Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)
This report is prepared according to the 2015 fiscal year of the Royal Thai Government, from 1 October 2014 – 30 September 2015.
2 Annual Report 2015 3 National Center for Genetic Engineering and Biotechnology (BIOTEC) International Collaboration
Annual Report 2015 CONTENTS National Center for Genetic Engineering and Biotechnology (BIOTEC) Message from the BIOTEC Executive Director 5 Facts and Figures 7 Research and Development 11 ISBN 978-616-12-0431-0 Technology Transfer 35 First Edition January 2016 February 2016 Human Resources Development 41 Number of copies printed 500 Public Awareness 47 International Collaboration 51 Copyright ©2016 by Impact of BIOTEC’s Output 57 National Center for Genetic Engineering and Biotechnology (BIOTEC) Appendices 63 National Science and Technology Development Agency (NSTDA) List of Publications 64 List of Intellectual Properties 80 Honors and Awards 86 Executives and Management Team 88
Annual Report 2015 National Center for Genetic Engineering and Biotechnology/ National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (NSTDA). -- Pathum Thani : National Center for Genetic Engineering and Biotechnology, 2016. 90 p. : ill. ISBN : 978-616-12-0431-0 F
1. Biotechnology 2. Genetic Engineering I. National Center for Genetic Engineering and Biotechnology II. National Science and Technology Development Agency III. Title
TP248.2 660.6
Published by National Center for Genetic Engineering and Biotechnology (BIOTEC) National Science and Technology Development Agency (NSTDA) Ministry of Science and Technology 113 Thailand Science Park Phahonyothin Road Khlong Nueng, Khlong Luang Pathumthani 12120 THAILAND Tel: +66 (0) 2564 6700 Fax: +66 (0) 2564 6701-5 Website: http://www.biotec.or.th International Collaboration 3
CONTENTS
Message from the BIOTEC Executive Director 5 Facts and Figures 7 Research and Development 11 Technology Transfer 35 Human Resources Development 41 Public Awareness 47 International Collaboration 51 Impact of BIOTEC’s Output 57 Appendices 63 List of Publications 64 List of Intellectual Properties 80 Honors and Awards 86 Executives and Management Team 88
F 4 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC) Message from the BIOTEC Executive Director 5
MESSAGE FROM THE BIOTEC EXECUTIVE DIRECTOR
BIOTEC has always strived to find a balance between Human capital is the most important asset of any excellence and relevance. In Thailand, science is not organization. This year, a number of our staff have intrinsically perceived as relevant, so it is always brought pride to BIOTEC. Just a few examples gratifying when we can see our research results make illustrating the strength and diversity of our human their ways to actual users. This year, we launched a resources; Dr. Bunpote Siridechadilok was named the collaborative project with the Department of 2014 Young Technologist by the Foundation for the Agricultural Extension to transfer the effective Promotion of Science and Technology under the Beauveria bassiana strain and the cultivation Patronage of His Majesty the King for the development technology to the field stations of the Department, of a quick and efficient method to construct mutations as well as the training of farmers to effectively produce of dengue virus which will enable high throughput the fungal spores from the inoculum distributed by screening of drug targets as well as understanding the Department. The effective strain and cultivation gene functions. Dr. Noppol Kobmoo won the Marie process were the fruit of our research program to find Skłodowska-Curie Individual Fellowship to work with useful applications for the wealth of the country’s Prof. Titiana Giraud at the University of Paris-Sud, to biodiversity. B. bassiana has proven to be a potent use population genomics approach to explain the host biopesticide against insect pest larvae. specificity in insect fungi. In addition, Dr. Chalermpol Kirdmanee and Ms. Phongphan Ek-arwut were BIOTEC is currently undertaking two large-scale recognized for their work in the rural areas. Dr. projects under NSTDA Giga Impact Initiative, a new Chalermpol received the Siam Cement Group Chair Fprogram set up in 2013 providing funds to projects Professor Scholarship for the work on land with high potential to generate a significant impact rehabilitation in northeastern Thailand. The National to the economy. One project aims to use genomic Office of Buddhism honored Ms. Phongphan for her technologies in a sugarcane breeding program to support in education of monastic schools in the rural increase yield and sugar level. The other project aims area. Indeed, we never forget to be relevant. to develop a platform for large-scale production of industrial enzymes, concentrating on three This year, the Ministry of Science and Technology has recombinant enzymes for three distinct industries, been regrouped with the economic ministries, a good namely animal feed, textile and pulp and paper. In indication that our policy makers see the potential of addition to the R&D component, both projects are science and technology. It also comes with engaging industrial partners to expedite the transfer responsibilities to deliver innovations to support our of technology to the private sector once completed. local industry. At BIOTEC, we are looking forward to The sugarcane project is co-funded by and co- making this contribution in the years to come. investigated with an industrial partner, Mitr Phol Group, a key player in sugarcane and sugar business in Thailand and the Asia Pacific; whereas the enzyme team is now in discussion with various enzyme users for field validation and commercial enzyme production in Thailand.
