btu

Bioresources Technology Unit

Research Profi le

National Center for GeneticEngineering and Biotechnology

Foreword

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The Bioresources Technology Unit was founded in 2007 and includes researchers each with at least 10 years of research experience in biodiversity utilization at BIOTEC. These scientists had established themselves internationally in the fields of microbial taxonomy and natural product chemistry and as members of this new unit their initial task was to strengthen cooperation and realign the focus of the members of this new group to work on the ex situ conservation and utilization of microorganisms. Although world-wide, survey, collection, identification and utilization of microorganisms are common research activities of research institutes, systematic exploration and subsequent discovery of useful compounds require a highly coordinated work environment to achieve rapid results.

The Unit now consists of scientists with expertise in a wide variety of fields ranging from microbial taxonomy, ecology, molecular biology, chemistry and information technology. In addition, because government regulators are currently struggling to keep up with rapid changes in biotechnology and microbial resource management, legal experts are also incorporated into the Unit in order to define guidelines and conduct research on bioresources management in aspects that are not currently regulated by existing Thai laws.

Understanding biodiversity while advancing its utilization is a complex task. The Bioresources Technology Unit has demonstrated that with the efforts of many scientists working as a team and with partners both local and international, Thailand can discover under-explored and under-utilized microorganisms, as well as conserve them and harness their useful properties for the benefit of Thailand and others.

(Dr. Kanyawim Kirtikara)Director

National Center for Genetic Engineering and Biotechnology (BIOTEC)

Foreword

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Bioresources Technology Unit (BTU) Microbes are important sources of various biological compounds and enzymes that are useful for humans, from food to essential therapeutic proteins. The Bioresources Technology Unit (BTU) was established in recognition of the need to explore the rich diversity of microorganisms in Thailand. Our main interests are thus to collect, identify, preserve and systematically utilize Thai-indigenous microorganisms. Several environmental challenges, including global warming underlie the urgency to collect and identify new microbial strains from environmental habitats all over Thailand. In the near future, the BIOTEC Culture Collection (BCC) which currently houses more than 30,000 microbial strains will act as a Biological Resource Center (BRC). The BRC will not only continue its previous role as a depository of microbial strains, but will also provide other types of biological materials such as DNA, and all associated information.

To maximize the bioresources potential, several systems for systematic storage of biological extracts and chemical libraries, together with semi-high throughput capability in conducting various biological assays have been established. Our bioresource management system set up in the unit allows us to speed up the vast amount of information. This will not only facilitate us in utilization of our resources efficiently, but also in connecting us to other leading research institutes. Over the past few years, BTU has also established several collaborations with world-leading pharma and biotechnological companies, e.g. Novartis for the scientific exchange of techniques and expertise. BTU also plays a role as a training hub for our neighboring countries in microbial preservation and identification. Our vision is to be a Center for bioresources research for this region.

(Dr. Lily Eurwilaichitr)Director


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BIORESOURCES TECHNOLOGY UNIT

Introduction VI

BIORESOURCES MANAGEMENT SYSTEM PROGRAM

1. BIOTEC Culture Collection Laboratory (BCC) 15 The BCC Collections 15 Database Management 16 Services Provided 16 Award 17 Collaboration with Local Scientists 17 Collaboration with International Scientists 17 Research Staff 17

2. Biotechnology Law 18 Research Experiences 18 International and National Legal Developments 19 Collaboration with Local Scientists 19 Collaboration with International Scientists 19 Research Staff 19 DISCOVERY PROGRAM

1. Bioresources Research Laboratory (BRL) 21 Award 23 Collaboration with Local Scientists 23 Collaboration with International Scientists 23 Technical Services 24 Research Staff 24

2. Bioassay Laboratory 25 Collaboration with Local Scientists 26 Collaboration with International Scientists 26 Research Staff 26

3. Enzyme Technology Laboratory 27 Enzyme Discovery 28 Optimization of Large-Scale Enzyme Production 29 Enzyme Application in Industry 29 Awards 30 Collaboration with Local Scientists 31 Collaboration with International Scientists 31 Research Staff 31

4. Fermentation Technology and Biochemical Engineering 32 Laboratory

Cultivation and Media Development Technology 32 Metabolic Product Optimization Technology 33 Bioprocess Development Technology 34 Collaboration with Local Scientists 35 Collaboration with International Scientists 35 Collaboration with Privates Sectors 35 Research Staff 35

5. Microbial Cell Factory Laboratory 36 Our Current Work in the Laboratory Involves: 36 Development of Cell-Surface Display Expression System Improvement of Protein Production 37 Development of Thai-Isolated New Yeast Strains as Alternative Host 37 Gene Expression in Lactobacillus 37 Collaboration 37 Collaboration with Local Scientists 38 Collaboration with International Scientists 38 Collaboration with Private Sector 38 Research Staff 38

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6. Microbial Engineering Laboratory 39 Exploration of Biocontrol Agents 40 Molecular Mechanism of Mosquito Larvicidal Toxins 40 Resistance Mechanism in Mosquito Larvae 41 Development of a host cell for Production of Insecticidal Proteins 41 Production of VIP3Aa for Effective Control of Insect Pests 42 Collaboration with Local Scientists 42 Collaboration with International Scientists 42 Research Staff 42 MICROORGANISM PROGRAM

1. Mycology laboratory 44 Biodiversity of Fungi in Thailand 44

Insect Fungi 45 Seed Decay Fungi 45 Coelomyceteous Fungi 45 Palm Fungi of a Peat Swamp Forest 46 Fungal Herbarium 46 Collaboration with Local Scientists 47 Collaboration with International Scientists 47 Research Staff 47

2. Phylogenetics Laboratory 48 Invertebrate-Pathogenic Fungi 48 Marine Fungi 48 Molecular Identification of Endophytic and Non-Sporulating Fungi which 49 Produce Antimicrobial Subtances Saprophytic and Endophytic Fungi from Nypa fruticans 49 Aquatic Fungi 50 Polyketide Synthases (PKS) 50

Awards 50 Collaboration with Local Scientists 51 Collaboration with International Scientists 51 Research Staff 51

INFORMATION SYSTEMS PROGRAM

1. The Information Systems Laboratory 53 Bioresource Data Management 53 Sharing and Networking Microbial Information Resources 55 Omics Data Analysis and Tools 56 Development of Interactive Training Digital Media 57

Award 58 Collaboration with International Scientists 58 Research Staff 58

2. Geoinformatics Laboratory 59 Ecosystems of the Bala Wildlife Sanctuary and Their Impact Assessment 59 Flora Survey in the Nature Trail of Hala-Bala Wildlife Sanctuary 60 The Sugarcane (Saccharum) Spatial Database Development for Production 61 Improvement and Supply Management Biology Study and Pollen Distribution of Papaya by Computer Modeling with GIS system 61 Collaboration with Local Scientists 62 Collaboration with International Scientists 62 Research Staff 62

3. Ecology Laboratory 63 The Mo Singto Research Site 63 Smaller Research Plots 65 Future Research Directions 65 Awards 66 Collaboration with Local Scientists 66 Collaboration with International Scientists 66 Research Staff 66 Publication (2004-September 2009) 67


The Bioresources Research Program was established in 1996 with the focus on research and development of the preservation, utilization and conservation of bioresources. The program has a special emphasis on the utilization of biotechnology as a core technology to increase the value of commercial products, such as food and feed products, enzyme products, drug and bioactive compounds and bio-control products. Since the Program was established over a decade ago under the BIOTEC Central Research Unit, the program has generated a substantial amount of research output. In 2007, the Bioresources Research Program was separated from the BIOTEC Central Research unit to set up a new research unit called the Bioresources Technology Unit with the aim of conducting research focused on conservation and effective utilization of bioresources. The Bioresources Technology Unit is now the major research program of BIOTEC, taking up one-third of BIOTECs research staff whose research is coordinated to strongly support BIOTECs Bioresources Technology Program.

The mission of the Bioresources Technology Unit is: i) to exploit the unique natural resources available in Thailand using biotechnologies to convert into value-added products required in the pharmaceutical, food and feed, and enzyme industries ii) to develop both platform and innovative technologies to build national infrastructure and research and development on the utilization of bioresources.

The vision is to become a leading bioresources technology research unit that promotes sustainable use of biodiversity for deriving high value added products and economic development.

Objectives

The utilization of bioresources for high value added products such as enzymes, bio-control, food and feed for the benefit of agriculture and industry in the country.

