PARTNERSHIPS FOR ENHANCED ENGAGEMENT IN RESEARCH (PEER) Cycle 5 (2015 Deadline) Enhancing research capacity through a biotechnology-driven investigation of novel Gram-negative bacteria from Indonesian sponges PI: Ocky Radjasa (ocky.radjasa@ristekdikti.go.id), Diponegoro University U.S. Partner: Phillip Crews, University of California, Santa Cruz Project dates: December 2016 - May 2020 Project Overview: The team pauses for a group photo during a field expedition to collect samples in May 2017 (photo courtesy of Dr. Radjasa). This team’s hypothesis was that an experimental design based on so-called “integrative chemical biology” will provide new insights to better describe life processes in Oceana. This project leveraged an existing Indonesian-U.S. collaboration to make advances in marine microorganism biology, stimulate chemistry discoveries, and engage in outreach to STEM students. The focus was on using biodiverse Indonesian sponges as a source of new halotolerant Gram-negative bacteria. The project was based at Diponegoro University (UNDIP, Indonesia) and was led by Prof. Radjasa, working in cooperation with the natural products lab team of Prof. Crews and leveraging his National Institutes of Health-sponsored project “Merging Marine-Derived Natural Products With Experimental Therapeutics.” The goals were (1) to leverage the aims and expertise of the Crews group to forge a marine sponge-inspired initiative with the Radjasa group at UNDIP; (2) to isolate, characterize, and culture at least 30 strains of Gram-negative bacteria from Indonesian marine sponges; (3) to discover at least 10 novel secondary metabolite scaffolds through the culturing of chemically prolific microorganism strains; (4) to establish a productive research partnership between scientists at UNDIP and UCSC through exchange visits; and (5) to play a part in USAID/Indonesia higher education objectives by providing undergraduate training for at least five UNDIP students using STEM programs in place at UCSC, training on marine biotechnology for four undergraduate students, and curriculum development. The exploitation of symbiotic bacteria as a source for novel secondary metabolites is considered to be in its infancy; however, the discovery rate of novel active metabolites from marine Gram-negative bacteria could surpass that of their terrestrial counterparts. Sponges represent an unusual niche for novel microbes, as they host hundreds of different bacterial groups and contain diverse symbionts. However, the marine environments in which they are found still remain largely unexplored, understudied, and underexploited. The urgent need for novel substances for the treatment of severe human diseases combined with the recognition that marine organisms provide a rich potential source of such substances support the intensive exploration of new substances from marine organisms. This project used organisms and methods to overcome existing bottlenecks by using halotolerant Gram-negative bacteria, which can be grown in large scale in the laboratory and which are potent producers of bioactive compounds. Final Summary of Project Activities Indonesia is the global epicenter of marine biodiversity and is one of the megadiverse countries that harbor the majority of the Earth’s species. Marine biotechnology aims toward development of products and other benefits for humankind from marine biodiversity through the application of biological knowledge and cutting-edge techniques. However, marine biotechnology is considered to be in its infancy. Indonesia has fewer than 40 universities with faculties of fisheries and marine sciences and fewer than 10 faculties with a research focus on marine biotechnology, as a result of which Indonesian universities offer fewer courses on the subject. Ultimately, Indonesia faces a lack of both human resources and research capacity on marine biotechnology. This project addressed both these issues, as well as a serious bottleneck in developing natural products from marine sources, namely the availability of biomass and/or of optimized cultivation conditions to gain sufficient amounts of compound for pre-clinical and clinical studies. The PI and his team focused on one way of supplying marine metabolites for testing, specifically by collecting, culturing, and testing bacteria collected from sponges. The high potential of sponge-derived therapeutics is clear, but there is no effective sustainable production method yet developed, despite decades of trial-and-error research. Gram-negative bacteria are a rich source of unique bioactive molecules that are rarely produced by other bacteria, actinomycetes, or fungi. This PEER project specifically used organisms and methods that address this issue by using bacterial symbionts, which can be grown in large scale in the laboratory and are potent producers of bioactive compounds. In his final report on the project, the PI Dr. Radjasa indicates that he and his team achieved all five aims of the project: Collaboration with U.S. partner Dr. Philip Crews and his group at the University of California, Santa Cruz was strong, with the UCSC researchers providing training, mentorship, and collaboration on all aspects of the project. The PI and his colleagues isolated, characterized, and cultured halotolerant bacteria from Indonesian marine sponges, implementing techniques transferred by Dr. Crews’s group to estimate the genetic diversity of these gram-negative bacteria. Also with guidance from the U.S. partner and his group, the Indonesian PEER researchers worked to discover new secondary metabolite scaffolds from sponge-derived halophilic bacteria using a powerful combination of approaches, analytical techniques, and strategies. The crude extracts obtained from liquid cultures were pre-fractionated into several samples and, when appropriate, followed by peak library generation. Based on results of mass spectroscopy screening, the researchers selected promising compounds