Kanyawim Kirtikara, PhD. Executive Director, BIOTEC 6 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC) Facts and Figures 7
FFACTS AND FIGURES
BIOTEC was first set up under the Ministry for As a premier research institute in Thailand and Asia, Science, Technology and Energy on 20 September BIOTEC operates research units located at Thailand 1983. After the establishment of the National Science Science Park and specialized laboratories hosted and Technology Development Agency (NSTDA) on 30 by various universities, covering a wide spectrum December 1991, BIOTEC became one of the NSTDA of research topics from agricultural science to centers, operating outside the normal framework of biomedical science and environmental science. In civil service and state enterprises. This enabled the addition to research units, development units have Center to operate more effectively to support and been established for activities with high commercial transfer technology for the development of industry, potential. These are full scale business and agriculture, natural resources, environment and production operations designed to demonstrate the consequently the social and economic well-being commercial viability of technologies to prospective of Thai people. Other centers under the NSTDA investors. family include National Metal and Materials Technology Center (MTEC), National Electronics and Apart from research and commercialization, BIOTEC Computer Technology Center (NECTEC), National activities also include policy research, an outreach Nanotechnology Center (NANOTEC) and Technology program, human resource development and Management Center (TMC). international relations. 8 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC) Organization
BIOTEC Research Units at Thailand Science Park • Bioresources Technology Research Unit • Animal Biotechnology Research Unit • Food Biotechnology Research Unit • Medical Molecular Biology Research Unit • Genome Technology Research Unit • Biosensing Technology Research Unit
Collaborative Research Laboratories at universities and government organization • Biochemical Engineering and Pilot Plant Research and Development Laboratory - at King Mongkut’s University of Technology Thonburi (KMUTT) • Waste Utilization and Management Laboratory - at King Mongkut’s University of Technology Thonburi (KMUTT) • Cassava and Starch Technology Research Laboratory - at Kasetsart University • Rice Gene Discovery Laboratory - at Kasetsart University • Medical Biotechnology Research Laboratory - at Faculty of Medicine Siriraj Hospital and Chiang Mai University • Biomedical Technology Research Laboratory - at Chiang Mai University • Marine Biotechnology Laboratory - at Chulalongkorn University • Molecular Biology and Genomics of Shrimp Laboratory - at Chulalongkorn University • Shrimp Molecular Biology and Biotechnology Laboratory - at Mahidol University • Peat Swamp and Hala-Bala Rain Forest Research Laboratory - jointly established with the National Park, Wildlife and Plant Conservation Department and located in Narathiwat Province
Translational Administration Research Facilities and Multi-disciplinary • Policy Study and Biosafety Unit Laboratory • Rural Development Technology Service Unit • Biotechnology Business Development Division • Shrimp Genetic Improvement Center • Human Resources and Platform Technology • Nuclear Polyhedrosis Virus Pilot Development Division Plant for Insect Pest Control • Strategic Planning and Organization • Thailand Bioresource Research Center Development Division • Food and Feed Innovation Center • Evaluation and Monitoring Division • Integrative Biorefinery Laboratory • Research Unit Coordination Division • National Biopharmaceutical Facility • Management Information System Division (in collaboration with King Mongkut’s • International Cooperation and Public University of Technology Thonburi) Relations Division • Building Management and Equipment Service Division • General Management Division Facts and Figures 9
Expenditure, 775.7 m THB Human Resources, 579 Technology Transfer, 3% Research Management, 3% Human Resource Development, 4% Platform Technologies, 11% Infrastructure, 8% Cross-cutting Technologies, 0% 26 Internal Management,15% Bioresources and Community, 22% (5%) 3% Energy and Environment, 1% 4% 3% Health and Medicine, 9% 8% Agriculture and Food, 24% 158 (27%) 180 (31%) 11%
15%
22% 215 (37%) 24% 1% 9% PhD., 31% B.Sc., 27% M.Sc., 37% Below B.S., 5%
Socio-economic impact Income from sources outside NSTDA, 107.74 m THB of 5,270 m THB 2% generated by 49 1% completed project 1% F 15% Investment generation Research Fund 62.73 m THB 127 Contract/Collaborative m THB 107.74 58% Research 24.84 m THB m THB Technical Services 16.10 m THB Revenue generation 23% Workshop/Conference 1.32 m THB 4,540 Licensing Activity 1.34 m THB m THB Others 1.41 m THB
Cost reduction 509 m THB Import replacement Major Outputs 91 m THB Intellectual Properties
granted patent overseas Publications 1 233 papers, including 16 granted patent in Thailand papers in non-citation index 1 journals 30 granted petty patents in Thailand 27 patent applications in Thailand Honors and Awards 12 awards and honors 23 petty patent applications in Thailand 5,270 m THB 2 trade secret applications in Thailand 10 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC) Research and Development 11
RESEARCH AND FDEVELOPMENT
BIOTEC’s R&D and technical program covers a wide range of topics. Plant biotechnology focuses on three economically important plants: rice, cassava, and oil palm. Animal biotechnology focuses on shrimp and dairy cows, whereas food biotechnology aims to improve and upgrade the processing and quality of fermented food, including topics such as food safety and risk assessment, food chemistry, and starter culture technology. Medical biotechnology focuses on tropical and emerging diseases such as malaria, tuberculosis, dengue fever and influenza. On environmental issues, BIOTEC gives emphasis to the study of microbial diversity and the preservation, use and conservation of bioresources. Biogas and other renewable energies are the focus in the energy research theme. 12 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)
Plant and grain types of pseudo-backcross inbred lines PinK + 4 (A-C) Morphology of paddy and milled rice grains compared with pseudo-recurrent parent PinK3 (D). of Myanmar Paw San Hmwe accessions.