To set up the state-of-the-art biotechnology research facilities for sustainable development of bioresources.

To build technology platforms to strengthen the national bioresources research and development capability.

To collaborate with other institutions/ organizations/ universities nationally and internationally in bioresources.

The Bioresources Technology Unit houses 13 laboratories ranging from survey and identification of microorganisms, preservation, utilization, conservation and legal management of bioresources. This research unit is the most advanced bioresources research unit in Thailand as it is made up of researchers in different fields in order to utilize and scientifically manage biodiversity. Researchers in the unit are working together with the aim of using biotechnology to explore novel high value products, such as drugs and enzymes (Figure 1, 2). Areas of expertise include:

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Taxonomy of microorganisms especially fungi, yeasts and actinomycetes

Developing throughput biological assays

Natural product chemistry

Pre-pilot scale production of commercially valuable bio-products

Enzyme discovery

Recombinant expression systems

Biodiversity and ecology of plant, animal and microorganism

Structure Elucidation and Modification (Chemistry laboratory)

Enzymes for IndustryThailand National CultureCollection (TNCC)

BIOTEC Culture Collection (BCC) Screening for Enzymes(Enzyme technology laboratory)

Microbe collection (Mycology laboratory

and BCC)

Phylogeny study (Phylogenetics laboratory)

Growing fungi (Fermentation technology and biochemical

engineering laboratory)

Screening for Active Compounds(Bioassay laboratory)

Potential Lead Compounds forFurther Drug Development

Figure 1 Diagram illustrating the connection of research activities between laboratories under the Bioresources Technology Research Program

Commercial Benefit

Structure Elucidation and Chemical Modification of Bioactive Compounds

Bioactivity Screening

Fermentation Technology for Culturing Microbes

Study on Phylogenetics

Culture Collection

Taxonomy

Microbes from the Environment

MIC

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Figure 2 Process of increasing microbial value

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Research and Development Programs

The Bioresources Technology Unit aligns with the goal of Thailands National Biotechnology Policy Framework on Biotechnology and NSTDAs Cluster for Environment by research and development for sustainable social and commercial use of biological resources. The Bioresources Technology Unit aims to build National infrastructure and research and development on the utilization of bioresources.

The Bioresources Technology Unit has four core research programs, namely Bioresources Management System Program, Discovery Program, Microorganism Program and Information System Program (Figure 3). These research programs are categorized as follows:

Bioresources Management System Program aims to collect and preserve microbial cultures and their relevant data for BIOTEC in house research programs and for the countrys official depository and to conduct legal and policy studies with the focus on legal management of bioresources and intellectual property management. The Bioresources Management System Program consists of the following two laboratories.

- BIOTEC Culture Collection Laboratory

- Biotechnology Law

Discovery Program aims to utilize bioresources for high value added products such as enzymes, bio-control, food and feed for the benefit of agriculture and industry in the country, to build up research facilities on screening for bioactive compounds which are typically present in very low concentrations, with rapid, low cost and highly sensitive detection methods, to develop cultivation technology and bioprocesses for efficient production of various valuable bio-products from microorganisms and to isolate and identify bioactive substances produced from various microorganisms, especially insect pathogenic fungi by using activity-guided fractionation and structure modification for increased biological activity. The Discovery Program consists of the following six laboratories.

- Bioresources Research Laboratory

- Bioassay Laboratory

- Enzyme Technology Laboratory

- Fermentation Technology and Biochemical Engineering Laboratory

- Microbial Engineering Laboratory

- Microbial Cell Factory Laboratory

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Microorganism Program aims to provide data on the biodiversity of fungi in Thailand, to isolate fungi for the BIOTEC culture collection and screening program and to develop and expand the advancement of molecular phylogeny for a better understanding of the evolutionary relationships of fungi and other organisms. The Microorganism Program consists of the following two laboratories.

- Mycology Laboratory

- Phylogenetics Laboratory

Information Systems Program aims to develop information technology in order to establish and enhance the information infrastructure such as the systematic collection, preservation and distribution of bioresources and the search and discovery of exploitable bioresources using our custom-designed program namely, Microbial Information Management (MIMs) to develop high quality needed spatial database for bio-ecological resources and land resources management and monitoring and to study of the dynamics of and changes in ecosystem for ecological monitoring. The Information System Program consists of the following three laboratories.

- Information Systems Laboratory

- Geoinformatics Laboratory

- Ecology Laboratory

Figure 3 Organizational Structure of the Bioresources Technology Unit

Microbial EngineeringLaboratory

Director

Research Support

BioresourcesManagement

System Program

DiscoveryProgram

MicroorganismProgram

Informationsystems Program

BIOTEC Culture Collection Laboratory

Bioresources ResearchLaboratory

Mycology Laboratory Information SystemsLaboratory

Biotechnology Law

Bioassay Laboratory GeoinformaticsLaboratory

Enzyme TechnologyLaboratory

PhylogeneticsLaboratory

Ecology Laboratory

Fermentation Technologyand Biochemical

Engineering Laboratory

Microbial CellFactory Laboratory

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XPersonnel

As of September 2009, the Bioresources Technology Unit had 141 staff members in the research section, of these, 36 hold Doctoral Degrees, 48 hold Master Degrees, 49 hold Bachelor Degrees and 8 hold professional diplomas (Table 1).

Program

Academic standing

TotalPh.D. M.S. B.S. Professionaldiploma

Bioresources Management System Program

3 4 7 1 15

DiscoveryProgram

25 30 28 5 88

Microorganism Program

6 9 8 2 25

Information System Program

2 5 6 0 13

Total personnel 36 48 49 8 141

Table 1 Number of BIOTEC personnel working at the Bioresources Technology Unit in all four research programs classified according to academic standing

* Note: This table shows staff working at the various program in September 2009

Human Resources Development

The Bioresources Technology Unit recognizes the importance of personnel development when it comes to the transformation of young scientists into professional researchers who are able to achieve their career goals. To this end, the unit has introduced a mentoring system for new scientists, and periodically seeks assistance from renowned professors from universities to provide valuable suggestions for the researchers.

The Bioresources Technology Unit also offers research training for undergraduate, graduate and post-graduate levels to both local and foreign institutions. The unit currently has 50 undergraduate and 40 graduate students training each year. Most of the Master and Doctoral students receive scholarships supported by the Royal Golden Jubilee Program [Thailand Research Fund (TRF)], the Thai Graduate Institute of Science and Technology (TGIST) research fund and Biodiversity Research and Training Program (BRT).

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Research Funding

The period of October 2006-September 2009 showed a significant increase in the level of funding coming to the unit. The Bioresources Technology Unit receives research funding on the basis of technical merit from the National Science and Technology Development Agency (NSTDA) / National Center for Genetic Engineering and Biotechnology (BIOTEC). This accounts for the largest proportion of the units funding, making up around 72 percent of the total funding. Other funding comes from a wide variety of funding agencies, both local and international. Local funding bodies include the Biodiversity Research and Training Program (BRT) (accounting for 27 percent of the total) and other sources such as the Thailand Research Fund (TRF), and Mahidol University (which together account for approximately 1 percent of the total). International funding is derived from numerous agencies such as the United Nations Educational Scientific and Cultural Organization (UNESCO), which together provide around 1 percent of the budget as a whole (Table 2 and Figure 4).

Table 2 Overall funding by research program at the Bioresources Technology Unit (October 2006-September 2009)

Program Number of projects Funding (million Baht)

BioresourcesManagementSystem Program

21 12

Discovery Program 87 39.6


36 13

Information System Program

12 5.1

Total 156 69.7

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Industrial Col laboration and TechnologyTransfer

To effectively translate research advances into commercial products and processes, collaboration with industry is crucial. Bioresources Technology Unit is establishing relationships with enterprises in a range of industries, including pharmaceuticals, food, feed, energy, environment and cosmetics. These partnerships incorporate interactive contract and cooperative research. For cooperative research, industrial sponsors participate in specific research programs and projects. The result is mutually beneficial partnerships that stimulate research with marketable applications.

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BIOTEC72%

Note: NSTDA/BIOTEC = Cluster and Program Management Office (CPMO, NSTDA)

Platform Technology (BIOTEC)

Bioresource Research Network (BRN) International Org. = UNESCO Others = Biodiversity Research and Training Program (BRT), Thailand Research Fund, Private Sector

BIOTECOthersInternational Org.

