genus Oblongichytrium
because the oblongichytrids and aplanochytrids are shown as the major groups of labyrinthuleans in the marine
environment by the metagenomic studies (e.g., TARA ocean project) and monitoring study by the culturing method. For
establishment of the method for quantifying the abundance of oblongichytrids by qPCR, we continue to work on thedesign for the specific primers of oblongichytrids, preparation for the standard samples of calibration curve, andestimation of the copy number of 18S rDNA gene sequence per a cell. By combining this study with the alreadyestablished qPCR method for measuring aplanochytrids, it should be possible to understand the ecological impact of thelabyrinthuleans.

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Contributed Paper (Poster Presentation) #17
Characterization of novel eicosapentaenoic acid producing thraustochytrid
Yui Takeuchi1, Ayako Matsuda¹, Daiske Honda2, Takayoshi Sekiguchi3, Li Han3, and Masahiro Hayashi¹¹University of Miyazaki, Miyazaki, Miyazaki, Japan; ²Konan University, Kobe, Hyogo, Japan; 3Nippon Suisan Kaisha,Ltd., Tokyo, Japan.
Thraustochytrids are known as marine heterotrophic unicellular protists that remarkably accumulate lipids in thecells. Their lipids are rich in polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA). On the other
and, although their lipids contain DHA and n-6 docosapentaenoic acid as PUFA, only trace amount of eicosapentaenoic
acid (EPA) is contained. Actually EPA is purified from sardine oil and has been utilize as a medicine forarteriosclerosis and hyperlipidemia. In the present study, a novel thraustochytrid producing EPA was isolated and its
characterization was investigated. The novel thraustochytrids were isolated by a pine pollen bating method. Afterisolation of novel thraustochytrids, their fatty acid compositions and contents were determined by GC analysis of their
total lipids. The phylogenic analyses based on the 18S rRNA gene sequences were also carried out for an identificationof the isolates.
As a result of screening, only the strain SEK704 contained significant amount of EPA. This thraustochytrid can growonly on the medium containing glycerol as a carbon source. The growth and productivity of the strain was greatly
affected by the condition of the culture such as agitation or aeration. The finding of the EPA producing thraustochytrid
and its characterization would be quite profitable for microbial production of EPA.

Contributed Paper (Poster Presentation) #18
Examination of the condition in the gene transfer into Parietichytrium cells by electroporation (Labyrinthulea,
Stramenopiles)
Haruka Umazume1, Yohei Ishibashi2, Makoto Ito2, Yasuhiko Hayakawa3, and Daiske Honda4,5
1

Graduate School of Natural Science, Konan University, Kobe, Hyogo, Japan; 2Department of Bioscience andBiotechnology, Kyushu University, Fukuoka, Japan; 3NEPA GENE CO.,LTD, Chiba, Japan; 4Faculty of Science andEngineering, Konan University, Kobe, Hyogo, Japan; 5Institute for Integrative Neurobiology, Konan University, Kobe,
Hyogo, Japan.
The strains of genus Aurantiochytrium have been used in applied molecular biology and industrial production of
PUFAs, carotenoids and so on because of their high growth ability and high gene transfer efficiency by electroporationor Agrobacterium-mediated plant transformation. While, Parietichytrium is also important for high PUFA productivity
by the different synthetic pathway of the fatty acids, but the gene transfer has been not successful except particle
bombardment method with relatively low efficiency. In this study, we examined the condition in the gene transfer into
Parietichytrium cells by electroporation. First, we attempted gene transfer with multiple pulses that square electricporing pulses for forming pores in cell membrane were applied, followed by multiple transferring pulses, for delivering
the target molecules into cells. In general, it is known that transformation efficiency tends to increase under pulse
condition that survival rate decrease rapidly. Then as the result of examination of the survival rates under the widevoltage condition (300–2,400 V), the survival rates in Parietichytrium cells decrease rapidly at relatively low voltage
conditions. However, no transformants have obtained in this condition yet. It has continued the examination of
conditions in low-voltage conditions such as using the pulses with voltage decay.