Ref: Ruengphayak, S., Chaichumpoo, E., Phromphan, S., Kamolsukyunyong, Highlights from Rice Biotechnology W., Sukhaket, W., Phuvanartnarubal, E., Korinsak, S., Korinsak, S., Vanavichit, A. (2015). Pseudo-backcrossing design for rapidly Using pseudo-backcrossing scheme to expedite pyramiding multiple traits into a preferential rice variety. Rice, 8:7 rice gene-pyramiding Genetic characterization of the world’s best rice, Rice production in irrigated areas of Thailand has been Myanmar Paw San Hmwe frequently and strongly affected by abiotic stresses resulting from unfavorable climatic changes, such as Paw San Hmwe (PSM) rice from Myanmar is among flooding and drought, as well as by biotic stresses the best rated rice varieties. It was named the World caused by bacterial leaf blight (BB), leaf/neck blast Best Rice 2011. It is cultivated in many areas of (BL) and brown planthopper (BPH). Therefore, new Myanmar. Strong aroma, good taste and its elongation successful breeding lines must possess multiple types during cooking are its key characteristics. However, of resistance to both biotic and abiotic stresses, as the molecular marker-based analysis of prominent well as demonstrating specific grain qualities and high traits and genetic/phenotypic characteristics in the yield. However, pyramiding multiple genes into a Paw San rice has not been well studied. desirable genetic background can take years to accomplish. The genetic characterization of PSM accessions obtained from the Myanmar germplasm bank was To shorten the time, researchers redesigned the gene- investigated. Thirty-one PSM accessions were pyramiding platform by integrating marker assisted genotypically characterized and their physical grain selection into pseudo-backcross breeding. With this and cooking quality traits were studied. Specific gene platform, they were successful in pyramiding five markers associated with aroma, apparent amylose functional genes (xa5, Xa21, Sub1A-C, SSIIa, TPS) and content (AAC) and alkali spreading value were used three QTLs (qBph3, qBL1, qBL11) into the ‘PinK3’ to determine the alleles carried by different PSM genome background within only seven breeding cycles accessions. The characterization and grouping data in four years. ‘PinK3’ is an aromatic, high-yielding, of PSM accessions posted benefits to Myanmar seed non-photoperiod-sensitive, high-amylose rice variety, banks, and these results can facilitate the utilization but it is susceptible to BPH, BB, BL and submergence of PSM rice as a genetic resource in rice breeding stresses. The new, improved lines have a high-yield programs. phenotype that confers submergence tolerance and resistance to BPH, BB and BL. This is the first report This study was jointly conducted by researchers from describing the application of pseudo-backcrossing to Rice Gene Discovery Laboratory, Kasetsart University, significantly shorten the time required for gene/QTL Myanmar Department of Agricultural Research and pyramiding in an annual crop (rice). Myanmar Agriculture Service.
This work was a collaborative effort between Rice ,Ref: Oo K.S., Kongjaimun, A., Khanthong, S., Yi, M., Myint, T.T., Korinsak, S., Siangliw, J.L., Myint, K.M., Vanavichit, A., Malumpong, C. Gene Discovery Laboratory and Kasetsart University. and Toojinda T. (2015). Characterization of Myanmar Paw San Hmwe Accessions Using Functional Genetic Markers. Rice Science, 22(2), 53-64. Research and Development 13
Insight into brown planthopper resistance To find an alternative to chemical bacteriocides, mechanisms in Thai Jasmine rice scientists explored the use of bacteriophages as biological control agents for R. solanacearum. In Thailand, brown planthopper (BPH; Nilaparvata Fourteen phages infecting R. solanacearum were lugens) infestation is one of the major annual disease isolated from soil samples collected in tomato fields outbreaks in rice crops, causing huge cultural and in Chiang Mai, Thailand. The phages showed different economic burdens to Thai Jasmine rice producers. host ranges when tested against 59 R. solanacearum Khao Dawk Mali 105 (KDML 105; KD), a well-known strains isolated from Thailand and Japan. These Thai Jasmine rice, is highly susceptible to BPH. phages were characterized as nine podoviruses and Planthoppers damage rice directly through feeding five myoviruses based on their morphology. The and also by transmitting two viruses, rice ragged stunt podoviruses isolated in this study showed strong lytic virus and rice grassy stunt virus. Up to 60% yield loss activity and wide host ranges. Therefore, these phages is common in susceptible rice cultivars attacked by BPH. have potential use in the decontamination of pathogen-infected field soils. Phage J2 demonstrated To better understand the biological mechanism of an ability to prevent bacterial wilt of tomato in pot infestation, researchers investigated the metabolomic experiments. responses to BPH infestation in Thai rice varieties.1 H NMR spectroscopy, combined with chemometrics, This study was a collaborative effort between Genome was used to analyze the polar metabolome from leaf Technology Research Unit, Kasetsart University and extracts of Thai Jasmine rice and its BPH resistant Hiroshima University (Japan). isogenic lines with and without BPH infestation at