International Org.1%

Others27%

Figure 4 Sources of research funding for the Bioresources Technology Unit (October 2006-September 2009)

Publications, Patents and New Technology

During the period October 2006-September 2009, the Bioresources Technology Unit has filed 13 patents and had one accepted, disclosed new inventions and had 170 international publications. At home and abroad, the unit continues to be recognized as an outstanding national science research unit, as evidenced by the fact that it has received several distinguished awards over the past few years. Notable pieces of research are outlined in more detail later in this profile.

International Col laboration

The unit collaborates with research centers across the world in the exchange of knowledge and scientists (Table 3).

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Table 3 Example of international collaborative partners of the Bioresources Technology Unit (September 2009)

Research Center Country Laboratory

Bioresources Management System Program

1 Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology

Korea

BIOTEC Culture Collection Laboratory

2 Culture Collection of Institute of Microbiology, Chinese Academy of Sciences

China

3 Institute of Tropical Biology Vietnam

4 Japan Collection of Microorganisms (JCM) Japan

5 Netherlands Culture Collection of Bacteria (NCCB) Netherland

6 NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation

Japan

7 NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation

Japan.Biotechnology Law

8 University of London UK

Discovery Program

1 Novartis Switzerland Bioresources Research Laboratory

2 Kunming Institute of Botany China Bioassay Laboratory

3 National Institute of Advanced Industrial Science and Technology (AIST)

Japan

Enzyme Technology Laboratory4 The University of Tokyo Japan

5 The University of Florida USA

6 Anhui Agricultural University China

Fermentation Technology and Biochemical Engineering Laboratory

7 Chalmer University Sweden

8 Copenhagen University Denmark

9 Technical University of Denmark Denmark

10 University of Maryland USA

11 Vinh University Vietnam

12 Laboratories of Applied Bioscience, Niigata University

Japan

Microbial Engineering Laboratory

13 Public Health Entomology Research & Education Center, Florida A&M University

USA

14 University of Maryland USA

15 Lawrence Berkeley National Laboratory, University of California

USA


1 Portsmouth University UK

Mycology Laboratory

2 Oregon State University USA

3 Bhutan Government Bhutan

4 City University of Hong Kong, Hong Kong SAR China

5 Hong Kong University, Hong Kong SAR Taiwan

6 Food Research Institute China

7 Institute of Fermentation, Osaka Japan

8 Landcare New Zealand

9 University Malaya Malaysia

10 National Taiwan Ocean University Taiwan

11 Oregon State University USAPhylogenetics Laboratory12 Vinh University Vietnam

13 National Taiwan Ocean University Taiwan

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National Center for Genetic Engineering and Biotechnology

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Microorganisms are the largest and most diverse group of all living organisms. They are generally single celled and can not be seen by the naked eye. They are divided into four major groups: fungi, bacteria, protists and viruses. In nature, microorganisms play an important role in nutrient recycling. Their roles can be producers where organic compounds are synthesized from carbon dioxide (CO2) and other inorganic substances, or decomposers where accumulated or organic substances are degraded and mineralized. Numerous microorganisms are isolated from nature and exploited in several different areas such as the production of food, agricultural and pharmaceutical products. The isolated cultures need to be properly preserved in order to maintain their viability, purity and authenticity for future use.

1 . BIOTEC Culture Collection Laboratory (BCC)

Recognizing the importance of the microorganisms, especially those isolated from natural environments in Thailand, BIOTEC founded the BIOTEC Culture Collection (BCC), in 1996. The primary objective of BCC is to collect and preserve microbial cultures and their relevant data for BIOTECs in-house research programs, mainly the Bioresources Research Program which focuses on isolation of valuable products such as secondary metabolites, enzymes and bioactive short peptides from microorganisms. Besides the maintenance and distribution of cultures for in-house research, BCC provides training, identification, and lyophilization services to the public.

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percent of the cultures BCC hold are filamentous fungi, which are taxonomically and ecologically diverse. One quarter was isolated from insects (insect pathogenic fungi). The rest were isolated from soil, seeds, decayed wood, plants, lichens, dung, fresh water and sea water.

Almost all strains in the collection are cryo-preserved at 80oC as working cultures. Freeze drying, liquid drying or storage in vapor phase of nitrogen is also used for long term preservation of strains with special characteristics, such as new species and those that produce biologically active compounds. Duplicate collections of valuable strains are held at different locations in Thailand.


The BCC Collections

At present, BCC has more than 25,000 strains of filamentous fungi, yeasts and bacteria in the collection. Eighty


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Services Provided

1. BCC cultures BCC cultures are made available for

education and research in Thailand and overseas. The client can order BCC cultures electronically through BCCs on-line catalogue (http://bcc.biotec.or.th).

2. Deposit cultures to BCC Researchers are encouraged to make

microbial cultures available for public access through deposition of the cultures into BCC. BCC accepts most groups of bacteria, filamentous fungi and yeasts classified in Hazard Group 1 or 2. BCC also provides safe and patent deposit facilities by preserving microbial cultures in optimal conditions with all proprietary rights retained by the depositor or the patent owner.


and through the internet for the general scientific community. MIMS and e-BCC assist BCC staffs not only to rapidly retrieve general microbial information, but select strains for cataloging. Using two different sets of criteria, two different lists of cultures are published electronically, one through the intranet for BIOTECs in-house use, the other through the internet for public access.

Database Management

Microbial Information Management Systems (MIMS) and e-BCC have been developed by the Information Systems Laboratory in order to ease the management and utilization of the large amount of data generated by BCCs researchers. MIMS is used to manage strain data, mainly for storage and distribution, while e-BCC is used to facilitate access to BCC strains through the intranet for BIOTEC researchers

3. Culture preservation BCC offers a culture preservation service

in which cultures are expertly preserved and returned to the clients for storage by themselves. BCC offers freeze-drying and preparation of cultures for storage in freezers and in the vapor phase of nitrogen of most groups of bacteria, yeasts and sporulating (in vitro) fungi classified in Hazard Group 1 or 2.

4. Identification BCC offers a service to identify

isolates of eubacteria, sporulating (in vitro) fungi, and ascomycetous and basidiomycetous yeasts. BCC also offers a service to purify microbial strains from contaminated sources.

5. Training BCC offers personalized training on

preservation techniques, culture collection management and identification of eubacteria and yeasts based on molecular techniques upon request.

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1. Department of Agriculture (DOA), Ministry of Agriculture and Cooperatives

2. Department of Medical Sciences (DMST), Ministry of Public Health

3. Thailand Institute of Scientific and Technological Research (TISTR), Ministry of Science, Technology and Energy

Research Staff

1. Wanchern Potacharoen, M.Sc. (Microbiology, Kasetsart University, Thailand)

2. Pattaraporn Rattanawaree, Ph.D. (Bioresources, Gifu University, Japan)

3. Sasitorn Jindamorakot, Ph.D. (Microbiology, Kasetsart University, Thailand)

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Award

1. Best Dissertation Award 2006 from Graduate School of Kasetsart University for the project entitled The Species Diversity of Yeasts in Some Natural Habitats of Thailand. (Dr. Sasitorn Jindamorakot)

Collaboration with Local Scientists


1. NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Japan

2. Japan Collection of Microorganisms (JCM), Japan

3. Culture Collection of Institute of Microbiology, Chinese Academy of Sciences, China

4. Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Korea

5. Institute of Tropical Biology, Vietnam

6. Netherlands Culture Collection of Bacteria (NCCB), Netherland

Collaboration with International Scientists


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2. Legal management of bioresources: Recognizing the importance of bioresources and keeping pace with legal developments of biodiversity at international and national levels, BIOTEC invests its vast efforts on not only the utilization of bioresources but also the legal management. Legal management of plant genetic resources for biotechnology research is an on going research project with the aim of developing practical guides for researchers who utilize plant genetic resources.

Research Experiences

1. Legal protection of traditional knowledge: BIOTECs BRS conducted an important study legal framework of protection of traditional knowledge with the commission of the Department of Intellectual Property. The study aims at solving the problems of misappropriation and misuse of Thai traditional knowledge. The knowledge gained from this study leads to the drafting of law on promotion and protection of traditional knowledge.

Advances in biotechnology in the 20th century offer promises of technological innovation in a wide array of applications, viz., agriculture, health, environment and energy. With rapid technological development, the potential applications of biotechnology will be beyond estimation. Keeping pace with technological development, more legal issues disputes and challenges concerning biotechnology have been witnessed. BioLaw issues cover growing areas of intellectual property, environmental law, human right, and most importantly

biodiversity law. In Thailand the knowledge of legal aspects and management is critically lagging behind.