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Contributed Paper (Poster Presentation) #19
Research on a dehydratase gene which is responsible for DHA synthesis in Aurantiochytrium limacinum
Mai Shiroi, Yuki Kubo, Saki Tokunaga, Takahisa Koyama, Daichi Morimoto, Satoshi Nakagawa, and Shigeki
Sawayama
Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Omega-3 polyunsaturated fatty acids (PUFAs), such as DHA and EPA are known to show beneficial effects on ourhealth. It has been reported that PUFAs reduce blood lipid levels, the risk of cardiovascular disease, the symptoms ofrheumatoid arthritis, and the risk of ALS. Aurantiochytrium limacinum, a Labyrinthulean, has received great attention
because of its high accumulation of PUFAs, especially docosahexaenoic acid (DHA). Previous works have revealed that
Thraustochytrium sp., a related species of A. limacinum relies on both a ∆4-desaturation-dependent pathway and a PKSlike pathway to synthesize DHA. PKS-like pathway is catalyzed by PUFA-synthases, which comprise threemultifunctional genes PKS-A, -B, and -C. According to previous studies, overexpression of the dehydratase gene in
Schizochytrium limacinum is predicted to improve the ability of DHA synthesis in silico analysis, and the reduced
amount of biomass is also smaller than when overexpressing other genes. In this study, to accelerate the accumulationof DHA, we have tried to overexpress dehydratase (DH) region which is located on the PKS-C. PKS-C of A. limacinum
was searched using the amino acid sequence of Thraustochytrium sp. as a query, and then DH region was deduced. The
corresponding region was amplified by PCR and ligated to a protein expression vector for A. limacinum. Afterscreening with antibiotic G418, we obtained 24 transformant colonies. These transformants had 5-folds higher DHA
accumulation than that of wild strain at most, suggesting that the overexpression of the DH region of PKS-C could
improve the DHA content in A. limacinum.

Contributed Paper (Poster Presentation) #20
Utilization of plant biomass wastes (soybean whey and molasses) for cultivation of thraustochytrids
Ryu Tsukasaki¹, Ayako Matsuda¹, Takuma Inoue¹, Kenta Takagi², and Masahiro Hayashi¹¹University of Miyazaki, Miyazaki, Miyazaki, Japan; ²Fuji Oil Co., Ltd., Izumisano, Osaka, Japan.
For the production of biofuels and functional lipids, thraustochytrids has the advantage of being remarkably highgrowth and lipid accumulation. Since thraustochytrids is, however, heterotrophic oleaginous microorganisms, it isrequired to consume a large amount of carbon sources for the mass cultivation and the production of biofuels andfunctional lipids. As for the cheap carbon sources, plant biomass wastes have been one of the candidates in the near
future. Although saccharified cellulosic and lignocellulosic plant biomass are investigated as carbon sources for biofuel
production, the cost of saccharification is still high for the actual utilization. On the other hand, molasses and soybean
whey are also plant biomass wastes and consist of disaccharide and/or oligosaccharides. Although it was reported thatthe activity of sucrase or α-glucosidase were detected in the supernatant of the culture of some thraustochytrids, theactivities were very weak, and thraustochytrids could not assimilate as a sole carbon source actually. As these plantbiomass wastes are smaller molecules than cellulosic and lignocellulosic biomass, these are much easier hydrolyzed tomonosaccharides. In this study, utilization of soybean whey and molasses as carbon sources for the cultivation ofthraustochytrids were investigated.
As a results, molasses and soybean whey were easily hydrolyzed to monosaccharides such as glucose, fructose,galactose and the others. These hydrolysates could be assimilated as carbon sources for thraustochytrids. These results
suggest that the utilization of molasses or soybean whey provided cheap carbon sources for mass cultivation ofthraustochytrids