various time points. The study identified, for the first Ref: Bhunchoth, A., Phironrit, N., Leksomboon, C., Chatchawankanphanich, time, several potential metabolic pathways for O., Kotera, S., Narulita, E., Kawasaki, T., Fujie, M. and Yamada, T. acclimatization and defense mechanisms against BPH (2015) Isolation ofRalstonia solanacearum-infecting bacteriophages from tomato fields in Chiang Mai, Thailand, and their experimental use infestation. These findings provide a valuable, first as biocontrol agents. Journal of Applied Microbiology, 118(4), 1023-33. insight into BPH resistance mechanisms in Thai Jasmine rice. Large-scale SNP discovery in cassava via F transcriptome sequencing This study was conducted by researchers from BIOTEC, Cassava is one of the most important crop species, Kasetsart University, Queen’s University Belfast (UK) having multiple uses including starch and being the and Beaumont Health System (USA). main source of dietary energy in several less developed Ref: Uawisetwathana, U., Graham, S.F., Kamolsukyunyong, W., Sukhaket, countries. Marker-assisted selection has become an W., Klanchui, A., Toojinda, T., Vanavichit, A., Karoonuthaisiri, N. and essential tool in plant breeding. Single nucleotide Elliott, C.T. (2015). Quantitative 1H NMR metabolome profiling of Thai Jasmine rice (Oryza sativa) reveals primary metabolic polymorphism (SNP) discovery via transcriptome response during brown planthopper infestation. Metabolomics, sequencing is an attractive strategy for genome 11, 1640-1655. complexity reduction in organisms with large genomes.
Highlights from Plant and Animal Biotechnology
The use of bacteriophage to control bacterial wilt Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating diseases of many economically important crops in Thailand such as ginger, pepper, tomato, potato and Curcuma alismatifolia Gagnep. At present, protection from losses by bacterial wilt is achieved mainly by early detection and subsequent eradication by destroying
the host, usually by using chemical bacteriocides. Cassava is a major staple food in the developing world, as well as a source for producing ethanol. 14 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)
Researchers sequenced the transcriptome of 16 placed on the genetic map. This SNP-based linkage cassava accessions using the Illumina HiSeq platform map was subsequently employed in a QTL analysis to and identified 675,559 EST-derived SNP markers. A detect markers associated with fruit bunch weight subset of those markers was subsequently genotyped and trunk height. These markers will be useful for by capture-based targeted enrichment sequencing in selecting individual palms with desirable characteristics
100 F1 progeny segregating for starch viscosity in breeding programs. Furthermore, the high-density phenotypes. A total of 2,110 non-redundant SNP map will contribute to a fundamental knowledge of markers were used to construct a genetic map. This genome structure and will be valuable for mapping map encompasses 1,785 cM and consists of 19 linkage other economically important genes for marker- groups. A major quantitative trait locus (QTL) assisted selections. controlling starch viscosity was identified and shown to coincide with the QTL previously reported for this This study was performed by Genome Technology trait. This work represents the first effort to perform Research Unit. capture-based targeted enrichment sequencing in cassava and illustrates the attractiveness of this Ref: Pootakham, W., Jomchai, N., Ruang-Areerate, P., Shearman, J.R., Sonthirod, C., Sangsrakru, D., Tragoonrung, S approach for genotyping SNPs in predetermined and Tangphatsornruang, S. (2015). Genome-wide SNP discovery and genomic regions. identification of QTL associated with agronomic traits in oil palm using genotyping-by-sequencing (GBS). Genomics, 105(5-6), 288-95. This study was a collaborative effort by researchers Finding genes responsible for oil palm yield from Mahidol University and Genome Technology Research Unit. Oil palm (Elaeis guineensis), the tropical perennial and cross-pollinated crop, is the most productive oil- Ref: Pootakham, W., Shearman, J.R., Ruang-Areerate, P., Sonthirod, producing crop. Improvement of oil palm yield could C., Sangsrakru, D., Jomchai, N., Yoocha, T., Triwitayakorn, K., Tragoonrung, S. and Tangphatsornruang, S. (2014). Large-scale significantly contribute to the overall oil production SNP discovery through RNA sequencing and SNP genotyping by in the world. The number of fruit bunches is one yield targeted enrichment sequencing in cassava (Manihot esculenta Crantz). PLOS ONE, 9(12):e116028. doi: 10.1371/journal. component that can be improved by increasing sex pone.0116028. ratio, the ratio of female inflorescences to total inflorescences. Genome-wide SNP discovery in oil palm using genotyping-by-sequencing approach Researchers attempted to identify gene(s) by making a physical map of a specific locus responsible for sex With rapid advancement in sequencing throughput, ratio. A putative aldo-keto reductase gene together with an overall decrease in sequencing cost, (named EgAKR1) was revealed to be a promising next generation sequencing technologies have been candidate for sex ratio determination, via controlling applied to SNP identification in various plant species. female inflorescence number. This was predicted from However, it remains costly to employ whole-genome the two newly identified polymorphic marker loci sequencing to evaluate multiple individuals in a (mEgSSRsr8-21LB