Recognizing the importance of biolaw knowledge, BIOTEC established BioLaw Research Section (BRS) in 2008. The BRS aims to conduct legal and policy studies with the focus on legal management of bioresources and intellectual property management. BIOTEC hopes that the newly developed BioLaw knowledge will be useful for biotechnology research organizations.

2. Biotechnology Law (BioLaw)


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1. Chulalongkorn University

2. Thammasart University

3. Naresuan University

4. Government institutes such as Central Intellectual Property and International Trade Court, Department of Intellectual Property, Department of Agriculture

Experienced in legal management of bioresources and intellectual property, BIOTECs BRS has involved in several international legal negotiations and advancements. BIOTEC is one of Thailand delegations in the negotiation of an

international regime of access and benefit sharing under the Convention on Biological Diversity. BRS also takes part in developing access and benefit sharing regulations for Thailand.

3. Intellectual property management: With firm belief of intellectual property driving innovation in Thailand, BIOTEC has long committed in building human resources in the knowledge of intellectual property law. Intellectual property law is the core BRS

knowledge. BRS conducts several research projects on intellectual property law and management. One vital project is on the development of intellectual property management office for universities and innovative organizations in Thailand.

Research Staff

1. Tanit Changthavorn, Ph.D. (Intellectual Property, University of

London, UK)


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International and National Legal Developments

1. University of London, United Kingdom

2. NITE Biological Resource Center (NBRC), Japan

3. The International Service for the Acquisition of Agri-biotech Applications (ISAAA)



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The Bioresources Research Laboratory (BRL) aims at utilization of Thai bioresources, especially microorganisms. Since 1996, over 60,000 crude extracts from microorganisms have been evaluated for biological activities, including anti- malarial, anti-fungal, anti-herpes simplex virus, anti-mycobacterial, anti-inflammatory and anti-tumor against three cancerous cell types (MCF-7, KB, and NCI-H187). The on-going search for bioactive substances produced by various microorganisms, especially insect pathogenic fungi involves first biological activity-guided fractionation. Spectroscopic techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, mass spectrometry, ultraviolet-visible (UV), and infrared (IR) spectroscopy are then routinely employed for structure determination, which have led to the development of a chemical profile library of isolated metabolites.

The major successes on insect pathogenic fungi were reviewed by us in the journal Accounts of Chemical Research in 2005. Since then, many new compounds have been isolated and their chemical structures have been published. These include hirsutellones A-E and hirsutatins A-B from Hirsutella nivea, hirsutellic acid A from Hirsutella sp., ascherxanthone A from Aschersonia sp., verihemiptellides

A-B from Verticillium hemipterigenum, cordyheptapeptide A, bioxanthracenes, and cordyols A-C from Cordyceps spp. The results show that insect fungi (entomopathogenic fungi) are an excellent source of chemical diversity, both in terms of structures and biological activities. Other organisms such as seed fungi, endophytes, marine fungi, etc. have also been investigated, which have also proven to be excellent sources of bioactive substances; for example, sesquiterpene connatusins A-B from Lentinus conatus, hirstutellone F from Trichoderma sp., lachnones A-E from Lachnum sp., depsidones from the unidentified endophytic fungus BCC8616, bioxanthracenes from Cordyceps sp., hamevellones from Hamigera avellanea, and new benzofuran derivatives from Hypocrea sp. were amongst isolated compounds from these groups.

1 . Bioresources Research Laboratory (BRL)

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Production of natural product analogues has successfully been carried out, including beauvericin analogues from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614 by precursor-directed biosynthesis. Structure modifications and structure-activity relationshi study of these modified compounds have been undertaken.

In addition, in order to find lead compounds to be used as novel anti- malarial drugs, our scientists have synthesized several new dihydrofolate reductase inhibitors, (dihydrotriazine and diaminopyrimidine derivatives), and evaluated their activity against parasite

enzymes resistant to current anti-malarial drugs targeting the same enzyme. Many synthesized compounds exhibited very good binding affinity and showed promising anti-plasmodial activity. Consequently, large scale synthesis has become of great significance in order to prepare potential compounds in sufficient amount for further testing. Accordingly, the facility for large scale synthetic chemistry has been set up. This facility will be used in the scale up process, not only for antimalarial compounds but also for other active pharmaceutical ingredients (API), which will be beneficial to all of the Thai pharmaceutical community.

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1. Novartis, Switzerland Since 2005, the Bioresources Research Laboratory (BRL) has collaborated with Novartis, Switzerland, on the search for new bioactive compounds from insect fungi. The primary aim of the BIOTEC-Novartis collaboration is to find potential compounds suitable for further drug development.

2. Nagasaki University, Japan BRL researcher Dr. Smanmoo has collaborated with Professor Masaaki Kai, graduate school of pharmaceutical science, Nagasaki University on the chemiluminescent detection of biologically significant compounds.

1. Chulalongkorn University

2. Mahidol University

3. King Mongkuts Institute of Technology Ladkrabang (KMITL)

4. Asian Institute of Technology (AIT)


UNESCO-LOREAL International Fellowships 2007 from LOreal Thailand Company Limited and Thai National Commission for UNESCO for the project entitled Scale-up Process for the Anti-malarial Leads Synthesis (Dr. Chawanee Thongpanchang)

CST-Wiley Outstanding Publication Award 2009 from Chemical Society of Thailand (C.S.T.) for the project entitled Immobilization of Malarial (Plasmodium falciparum) Dihydrofolate Reductase for the Selection of Tight-binding Inhibitors from Combinatorial Library (Dr. Chawanee Thongpanchang)

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Award

To date, our on-going research is focused in many areas concerning the utilization of bio-resources, namely:

1) The search for novel bioactive substances from microorganisms,

2) Synthesis of compounds with good anti-malarial activity against Plasmodium falciparum,

3) Large-scale synthesis of potential compounds for further testing (API included), and

4) Development of compounds for other applications, for example, compounds for use in bio-sensors



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7. Srung Smanmoo, Ph.D (Organic Synthesis, University of

Sheffield, UK) 8. Chollaratt Boonlarppradab, Ph.D (Oceanography (Marine Chemistry),

University of California, San Diego, USA)

Research Staff

1. Prof. Yodhathai Thebtaranonth, Ph.D (Organic Chemistry, University of

Sheffield, UK)2. Masahiko Isaka, Ph.D (Organic Chemistry, Tokyo Institute of

Technology, Japan)3. Pattama Pittayakhajonwut, Ph.D (Organic Chemistry, University of

Bradford, UK)4. Chawanee Thongpanchang, Ph.D (Organic Chemistry, Mahidol University,

Thailand)5. Taridaporn Bunyapaiboonsri, Ph.D (Chemistry, Louis Pasteur University,

France)6. Sasithorn Teeveerapanya, Ph.D (Organic Chemistry, University of

Oxford, UK)


BRL also gives support to universities in Thailand and the private sector in terms of technical services including NMR, IR, MS, and SFE techniques.

1. Nuclear Magnetic Resonance (NMR) Spectroscopy

2. Mass Spectrometer / LC-MS spectrometer

3. Infrared (IR) Spectrophotometer

4. Supercritical Fluid CO2 Extraction (SFE)

Technical Services

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Since its establishment as part of the Bioresources Program in 1996, the primary goal of the Bioassay Laboratory has been to evaluate the medicinal potential of Thailands biological resources, with particular emphasis on microbes. As the program progresses into its second decade, the value of microbes in the BIOTEC culture collection (BCC) has been increasingly recognized by us. Therefore, the laboratory directs its effort toward the identification of potential lead compounds for further development in specific areas of utilization.

For the medicinal purpose, screening assays that target whole organisms are carried out routinely to identify microbial-derived compounds with activities against disease targets that are major health problems in Thailand, including malaria and tuberculosis, as well as opportunistic infectious pathogens, such as Candida albicans and Bacillus cereus. To increase our capacity for the identification of potential lead compounds against specific enzyme targets, we have also developed target-based assays. Assays for compounds inhibitory to the malaria pathogens plasmepsin II and dihydrofolate reductase enzymes are in hand, while assays are being developed for the avian influenza virus neuraminidase enzyme and the enzymes for cell wall synthesis in Candida albicans.