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Contributed Paper (Poster Presentation) #21
Genomic analysis of Sicyoidochytrium minutum DNA virus (SmDNAV)
Yoshitake Takao1,2, Takayuki Shimeki2, and Yumi Murakoshi1
1Department of Marine Science and Technology, Fukui Prefectural University, Obama, Fukui, Japan; 2Graduate Schoolof Bioscience and Biotechnology, Fukui Prefectural University, Obama, Fukui, Japan.
Viruses are highly abundant in marine environments and are recognized as important factors in controlling
bacterial and algal biomass, in nutrient cycling, and in maintaining the bio-diversity of bacteria and microalgae. Until
today, many viruses infecting marine microalgae and bacteria have been isolated and characterized. As for
Thraustochytrids, we reported two distinct viruses and their ecological dynamics: AuRNAV (Aurantiochytrium RNAvirus: reported as SssRNAV) and SmDNAV (Sicyoidochytrium minutum DNA virus), previously.
Recently, we succeeded in isolating new viral strains infecting Sicyoidochytrium sp. FPUMEL09M18 from Obamabay, Japan Sea. On the basis of morphological features, host range and genome type, we identified them as the
SmDNAV. In this study, we determined and compared full-genome sequences of five SmDNAV strains (SmDNAV
001T, Ns-Ss, Os-Ss, Ks-Ss, Nm-Ss).
The genomes of SmDNAV strains were dsDNA sized in 234,007 ~ 250,349 bp which included nucleotide
compositions of 50.3 ~ 51.63 % G+C and 129 ~ 175 protein cording sequences (CDSs), respectively. However, only ca.
10% of CDSs had been annotated with functional product predictions on the basis of sequence similarity or protein
domain matches. Fewer CDSs were found to be homologous to known viral sequences, those of predicted function
included those encoding virion packaging ATPase, lambda-type exonuclease, zinc finger/DNA-binding protein,deoxynucleoside monophosphate kinase. From the results of comparative analysis among SmDNAV strain genomes,
many insertions, deletions and single short inversion were found in strain Nm-Ss.

Contributed Paper (Poster Presentation) #22
Examination of the condition of CARD-FISH for specific detection of the aplanochytrid cells
Haruka Nakamura1, Runa Mori2, Yoko Hamamoto2,3, Takanori Shono2, Akira Kuwata4, and Daiske Honda2,3
1
Graduate School of Natural Science, Konan University, Kobe, Hyogo, Japan; 2Department of Biology, Faculty of
Science and Engineering, Konan University, Kobe Hyogo, Japan; 3Institute for Integrative Neurobiology, Konan
University, Kobe, Hyogo, Japan; 4Tohoku National Fisheries Research Institute, Shiogama, Miyagi, Japan.

The labyrinthuleans have been detected as ubiquitous and abundant heterotrophs in the marine and brackish water.
Recent studies suggested that each phylogenetic group has different ecological roles such as decomposers and predators,
so that the catalyzed reporter deposition – fluorescence in situ hybridization (CARD-FISH) method is effective to stain
each phylogenetic group of labyrinthuleans specifically. In this study, we focused on the phylogenetic group including
the genus Aplanochytrium because the aplanochytrids is one of the major groups in the marine environment by themetagenomic studies and it was revealed that the aplanochytrid cells intake the nutrients from the important primary
producer diatoms. Initially specific fluorescence of aplanochytrids was not confirmed, but non-specific fluorescence
was observed in microalgae and zooplankton. After the examination of the condition of the hybridization temperatureand endogenous peroxidase inactivation, specific fluorescence of the aplanochytrids was successively confirmed. The
observation of the environmental samples with this method will be provide the important information for understanding
the role and impact of the aplanochytrids in the marine ecosystem.

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First International Conferenceon Labyrinthulean Protists

28-30 August, 2019
in Konan University

http://syst.bio.konan-u.ac.jp/icolp/index_en.html

お問い合わせ：100周年記念平生国際科学シンポジウム事務局（経営企画室内）
Tel: 078-435-2607, E-mail: icolp@ml.konan-u.ac.jp