and mEgAKR1-9) designed from mapping population, especially for organisms with EgAKR1. The functions of AKR gene families in other large genomes such as oil palm. Reduced representation plant species and the promoter analysis suggested methods are extremely useful, not only because of that EgAKR1 may contribute to the sex ratio through their cost-reducing aspects, but also because many abiotic stress responsiveness. research questions can be answered with a small set of markers and do not require every base of the This investigation was made by researchers from genome to be sequenced. Genotyping-by-sequencing Genome Technology Research Unit and Thammasat (GBS) is an efficient strategy that can simultaneously University. detect and score tens of thousands of molecular markers. Ref: Somyong, S., Poopear, S., Jomchai, N., Uthaipaisanwong, P., Ruang- areerate, P., Sangsrakru, D., Sonthirod, C., Ukoskit, K., Tragoonrung, Researchers employed GBS approach to perform a S. and Tangphatsornruang, S. (2015). The AKR gene family and modifying sex ratios in palms through abiotic stress large-scale SNP discovery and genotyping of a responsiveness. Functional & Integrative Genomics, 15(3), 349-362. mapping population in oil palm. Over 21,000 SNP markers were identified and 1085 markers were Research and Development 15
generating a common set of genome-wide SNP data suitable for constructing integrated linkage maps from multiple populations in a highly heterozygous agricultural species.
This was a collaborative work between Genome Technology Research Unit and Rubber Research Institute of Thailand.
Ref: Pootakham, W., Ruang-Areerate, P., Jomchai, N., Sonthirod, C., Sangsrakru, D., Yoocha, T., Theerawattanasuk, K., Nirapathpongporn, K., Romruensukharom, P., Tragoonrung, S. and Tangphatsornruang, S. (2015). Construction of a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis) using genotyping-by- sequencing (GBS). Frontiers in Plant Science, 6, 367.
GBS-based genetic linkage maps of rubber tree derived from F1 progeny from (A) BPM24 × RRIM600 and (B) BPM24 × RRIC110 crosses.
F Porcine epidemic diarrhoea virus has a substantial economic burden High-density integrated genetic linkage map of given that it is highly infectious, resulting in significant morbidity and mortality in piglets. rubber tree Construction of linkage maps is crucial for genetic studies and marker-assisted breeding programs. Development of porcine epidemic diarrhoea Recent advances in next generation sequencing vaccine technologies allow for the generation of high-density Porcine epidemic diarrhoea virus (PEDV) causes linkage maps, especially in non-model species lacking acute diarrhoea and dehydration in swine of all ages, extensive genomic resources. with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America Researchers employed the genotyping-by-sequencing warrants an urgent search for effective vaccines. (GBS) technique to perform a genome-wide SNP However, PEDV vaccine research has been hampered discovery and genotyping of two rubber tree mapping by difficulties in isolating and propagating the virus populations. Single-population linkage maps were in mammalian cells, thereby complicating the generated and common SNP markers were used as recovery of infectious PEDV using a full-length bridges to merge them into a high-density integrated infectious clone. genetic map, possibly the most saturated genetic map on rubber tree to date. SNP markers reported in this In this study, researchers engineered VeroE6 cells to study will expand the existing repertoire of available stably express porcine aminopeptidase N (pAPN) and molecular markers in rubber tree, and the integrated used them as a platform to obtain a high-growth
genetic map presented will be useful for future variant of PEDV, termed PEDVAVCT12. Subsequently, breeding programs, association studies with desirable the full-length cDNA clone was constructed by agronomic traits, genetic diversity analyses and assembling contiguous cDNA fragments
phylogenetic studies. This study also demonstrated encompassing the complete genome of PEDVAVCT12 that GBS is a robust and cost-effective approach for 16 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)
in a bacterial artificial chromosome. Infectious PEDV Researchers investigated PmFAMeT gene which is could be recovered, and the rescued virus displayed believed to regulate growth and reproduction of P. phenotypic properties identical to the parental virus. monodon. Two full-length cDNAs of PmFAMeT were
Interestingly, PEDVAVCT12 was found to contain a identified, in order to examine molecular involvement C-terminal deletion of the spike gene, resulting in of PmFAMeT gene products on ovarian (and oocyte) disruption of the ORF-3 start codon. When a functional development. PmFAMeT was found to be differentially ORF-3 gene was restored, the recombinant virus could expressed during ovarian development of P. monodon. not be rescued, suggesting that ORF-3 could suppress Eyestalk ablation and exogenous serotonin injection PEDV replication in vitro. In addition, a high-growth can promote PmFAMeT expression. and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the In a separate study, researchers evaluated biological ORF-3 gene with the mCherry gene. Together, the roles of X-box binding protein 1 in reproduction and results of this study provide a means to generate growth of P. monodon by