For the agricultural purpose, our laboratory seeks to alleviate problems regarding the outbreak of crop diseases and the excessive use of toxic pesticides by developing assays to identify naturally-derived compounds that inhibit crop disease pathogens. At present, assays against rice disease pathogens, such as Magnaporthe grisea and Curvularia lunata are routinely performed;

a dual-assay that will simultaneously screen for compounds with activities against two chilli anthracnose pathogens, Colletotrichum glocosporoides and Colletotrichum capsici, is being developed for future use.

The role of the Bioassay Laboratory is not limited to in-house research; we also provide significant contributions to Thailands scientific infrastructure by providing screening services to the general scientific community. Aside from developing and improving screening assays, the Bioassay Laboratory staff also conduct research in different areas to serve the needs of Thailand.

For instance, in line with the laboratorys focus on the search for anti-infective agents, an initiative has been taken to develop a systematic approach for the detection of non-ribosomal peptides (NRPs), a group of fungal metabolites known to have diverse biological activities. We have shown that fungi in the BCC are potential sources of diverse peptide metabolites; therefore, we intend to isolate the peptides from these fungi to create a collection of fungal derived peptides. Subsequently, the chemical data generated

2. Bioassay Laboratory


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from these metabolites will be deposited in a chemical profile library, whose availability will expedite the search for novel peptide metabolites and ultimately lead to the discovery of novel antibiotic treatments.

In addition, we also investigate the control of prostaglandin biosynthesis enzymes in mammalian cells, which may have implications for the treatment of patients with anti-inflammatory drugs, and possibly alternative therapeutic targets for inflammatory diseases. Recently, our research interest has focused on the prostaglandin biosynthesis pathway in the giant tiger prawn (Penaeus monodon), which is Thailands most economically important aquaculture export. Although the farming of giant tiger prawns has been performed

6. Kwanta Thansa, Ph.D. (Animal Sciences, University of

Nottingham, UK)

Research Staff

1. Kanyawim Kirtikara, Ph.D. (Genetics, University of Connecticut,

USA)

2. Vanicha Vichai, Ph.D. (Biochemistry, University of Virginia,

USA)

3. Chanikul Chutrahul, Ph.D. (Microbiology, University of Nottingham,

UK)

4. Chotika Samarkchan, Ph.D. (Biomedical Sciences, Chulalongkorn University, Thailand)

5. Wananit Wimuttisuk, Ph.D. (Biochemistry, Brown University, USA)


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in Thailand for over two decades, the industry has experienced problems in propagating prawn larvae from captive broodstock. Based on previous knowledge that the prostaglandins and related metabolites could influence the ovarian development in other crustaceans, we have begun examining this pathway in the giant tiger prawn to shed light on the pathway that regulates ovarian development in this organism.


1. Novartis, Switzerland

2. Kunming Institute University


1. Prince of Songkla University

2. Ramkhamhaeng University


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Enzymes are produced by all living organisms, from microorganisms to plants and animals; enzymes are necessary for nearly all of lifes chemical reactions. These reactions include, but not limited to, the metabolic breakdown of complex molecules into simpler ones which often resulting in a release of energy (catabolism) and the biochemical synthesis of complex substances with the storage of energy (anabolism). With the advance of biotechnology, increasing numbers of enzymes have been identified and produced before being used in various industries including medicine, agro- industry, commodity production biofuel and modern biotechnology.

Currently, thousands of millions baht are spent for enzymes in Thailand each year. However, most of the enzymes used in Thai industries are imported. The Enzyme Technology Laboratory was established to identify and characterize novel enzymes with desirable characteristics by taking advantage of Thailands biodiversity, especially the hugely diverse variety of microorganisms. Research in Enzyme Technology Laboratory is focused on the identification and biotechnological utilization of enzymes for industrial processes. The laboratorys activities include all aspects of enzyme biotechnology from screening of enzymes from microbial isolates and from metagenomic libraries, gene isolation, enzyme production in wild-type microbes and recombinant systems to development of enzymatic processes in industry. As a part of BIOTEC, our research aims for establishment of platform technology for the country together with the application of our technology for supporting the biotechnological research of the local academic and industrial sectors. With

strong collaboration with a number of leading institutes in Thailand and overseas, we aim to play a major contribution on the development of enzyme biotechnology of the country.

At present, research in the laboratory focuses on enzymes used by major national industries. Current

3. Enzyme Technology Laboratory


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Enzyme Discovery The aim is to identify enzymes with desirable properties by two approaches:

(a) Culture-dependent approach: This approach is employed to identify potent enzymes from cultured organisms such as fungi and bacteria.

interest involves lignocellulose degrading enzymes, amylolytic enzymes and lipases/esterases as well as some other specialized enzymes of biotechnological value. Enzymes capable of working under extreme conditions applicable for industrial processes are also of special interest. Our work in the laboratory currently involves three aspects of research:

At present more than 28,000 microbial isolates at the BIOTEC Culture Collection provide a promising resource for enzyme screening.

(b) Culture-independent approach: This approach is employed to identify enzymes from microorganisms in the environment without the necessity of culturing. This approach is very useful to find a broad range of novel enzymes since only approximately 1% of microbes naturally residing in the environment can be grown in normal laboratory conditions. Advanced DNA technology is used to construct so-called metagenomic libraries, representing the genomes of all microbes from a particular environment. Then, genes encoding enzymes with desirable properties can be identified using several approaches, including activity-based screening, sequence-based screening and pyrosequencing. Metagenomic libraries have been constructed from various sources, including unique or extreme ecosystems e.g. hot springs, peat swamp forest, sugarcane bagasse compost and lignocellulolytic microbial

Lignocellulosic biomass degradation by a thermophilic lignocellulolytic microbial consortium

Enzyme discovery from metagenomes (Modified from K. J. Shelswell. 2009. Metagenomics: The science of biological diversity. (www.scq.ubc.ca/metagenomics-the-science-of-biological-diversity/)


METAGENOMIC DNA FRAGMENT

manipulation of DNA

Metagenomic library collection

Biodiversity conservation

Activity-based screening

Sequence-based screening

Pyrosequencing

isolation of DNA from environmental sample

EXPRESSION OF DIFFERENT PROTEINS

analysis

CULTURED E. coli COLONIES

Construct library

CLONEDMETAGENOMIC DNA

ligation of fragmentswith vectors

BAC VECTOR

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Optimizationof Large-ScaleEnzymeProduction: In close collaboration with the Fermentation and Biochemical Engineering Laboratory, BIOTEC, the research includes identification of appropriate conditions for enzyme production in large scale, so that particular enzymes can be produced in sufficient quantity by bioreactor to be used in industrial process optimisation and field trial. This includes both enzymes produced by native fungal strains and by recombinant systems. Various types of agricultural wastes are tested for their possible use in culturing media for high production of enzymes.

Enzyme Application in Industry: Current research and development projects with industrial sectors include application of enzymes in a range of industries. These include the use of non-starch polysaccharide hydrolyzing enzymes and phytases in the animal

consortium as well as particular biological niches including termite gut and cattle digestive tracts.

The two approaches enable us to obtain enzymes from both cultured and uncultured microorganisms with high activity and functional stability suitable for biotechnological application. The work also attributes to the awareness for conservation and sustainable use of the nations biodiversity.

Enzyme purification by using AKTA Explorer FPLC system

feed industry, the application of lignocellulolytic enzymes in biomass conversion process of agro-industrial by-products to value-added products, including biofuels, bio-plastics and chemicals and also application of cellulolytic/hemicellulolytic enzymes for the pulp and paper industry.

Current collaborative researches with academic institutions and industrial sectors include:

- Development of biomass pretreatmant process for agro-industrial by-products in collaboration with Advanced Industrial Science and Technology (AIST), Japan

- Collaboration with the Joint Graduate School of Energy and Enviroment (JGSEE) for production of alternative biofuels, including biodiesel and liquid alkane biofuel by thermocatalytic and biocatalytic processes

- Development of a non-thermal cassava feedstock saccharification process using fungal multi-enzyme for bioethanol production with Kasetsart Agricultural and Agro- Industrial Product Improvement Institute (KAPI), Kasetsart University

- Collaboration with the University of Tokyo, Japan to establish active lignocellulolytic microbial consortium with structural and functional

Quantitatively assays for various industrial enzymes


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stability for application in biomass degradation and discovery of lignocellulolytic enzymes

- Collaboration with Mahidol University and KMITL (King Mongkuts Institute of Technology Ladkrabang) to identify enzymes with special characteristics or enzymes suitable to be used in feedstuff

- Research collaboration with National Institute of Advanced Industrial Science and Technology (AIST), Japan on production of bio-based plastic/monomer

- Collaboration with Betagro Science Center, Asia Star Animal Health Co. Ltd. and Sunfeed on screening and production of enzymes for animal feed industry

- Collaboration with SCG Paper, Public Co. Ltd. to develop enzymes for pulp and paper production process

- Contract research with Electricity Generating Authority of Thailand (EGAT)

A database describing non-starch polysaccharide hydrolyzing enzymes and amylases obtained from screening fungi in the BIOTEC Culture Collection with high potential for various industries is available in the form of Enzyme Catalogue for interested private sectors and researchers. Enzyme Technology Laboratory also provides enzyme activity analysis service and consulting service for enzyme application in industries. In addition, several products aiding enzyme analysis, namely Enzhance Overlay Enzyme Detection Kit and Enzyme Assay Strip Test have been developed. These are considered important steps for the efficient advancement of enzyme application in Thailand.


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Awards

Taguchi award 2008 from Foundation for the Promotion of Biotechnology in Thailand for the project entitled Using molecular approach for enzyme discovery from microbial resource in Thailand. (Dr. Lily Eurwilaichitr)

Innovation award from National Research Council 2008 for the project entitled ENZhance Overlay Enzyme Detection Kit for screening of industrial enzymes.

Diploma from Universitatea de Stat Din Moldova (1st IIDC) for the project entitles ENZhance Overlay Enzyme Detection Kit for screening of industrial enzymes

Award Gold Medal from Belgrade Association of Inventors and Authors of Technical Improvements (1st IIDC) for the project entitles ENZhance Overlay Enzyme Detection Kit for screening of industrial enzymes

M.Sc. Thesis Distinction from Graduate School, Mahidol University 2007. (Ms. Benchamaporn Wonganu)


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Research Staff

1. Lily Eurwilaichitr, Ph.D. (Biochemistry, University of Kent at

Canterbury, UK)

2. Verawat Champreda, Ph.D. (Biochemistry/Biocatalysis, Imperial College London, UK)

3. Somchai Pongpattanakitshote, Ph.D (Flinders University, Australia)

4. Honglada Thoetkiattikul, Ph.D. (Entomology, University of Georgia, USA)


LOreal Thailand Fellowship For Women in Science 2006 from LOreal Thailand Company Limited and Thai National Commission for UNESCO for the project entitled Molecular approach for enzyme discovery from microbial resources in Thailand. (Dr. Lily Eurwilaichitr)


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1. The Joint Graduate School of Energy and Environment

2. Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University

3. Mahidol University

4. King Mongkuts Institute of Technology Ladkrabang

5. National Metal and Materials Technology Center

6. Kasetsart University

7. Electricity Generating Anthority of Thailand (EGAT)

1. National Institute of Advanced Industrial Scienc and Technology (AIST), Japan

2. The University of Tokyo, Japan


1. Betagro Science Center Co. Ltd.

2. Asia Star Animal Health Co. Ltd.

Collaboration with Privates Sectors

3. Sunfeed Co. Ltd.

4. SCG Paper, Public Co. Ltd.


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4. Fermentation Technology and Biochemical Engineering Laboratory

Our laboratory specializes in microbial cultivation technology and bioprocess development for efficient production of potential bio-products from microorganisms namely fungi, yeast and bacteria. Our research interests are i) Cultivation and Media development Technology aiming to facilitate the screening and production of bioactive compounds/bio-products of interest and mushroom cultivation, ii) Metabolic Product

Optimization Technology focusing on improving quantity/yield of the target metabolic product and iii) Bioprocess development Technology aiming to develop platform process of pre-pilot scale production while evaluating its commercial feasibility. In addition, our fermentation technologies and consultancy services are provided to support in-house research and to help solve private sector research problems.

Cultivation technology has been applied to improve production and efficiency of bioactive compounds produced by insect pathogenic fungi for being used as pest controlling agents. As the largest and most unique group of microorganisms in the BCC culture collection, it is also considered a powerful source of new and potent bioactive compounds. Many of these fungi are fastidious; hence, in-vitro cultivation conditions must be developed to promote fruiting body formation necessary for extraction of bioactive compounds. The ultimate aims are to develop new sources of potent bioactive compounds and to investigate the diversity of compound structures from fungi of differing morphology. In vitro cultivation studies entail testing nutritional supplements, modifying media recipes and testing different physicochemical conditions. New media have been developed broadening the diversity of available bioactive compounds, including a solid medium comprising brown rice and silk worm suitable for production of fruiting bodies of three insect pathogenic fungi, Cordyceps irangensis BCC 21484, Cordyceps sphecocephala BCC 23297 and Isaria tenuipes BCC 23112.

Besides cultivation for bioactive compounds, a group of mushroom fungi are also an important food. Therefore, the research interest also includes mushroom cultivation with the aim of developing economical cultivation technology which can be transferred to farmers or rural people, thus creating economic opportunity. In 2001, BIOTEC and the Arunyik

Cultivation and Media Development Technology


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This research group focuses on increasing quantity or yield of the target metabolic product while minimizing the level of other by-product(s). Laboratory- scale production has been achieved for various bioactive substances, including native enzymes from wild-type Aspergillus sp. and recombinant enzymes from other microorganisms. The groups highlight is the production of the animal feed ingredients which are docosahexaenoic acid (DHA) from Schizochytrium racemosum and fungal enzymes, namely, xylanase, -glucanase, and cellulase, from various microbial resources.

Both traditional optimization and experimental designs, such as Plackett Burman, Fractional factorial, and response surface methodology are applied to determine relevant factors and optimal range of concentrations which give maximum production. Once optimal conditions have been established, microbial cell physiology is monitored during cultivation to finalize the production yield and define the correct harvesting time in a laboratory scale bioreactor. Other products with potential for industrial use are biopolymers produced by insect pathogenic fungi (application in pharmaceuticals, cosmetic supplement, and prebiotics), bioethanol, and microbial cells for bioremediation. The production

and utilization of these potentially commercial products are on going as collaborative projects with other government institutes and the private sector.

Mushroom Center successfully developed a cultivation technique for the parasol mushroom [Macrolepiota gracilenta (krombh.) Moser]. Cultivation technology was applied to increase the initially low yield by optimizing the environmental factors and finding appropriate nutrients that stimulate both mycelial growth and fruiting body production. New mushroom varieties have been bred in our laboratory, where the productivity and morphology of fruiting bodies are initially assessed.

Different local agricultural wastes such as corncob were also tested to determine their potential as substrates.

MetabolicProductOptimizationTechnology


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This technology involves design and development of process for specific metabolic bio-product based on its application. In order to formulate a practicable bio-product for field-trial, the laboratory scale bioreactor fermentation is scaled up to pre-pilot scale. A specific process must be designed to minimize losses, and all downstream operation must be tailored to the products application. Our development covers any scale-up problems within all steps along the process. In collaboration with the Enzyme Technology laboratory and private sectors, we have developed an enzyme production and formulation process for chicken feed. This example demonstrates how technology established in the laboratory can then be transferred to industry.

Currently, our fermentation service could accommodate at laboratory scale (2-5L) and pilot scale (50-1000L). Our laboratory is also well-equipped with laboratory downstream units including cell separation, clarification, membrane concentration, and drying, all in good collaboration with Biochemical Engineering and Pilot Plant Research and Development Unit at King Mongkuts University of Technology Thonburi, Bangkuntien for large pilot-scale production. The procedures in practice from laboratory to pilot scale production, together with our experience thus serve as a platform technology for collaborative in-house research and private sector partnerships.