characterizing genetically defined PEDV as a promising vaccine the PmXbp1 cDNA sequence. Expression patterns of candidate. PmXbp1 during ovarian development in wild P. monodon broodstock were examined. The study This study was conducted by Animal Biotechnology showed that eyestalk ablation had an effect on the Research Unit. expression level of PmXbp1 during late ovarian development, whereas serotonin injection promoted Ref: Jengarn, J., Wongthida, P., Wanasen, N., Frantz, P.N., Wanitchang, the expression level of ovarian PmXbp1. A. and Jongkaewwattana, A. (2015) Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone. Journal of General Virology, 96(8), 2206-18. Both studies were conducted by Animal Biotechnology Research Unit in collaboration with Chulalongkorn University. Highlights from Shrimp Biotechnology Ref: 1. Buaklin, A., Jantee, N., Sittikankaew, K., Chumtong, P., Janpoom, S., Study of shrimp growth and reproduction Menasveta, P., Klinbunga, S. and Khamnamtong, B. (2015). Expression and polymorphism of farnesoic acid O-methyltransferase (FAMeT) Closing life cycle culture is crucial to the domestication and association between its SNPs and reproduction-related parameters of the giant tiger shrimp Penaeus monodon. Aquaculture, and genetic improvement of the giant tiger shrimp 441, 106-117. (Penaeus monodon). However, poor reproductive 2. Prasertlux, S., Yocawibun, P., Janpoom, S., Klinbunga, S., Menasveta, P. maturation of captive P. monodon females and low and Khamnamtong, B. (2015). Differential expression of X-box binding quality of spermatozoa of captive males have limited protein 1 during ovarian development and association between its SNP and growth-related parameters of the giant tiger shrimp Penaeus the potential of genetic improvement, which in turn, monodon. Aquaculture, 448, 531-538. resulted in remarkably slow domestication and selective breeding programs of P. monodon in Transmission of yellow head virus Thailand. Yellow head virus (YHV) is an economically important disease in farmed shrimp in South East Asia. Despite the continual search since its discovery in the early 1990s, the reservoir for YHV-1, the most virulent strain of YHV found in Thailand, has still not been identified. Outbreaks of the virus in cultivated, exotic white leg shrimp P. vannamei that originate from SPF stocks known to be free of the virus, suggested that the outbreaks occur via horizontal transmission from an environmental source. Since the Australian red claw crayfish, Cherax quadricarinatus and the marine penaeid shrimp are often cultivated in adjacent areas Examples of red claw crayfish and shrimp gill tissues immuno-stained in Thailand, it is of importance to know whether red with monoclonal antibodies against YHV nucleocapsid protein and claw crayfish is susceptible to YHV and whether YHV counter stained with hematoxylin and eosin. could be transmitted between these species. Research and Development 17
Researchers performed the tests whether red claw Ref: Rungrassamee, W., Kingcha, Y., Srimarut, Y., Maibunkaew, S., Karoonuthaisiri, N. and Visessanguan, W. (2014). crayfish was susceptible to endemic YHV and also Mannooligosaccharides from copra meal improves survival of the capable of transmitting it to black tiger shrimp. The Pacific white shrimp (Litopenaeus vannamei) after exposure to Vibrio results revealed that red claw crayfish is susceptible harveyi. Aquaculture, 434, 403–410. but highly tolerant to the virus. Infected red claw crayfish could transmit the virus without showing any signs of yellow head disease. These facts make them Highlights from Food Science and a perfect carrier for the virus. Biotechnology
This study was conducted by researchers from BIOTEC Discovery of a halotolerant yeast, a potential Shrimp Molecular Biology and Biotechnology starter culture for soy sauce fermentation Laboratory, Mahidol University, Institute of Veterinary Soy sauce is made by the fermentation of soybeans Research and Development of Central Vietnam and combined with wheat flour, rice flour and brine. The Institute of Biotechnology (Vietnam). production involves two steps: koji fermentation using Aspergillus oryzae and moromi fermentation by Ref: Soowannayan, C., Nguyen, G.T., Pham, L.N., Phanthura, M. and Nakthong, N. (2015). Australian red claw crayfish (Cherax adding brine solution into the koji. In moromi quadricarinatus) is susceptible to yellow head virus (YHV) infection fermentation, glutaminase produced by naturally- and can transmit it to the black tiger shrimp (Penaeus monodon). present yeast converts l-glutamine originated from Aquaculture, 445, 63-69. soy protein to l-glutamic acid, a compound responsible for “umami” taste. Given the high salinity condition Immunostimulant effect of copra meal MOS on of moromi fermentation, a halotolerant yeast would white shrimp be beneficial to the soy sauce fermentation. Mannooligosaccharides (MOS) are linear chains of mannose sugars. They are known to possess prebiotic Researchers investigated yeast isolates obtained from affect and therefore can be used as a feed additive to Thai soy sauce fermentation. The most interesting prevent pathogen colonization and modulate the yeast was identified as Meyerozyma (Pichia) Fimmune system of host animals. Most MOS used in guilliermondii EM2Y61. This strain is a salt-tolerant animal feed additives are derived from the cell wall yeast that could tolerate up