Bioprocess Development Technology


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1. Wai Prathumpai, Ph.D. (Process Biotechnology, Technical

Denmark, Denmark)

2. Vasimon Ruanglek, Ph.D. (Microbiology, University of Kent at

Canterbury, UK)

3. Panida Unagul, Ph.D. (Biotechnology, King Mongkuts

University of Technology Thonburi, Thailand)

1. Asia Star Animal Health Co. Ltd.

2. Betagro Science Center Co. Ltd.

3. Virbac S.A.

4. Higrimm Environmental Research

5. Mitrphol sugarcane and sugar R&D center

6. Banchong Farm

1. King Mongkuts University of Technology Thonburi

2. Institute for Scientific and Technology Research and Services [ISTRS], King

Mongkuts University of Technology Thonburi

3. Thammasat University

4. Chiang Mai University

5. Srinakharinwirot University

6. Ramkhamhaeng University

Collaboration with Privates Sectors


Research Staff


1. Anhui Agricultural University, China

2. Technical University of Denmark, Denmark

3. Vinh University, Vietnam

4. Chalmer University, Sweden3 5



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5. Microbial Cel l Factory Laboratory In molecular biology research, microbes are used extensively as production factories for proteins. Microbes are being increasingly employed for industrial manufacture of economically important proteins. Heterologous expression, in which a gene coding for the protein of interest is expressed in an experimentally tractable organism is one of the essential processes to produce target proteins efficiently, which is crucial when large scale production of proteins such as enzymes is required. The technologies for heterologous

protein expression have gained much interests in recent years. In addition, it has become clear that there is no single microorganism that can be utilized universally to produce all proteins of interest. Thus, the availability of several different host systems would help increase the possibility of success in target protein expression. In our laboratory, we utilize molecular techniques in order to develop and establish various heterologous expression systems including bacteria and yeast.

The power of biotechnology, especially genetic engineering, enables us to develop expertise on protein expression system. Taking advantage of Thailands vast biodiversity, several native enzymes from fungi have been isolated and characterized, some of which exhibited excellent properties with potential industrial applications. However, in general, the yield of endogenous enzyme is low. Thus, heterologous expression for more efficient production of these target enzymes in appropriate hosts is necessary. Pichia pastoris is a yeast which grows quickly in defined medium and can grow to very high cell densities. It is especially useful for the large scale production of target proteins. In addition, the target proteins are glycosylated, which make P. pastoris yeast a favorable host for expressing fungal, plant or human proteins. However, the need for further dowstream process such as purification and separation make the cost for large scale production prohibitive. We therefore exploit

cell surface display technology to express secreted target proteins on the yeast cell surface as anchored proteins. Production of enzymes that are immobilized on the cell surface thus obviates tedious purification processes. The yeast cells with anchored proteins can then be used further as a whole-cell biocatalyst.

OurCurrentWorkintheLaboratoryInvolves:DevelopmentofCell-SurfaceDisplayExpressionSystem


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xylanase and phytase. However, the current P. pastoris strains used are commercial strains under patent, which might cause further complications and additional cost when large scale production of target enzymes aimed for industrial applications is involved. Therefore, we aim to develop new alternative yeast strains with properties suitable and efficient for heterologous protein expression.

The methylotrophic yeast P. pastoris has become a widely studied host for heterologous protein expression, which has several advantages over E. coli including high density of the cultivation, gene stability, and high level of extracellular protein production under a controllable induction system. P. pastoris has been successfully exploited in our laboratory for heterologous expression of several enzymes including cellulase,

Improvement of Protein Production

Development of Thai-Isolated New YeastStrains as Alternative Host

Construction of in vivo multimers

Multi-copy expression of target enzymes has been proven to be a useful tool to increase the level of protein production. This technique is also employed in our laboratory to improve the level of protein expression both extracellularly and intracellularly. In addition, the viral cis-acting hydrolase element is being studied for its potential to improve the level of enzyme production. Alternative promoters from other organisms are also being investigated for strong and efficient expression of enzymes in yeast.

Lactobacillus has been used as GRAS starter in broad applications of food, functional food, agriculture and bioremediation. Hence, they are attractive hosts strains as desired properties for expansion their utilization. In our laboratory, we have developed Thai-isolated

Gene Expression in Lactobacillus

Lactobacillus to be used as host stain for heterologous protein expression. This host strain can be used for producing target proteins such as bacterial enzymes that are utilized in food or feed industrial applications.

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Collaboration

Intramural collaborations with Enzyme Technology Laboratory and Fermentation Technology and Biochemical Engineering Laboratory foster more complete and effective research in a tightly-knit environment. Collaborations with industrial sectors from local and overseas research institutes and universities have also been established for expertise sharing and technology transfer. These include:



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Research Staff

1. Sutipa Tanapongpipat, Ph.D. (Biochemistry, University of

Birmingham, UK)

2. Niran Roongsawang, Ph.D. (Material and Life Science, Osaka

University, Japan)

3. Piyanun Harnpicharnchai, Ph.D. (Biochemistry, Carnegie Mellon

University, USA)

4. Plearnpis Luxananil, Ph.D. (Engineering, Kyoto University, Japan)

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1. Mahidol University and KMITL (King Mongkuts Institute of Technology Ladkrabang) to identify enzymes with special characteristics or enzymes suitable to be used in feedstuff

2. Mahidol University, on the development of yeast strain for production of humanized proteins

3. Khon-Kaen University, on the expression of enzymes involved in geranylgeraniol biosynthetic pathway

4. Department of Biotechnology, Thammasat University to improve the efficiency of target enzyme by mutagenesis

5. Department of Microbiology, Faculty of Science, Chulalongkorn University, on the strain improvement of Bacillus subtilis for lipopeptide biosurfactants production

1. Institute of Sustainable Chemistry, AIST (Japan), for bioplastic degradation

2. Faculty of Engineering, Kobe University, for cell-surface display expression

Betagro Science Center to identify and improve enzymes for feedstuff supplementation

Collaboration with Private Sector



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Among disease carriers, mosquitoes are recognized as one of the most important vectors of human diseases. They are capable of transmitting serious, possibly even fatal diseases, such as mosquito-borne encephalitis, dengue, yellow fever, filariasis, and malaria. Transmission of disease occurs when an infected mosquito takes a blood meal. Each disease is transmitted by different species of mosquito. Anopheline mosquitoes are the main malaria vector. Encephalitis is carried by Culex spp., while dengue and yellow fever are transmitted by Aedes spp. The reason why each species carries different diseases might be because of molecular incompatibilities between the mosquito and the disease agent.

Each year, approximately 300 million people in developing countries are affected by malaria, with over 2 million deaths from the disease. Dengue and encephalitis also affect thousands of people in urban areas. There is a close relationship between disease outbreak and the number of mosquito carriers in the area. Therefore, to reduce the risk of disease, the mosquito population has to be controlled. The most widely used biological agents for controlling mosquito larvae are

Bacillus thuringiensis subsp. israelensis (Bti), and Bacillus sphaericus (Bs). These bacteria produce proteinaceous toxins that specifically kill certain species of mosquito larvae. The toxin is formed as a crystal in the bacterial cells. After ingestion by the mosquito larvae, the toxins are dissolved as a protoxin and activated by larval proteases. The active form of the toxin will then bind to the midgut membrane and destroy the midgut cells, leading to starvation and death of the larvae. However, application of both bacteria is limited by the longevity of these biocontrol agents. The degradation of microbial proteins in the field is also a main limitation. In addition, toxin resistance has been observed in Culex spp. when B. sphaericus was used. It is thought that the use of a single protein toxin for a long time contributed to the emergence of resistance. Therefore, application of bacteria containing various proteins might be a more effective broad-range biopesticide and could overcome the resistance in the mosquito larvae.

The main objective of our group is to improve efficacy and safety of microbial agents for controlling mosquitoes and major insect pests.

6. Microbial Engineering Laboratory

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We are currently studying structure-function relationships and molecular mechanisms of two mosquito larvicidal toxins; Cyt toxin from Bacillus thuringiensis and binary toxin from Bacillus sphaericus. Cyt toxins (Cytolytic-endotoxins) are a group of proteins produced by some strains of Bt. These proteins are lethal to larvae of Dipteran insects (mosquito and black fly). Current evidence indicates that Cyt toxins kill mosquito larvae by forming pores on the cell membrane in the larval gut. However, the detailed mechanism of this process is not clearly understood. The pore-forming mechanism and pore architecture of Cyt toxin integrated into biological membrane are under investigation in our laboratory. Another mosquito larvicidal toxin we are studying is binary toxin. This toxin consists of two components, 42 kDa (BinA) and 51 kDa (BinB). Both proteins function together to kill mosquito larvae. BinB acts as specificity determinant by binding to a specific receptor presented on the gut cell membrane. The toxic component (BinA) then binds to BinB and the complex translocates into the cell and exerts its toxicity through an unknown mechanism. We are now studying the molecular mechanism and structure- function relationships of both components.

Information obtained from these investigations will be useful for engineering the protein to improve its potency, the development of synergism with other toxins to broaden the host range, the design of new immunotoxins, and the delay or prevention of the emergence of resistance.

Exploration of Biocontrol Agents

Molecular Mechanism of Mosquito Larvicidal Toxins

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Bti and Bs are the most commonly used larvicide for mosquito control. Bti has potent toxicity toward Anopheles and Aedes mosquito, but low toxicity against Culex. In contrast, Bs is the most toxic agent for Culex, but not for Aedes. Difference in activity spectra of both bacteria is due to difference in the mosquito-larvicidal proteins produced in the bacterial cells. Furthermore, a variety of insecticidal proteins specific to different insects are produced in different

B. thuringiensis strains. Therefore, microbial agents containing novel mosquito larvicidal proteins as well as those toxic against major insect pests should be explored. Thailands rich biodiversity thus offers potential for this exploration.

Mosquito larvicide proteins, produced by Blidnd B3

Hemolytic activity of Cyt2Aa2 toxin and its mutants. We use this technique to access activity of the Cyt toxin


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Over 100,000-fold resistance to binary toxin in Culex has been found in Thailand and around the world when Bs is used continuously. Cross-resistance among different strains of Bs has also been observed. The larger component of binary toxin, 51-kDa protein (BinB), binds to the mosquito larval midgut. Alteration in binding might result in loss of activity of the toxin. Currently, we are investigating the receptors (alpha-glucosidases) for the 51-kDa proteins from susceptible and resistant mosquito larvae collected in Thailand. We have found differences in binding between susceptible and resistant mosquitoes. Mutations in the alpha- glucosidase gene might therefore be responsible for the binary toxin resistance. However, more information at molecular level of the receptors is required to test this hypothesis.

Resistance Mechanism in Mosquito Larvae

Immunohistochemistry detection of the binary toxin binding to mosquito larval gut cells

Development of a host cell for Production of Insecticidal Proteins

A gram-positive bacterium Bacillus subtilis has many beneficial features, including high capacity of protein secretion and non-pathogenicity, which allows its exploitation as a host for recombinant protein production. It offers a cost-effective alternative system for protein production. There are two major reasons hampering the use of B. subtilis as a cell factory:

structural instability of the expression plasmid and degradation of secreted recombinant proteins by native extracellular proteases. Therefore, an expression system in B. subtilis for heterologous secretory proteins is currently under development, focusing on stable expression vectors and protease-deficient B. subtilis strains.

Circular dichroism (CD) spectra of a mosquito-larvicidal Cyt2Aa2 toxin and its mutants. We use this technique to determine the secondary structure of a protein

Intrinsic fluorescent spectra of Bacillus sphaericus BinB toxin and its mutants. This technique is routinely used to follow conformational change of a protein

Recombinant plasmids for high production of mosquito- larvicidal toxins, BinA and BinB. Both plasmids were constructed in Microbial Engineering Lab


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1. Institute of Molecular Biosciences, Mahidol University

2. Faculty of Science, Mahidol University

3. Department of Entomology, Kasetsart University

4. Department of Agriculture, Ministry of Agriculture and Cooperatives

5. National Institute of Health, Ministry of Public Health

Research Staff

1. Boonhiang Promdonkoy, Ph.D. (Biochemistry, University of Cambridge,

UK)

2. Mongkon Audtho, Ph.D. (Biochemistry, Ohio state University,

USA)

3. Sumarin Soonsanga, Ph.D. (Microbiology, Cornell University, USA)

Bacillus thuringiensis is the most extensively used biopesticide worldwide. For decades, the insecticidal activity of this bacterium is thought to be associated with its ability to synthesize a group of crystal proteins, referred to as Cry and Cyt proteins. However, a group of proteins named vegetative insecticidal proteins (Vips) have been recently discovered and their essential roles in insecticidal activity of the bacterium have been demonstrated. One of the the most active Vip proteins is Vip3Aa, which is produced during the

Production of VIP3Aa for Effective Control of Insect Pests

vegetative stage and is highly toxic against several insect species, including beet armyworm (Spodoptera exigua) and S. litura. This protein is reportedly much more toxic to S. exigua and S. litura than Cry proteins. However, the application of this protein as an insect pest control has not been realized owing to inadequate production methods. Expression of Vip3Aa in B. thuringiensis is being developed in our laboratory. Our aim is to improve its expression level, stability and synergism with other insecticidal proteins.


1. Department of Pharmaceutical Science, University of Maryland, USA

2. Lawrence Berkeley National Laboratory, University of California, USA

3. School of Biological Sciences, Washington State University, USA

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Mic

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m

Pro

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1 . Mycology Laboratory It is currently estimated that the globe is home to approximately 1.5 million species of fungi, of which approximately 70,000-150,000 are found in Thailand. However, compared with other major groups (e.g. higher plants and animals) fungi are poorly researched, and to date only 80,000 species have been documented worldwide.

Although Thailand has a long tradition of studying the fungal pathogens of economically important plants, it is only in the last ten years that significant efforts have been made to survey the vast biodiversity of fungi in Thailands natural ecosystems. In 1993, BIOTEC established the Mycology Research Program to study the biodiversity of invertebrate pathogenic fungi in the Kingdom, and over the past ten years the activities of the program have

Biodiversity of Fungi in Thailand

Before 1990, less than 700 species of fungi found in Thailand had been reliably documented. Much of the published literature has been reviewed, and the list of fungi recorded from Thailand has been updated (A book of Thai fungal diversity, and also a project of Thai fungal checklist). Recently, the number of Thai species stands at around 6,000. Adding to records taken from the literature, the work of BIOTEC researchers, associated students, and visiting experts has placed

broadened to include other groups of fungi such as alkaline tolerant fungi, dung fungi, freshwater fungi, lichen fungi, litter Basidiomycetes, marine fungi, palm fungi and seed fungi.

More recently, numerous different taxonomic and ecological groups of fungi have been added to the research activities of the program, the overall aims of which are provided data on the biodiversity of fungi in Thailand, isolate fungi for the BIOTEC culture collection and screening programs, and develop an information resource on fungi which can be made widely available.

more than 1,000 newly documented species of fungi into the inventory over the last few years. New records are constantly being added from laboratory research and from continual reviews of the published literature. The last few years alone have seen the description of several genera: the yeast Siamia and the insect pathogen Hyperdermium, the marine ascomycete Thalespora, the freshwater basidiomycete Stauriella and ascomycete Megalohypha.


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Seed Decay Fungi

Seed decaying fungi are a group of fungi associated with seeds or any sexual propagules of plant that usually referred to as seeds. They are saprophytes and play an important role in recycling nutrients in the forest ecosystem. The study on seed decay fungi has recorded over hundred species including a number of new taxa. Many isolates of them have also shown to produce bioactive compounds with pharmaceutical potential. These results indicate that there are many more fungal

Insect Fungi

Insect fungi are difficult to find and isolate, which explains why so few are available, no matter where in the globe they originate. However, the Clavicipitaceae (a family accounting for many of the insect pathogenic fungi) is a recognized novel metabolite hotspot. The recent recognition of this hotspot feature, however, and a request for fresh isolates has resulted in more than 1,000 isolations being made in the last few years. In this regard, the mycology research program plays a critical role in novel metabolite screening research in Thailand.

There are now about 400 morphotaxa recognized in Thailand, of which approximately 30% are new species. The insect fungi have been isolated, and Thailand now has the richest collection of this group in the world, with 4,000-plus isolates representing 180-plus species deposited in the BIOTEC culture collection. This group of fungi has provided the most unique and potent biologically active compounds thus far investigated by BIOTEC scientists.

species on decaying seeds in the Thai forest waiting to be discovered and tested their bioactive properties. The intensive study of this fungal group is, therefore, continued on seeds of the Dipterocarpaceae, a major tree family of the principality, to gain the ecological information on microorganisms in relation to the major trees of the forest, and to provide the living cultures for further biotechnological research and utilization.

Coelomyceteous Fungi

Coelomycetes is a unique taxonomic group which has few experts and poor research worldwide. This fungal group plays an important role in the ecosystem as saprophytes, parasites, endophytes and mutualist organisms. Some coelomycetes have also possessed the ability in producing bioactive compounds with pharmaceutical potential and as biological control agents. This study is

continued to collect and isolate coelomycetes from the natural forest habitat and deposit them into BIOTEC Herbarium and BIOTEC Culture Collection. Any new species discovered from this study will be described and published. Herbarium specimens and living cultures yielded from this study will support further taxonomic research and biotechnology exploitation.

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An unusual habitat for fungi is the peat swamp forest in the southern province of Narathiwat, where species-colonizing

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