to 20% (w/v) NaCl and of yeast. However, copra meal, a waste product from produce extracellular and cell-bound glutaminases. coconut industries, is another rich source for MOS. The extracellular glutaminase activity was found to be much higher than that of cell-bound glutaminase. BIOTEC researchers investigated the effects of MOS This is the first report of glutaminase producing M. from copra meal as a dietary feed additive on growth guilliermondii isolated from the moromi of Thai soy performance, disease resistance, and immune sauce fermentation. M. guilliermondii EM2Y61 has enhancement of the Pacific white shrimp Litopenaeus high potential to be developed into starter yeast vannamei. The findings showed that MOS from copra culture to increase l-glutamic acid during soy sauce meal was able to significantly increase protection in fermentation. the Pacific white shrimp upon pathogen exposure, while it had no significant effect on shrimp growth This was a joint study between Mahidol University rate and feed conversion ratio. Moreover, MOS and Food Biotechnology Research Unit. supplementation induced crucial shrimp immune- related genes associated with antimicrobial peptides and the peritrophic membrane. The findings suggested that MOS can potentially be applied as an immunostimulant, particularly in aquaculture, and copra meal, which is considered a waste product of the coconut industry, can be an alternative source for MOS.
This study was conducted by researchers from Food Biotechnology Research Unit and Biosensing Technology Research Unit. Cassava pulp, waste from starch factory, can be a potentially good source of prebiotics, because of its high hemicelluloses content that promotes the growth of gut flora. 18 Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)
Ref: Aryuman, P., Lertsiri, S., Visessanguan, W., Niamsiri, N., Bhumiratana, Researchers isolated a phage from an infected culture A. and Assavanig, A. (2015). Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from Thai soy sauce fermentation. of B. amyloliquefaciens FB11, a strain used locally in International Journal of Food Microbiology. 192, 7-12. Thailand. The phage was classified morphologically into the Caudovirales order, Myoviridae family. In Novel bacteriocins: Natural antimicrobial agents order to eliminate phage infection, an early detection for food and feed industry of the phage and an inactivation measure must be applied in combination. Researchers therefore Contamination by food-borne pathogens is one of the developed a PCR-based method that can detect the major problems in food industry. Among the techniques phage with a 104 PFU/mL limit of detection, in less used to control the microbial contamination in food than 3 h including sample treatment, PCR analysis and is the use of natural antimicrobial agents. Bacteriocins gel electrophoresis. The study also demonstrated that or antimicrobial peptides produced by lactic acid the phage could be inactivated by either thermal bacteria (LAB) hold promise as natural antimicrobial treatment at 70°C for 5 min or treatment with agents. peracetic acid-based disinfectant (0.3 % v/v) for 5 min. Researchers discovered two small novel bacteriocins, This work was conducted by Food Biotechnology 7293A and 7293B, from Weissella hellenica BCC 7293, Research Unit. isolated from Thai fermented pork sausage. Both
bacteriocins had broad antimicrobial spectra and Ref: Pitaksutheepong, C., Abhisingha, M., Dumnin, J. and Visessanguan, exceptionally inhibited several important Gram- W. (2015). Isolation, detection and inactivation of a Myoviridae negative food-borne pathogens including bacteriophage infecting Bacillus amyloliquefaciens FB11. Annals of Microbiology, DOI 10.1007/s13213-014-1022-9. Pseudomonas aeruginosa, Aeromonas hydrophila, Salmonella Typhimurium and Escherichia coli. The Prebiotics from rice bran and cassava pulp sensitivity to proteolytic enzymes and stability in organic solvents, pH and temperature of both Agricultural wastes such as rice bran and cassava pulp bacteriocins suggested the potential use of bacteriocin are inherently rich in plant cell wall materials like 7293A and B in food and feed applications, replacing hemicelluloses, which are complex heteropolymers antibiotics. consisting of several kinds of monosaccharides connected by various glycosidic linkages. Due to this This study was a collaborative work between Food variety of glycosidic bonds between the monomers, Biotechnology Research Unit, Prince of Songkla hemicelluloses are usually not digestible by human University and Kyushu University (Japan). intestinal digestive enzymes, but could be utilized by members of the intestinal microbiota. The fact that Ref: Woraprayote, W., Pumpuang, L., Tosukhowong, A., Roytrakul, S., hemicelluloses can be utilized by some beneficial Perez, R.H., Zendo, T., Sonomoto, K., Benjakul, S. and Visessanguan, W. (2015). Two putatively novel bacteriocins active against Gram- microbes present in the intestine makes hemicellulose- negative food borne pathogens produced by Weissella hellenica BCC containing substances potential prebiotic sources. 7293. Food Control, 55, 176-184. Researchers investigated the prebiotic potential of Eliminating phage infection in animal feed rice bran and cassava pulp. Hydrothermal treatments production were used to extract hemicellulosic oligosaccharide Fermented soybean meal is commonly used as a mixtures from rice bran and cassava pulp. Microbial protein source in animal feed, with Bacillus utilization of the obtained oligosaccharide mixtures amyloliquefaciens primarily used as a starter culture showed that they were able to promote the growth for fermentation. The fermentation industry considers of two from three Lactobacillus, as well as three from phage attacks a major threat as they can cause serious five Bifidobacterium species tested. From the three yield losses. Thus, characterization and inactivation tested Bacteriodes strains, one utilized the cassava of these phages would be essential in managing phage pulp oligosaccharide mixture better than inulin, while decontamination in fermented feed production two grew better on rice bran oligosaccharide mixture factories. than on inulin. Furthermore, the two oligosaccharide mixtures were found to be stable at 85°C for 30 min. Similarly, these mixtures were able to withstand their exposure to simulated human gastric juice (pH 1–5) Research and Development 19
and to pancreatin treatments for up to two hours. Molecular characterization of Plasmodium These findings suggested that rice bran and cassava falciparum Bruno/CELF RNA binding proteins. pulp can be a potentially good source of prebiotics. The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory This research was a joint study between Mahidol mechanisms in different stages of its life cycle. Despite University and Food Biotechnology Research Unit. the importance of post-transcriptional regulation, key
Ref: Kurdi, P. and Hansawasdi, C. (2015). Assessment of the prebiotic elements of these processes, namely RNA binding potential of oligosaccharide mixtures from rice bran and cassava proteins (RBPs), are poorly characterized. pulp. Lwt-Food Science and Technology, 63(2), 1288–1293. Researchers characterized the RNA binding properties of P. falciparum proteins, including two putative Highlights from Malaria Research members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate Bacterial surrogate system: A tool to explore synthase (PfDHFR-TS), and adenosine deaminase antimalarial drug interaction (PfAda). RNA binding activity was tested using UV- With continuing global threat of malaria, there is an crosslinking and electrophoretic mobility shift assays. urgent need to search not only for new drugs, but also PfCELF1 and PfDHFR-TS demonstrated RNA binding for effective drug combinations. Inhibitors of activity, whereas PfAda and PfCELF2 were RBP- dihydrofolate reductase (DHFR) such as pyrimethamine negative. Intracellular protein localization of RBPs was and of dihydropteroate synthase (DHPS) such as sulfa studied using GFP-tagged transgenic parasite drugs are known to have synergistic interactions. lines. PfCELF1 protein may shuttle between nucleus However, studies of the synergism are complicated and cytoplasm, as shown by a predominantly by the fact that the malaria parasite can also salvage nuclear PfCELF1 cell population and another exogenous folates, and the salvage may also be predominantly cytoplasmic. In contrast, PfDHFR-TS affected by the drugs. It is desirable to have a protein is predominantly cytoplasmic. PfCELF1 may convenient system to study interaction of DHFR and thus have several roles, including pre-mRNA FDHPS inhibitors without such complications. processing. The mRNA targets of these P. falciparum proteins were investigated by ribonomics Researchers used Escherichia coli transformed with using DNA microarrays. A sequence motif similar to malarial DHFR and DHPS, while its own corresponding that recognized by CELF proteins in other species is genes were inactivated by optimal concentration of common in the introns of target mRNAs identified for trimethoprim and genetic knockout, respectively, to PfCELF1, suggesting that nuclear-localized PfCELF1 study the interaction of the inhibitors. Marked may regulate pre-mRNA splicing in P. falciparum, as synergistic effects are observed for all combinations has been found for CELF proteins in other species. In of pyrimethamine and sulfa inhibitors in the presence contrast, none or very few mRNA targets were found of trimethoprim. The results show synergism between for the other proteins, suggesting that they do not inhibitors of the two enzymes even in the absence of have biologically relevant roles as RBPs in the asexual folate transport and uptake. This bacterial surrogate stages of P. falciparum. system can, therefore, be used as a tool for assessing the interactions of drug combinations between the This work was conducted by Medical Molecular DHFR and DHPS inhibitors. Biology Research Unit in collaboration with the University of Edinburgh. This study was conducted by researchers from Mahidol University and Medical Molecular Biology Ref: Wongsombat, C., Aroonsri A., Kamchonwongpaisan, S., Morgan, H.P., Walkinshaw, M.D., Yuthavong, Y. and Shaw P.J. (2014). Molecular Research Unit. characterization ofPlasmodium falciparum Bruno/CELF RNA binding proteins. Molecular and Biochemical Parasitology, 198(1), 1-10. Ref: Talawanich, Y., Kamchonwongpaisan, S., Sirawaraporn, W. and Yuthavong, Y. (2015). Use of bacterial surrogates as a tool to explore antimalarial drug interaction: Synergism between inhibitors of malarial dihydrofolate reductase and dihydropteroate synthase. Acta Tropica, 149, 64-69. Annual Report 2015 National Center for Genetic Engineering and Biotechnology (BIOTEC)