for isolation, characterization, and structure elucidation. The productive research partnership between scientists at Diponegoro University and UCSC was facilitated through exchange visits in both directions, during which the participants took part in joint sample collection voyages, co-taught training courses for Indonesian students, and worked side by side in the lab. The project also contributed to USAID/Indonesia higher education objectives by providing training to undergraduate students (the majority of them female). Special attention was given to students and young scientists from the eastern part of Indonesia, such as Kupang, East Nusa Tenggara, and Papua. To support gender equality and social inclusion, the PI and his colleagues we allocated more internships for female students, as well as students from minority ethnic backgrounds. One of the highlights of the project was capacity building in the field of marine natural products chemistry and marine biotechnology through internships and workshops for undergraduates, with some of them getting the opportunity to visit UCSC for additional training and hands-on research experience. Specific significant outputs of the project included the following: Six peer-reviewed articles in reputable international journals, one of which was co-authored with their U.S. partner, along with one book chapter to appear in 2020. Indonesian patent applications submitted in 2019 for two different Gram-negative bacteria, namely Vibrio parahaemolyticus and Erythrobacter flavus. Bacterial sequence data submitted to public online database of the National Center for Biotechnology Information of the U.S. National Institutes of Health (www.ncbi.nih.gov). Capacity building on marine biotechnology through trainings, internships, workshops and short training visits at UCSC, as well as the upgrading of curricula for three courses (Marine Biotechnology, Marine Natural Products, and Marine Chemical Ecology) benefiting 91 students as of June 2020. Six students obtained their PhDs with partial support from the PEER grant, of which three have been hired at UNDIP, one directs an environmental NGO in Bali, and one is a junior researcher in The Netherlands. Improved scientific performance and recognition for the UNDIP team, as indicated by the selection of Prof. Ocky Radjasa and two key project team members (Prof. Agus Sabdono and Dr. Agus Trianto) among the 500 best Indonesian researchers out of 194,000 researchers announced by the Indonesian Minister of Research and Technology on May 28, 2020. Their ratings were numbers 168, 135, and 179, respectively. Prof. Sabdono has been re-elected by the Dean as the head of the Tropical Marine Biotechnology Laboratory, while Prof. Trianto has just been elected as the Vice Dean for Academic Affairs, who is also responsible for managing research and innovation in the faculty. Prof. Radjasa has been appointed to the Governing Board of MERO (Marine Education and Research Organization), a non-profit marine research foundation in Bali. Six competitive research grants received from Indonesian Ministry of Research and Technology in 2020, with possible extension up to three years. In addition, the team has obtained two additional international joint research grants from the UK Newton Fund and the Zoological Museum of Moscow State University, and they have submitted an international joint research proposal with partners from the Netherlands to the Indonesian Endowment Fund for Education, with the results expected in October 2020. Contacts established with NGOs working in the marine environment sector. The PI and his team met with the World Wildlife Fund and The Nature Conservancy to explore possibilities for collaboration on sustainable use of Indonesian coral reefs, especially in the Raja Ampat region of Papua. They also built a working relationship with the MERO Foundation in Bali to help expand their research capacity for bioprospecting of marine microbial symbionts. Prof. Radjasa emphasizes that the PEER project provided much-needed human and lab infrastructure capital for strengthening research capacity. With partial support from PEER and complementary support from UNDIP, he and his team now have an established tropical marine biotechnology laboratory that can serve as a vehicle for carrying out world class research going forward. The laboratory is now more capable of obtaining external research grants as well as generating revenues needed for its operations. Although the PEER grant has now ended, the young research team created as part of the project will coordinate planned projects from 2020 to 2022. The team includes three scientists who recently received their PhDs under the PI’s supervision. Prof. Radjasa and his group will continue to leverage and build their network of international research counterparts from the United States, Germany, France, The Netherlands, and Japan. Publications (as of June 2020) Sibero, M.T. T. Zho, Y. Igarashi, O.K. Radjasa, A. Sabdono, A. Trianto, T. Ulfa, Bachtiarini, and M.S. Bahry. 2020. Chromanone-type compounds from marine sponge-derived Daldinia eschscholtzii KJMT FP 4.1. Journal of Applied Pharmaceutical Science 10(1):001-007 https://www.japsonline.com/admin/php/uploads/3052_pdf.pdf   Kristiana, R., G. Bedoux, G. Pals, I.W. Mudianta, L. Taupin, C. Marty, M.A. Asagabaldan, D. Ayuningrum, A. Trianto, N. Bourgougnon, O.K. Radjasa, A. Sabdono, and M. Hanafi. 2020. Bioactivity of compounds secreted by symbiont bacteria of Nudibranchs from Indonesia. PeerJ 8:e8093 DOI: 10.7717/peerj.8093 Trianto, A., Nirwani, O. Susanti, D. Maesaroh, and O.K. Radjasa. 2019a. The bioactivity of bacterium and fungi living associate with the sponge Reniera sp. against multidrug-resistant Staphylococcus aureus and Escherichia coli. Biodiversitas. 20(8) 2302-2307. https://www.researchgate.net/publication/338452028_The_bioactivity_of_bacterium_and_fungi_living_associate_with_the_sponge_Reniera_sp_against_multidrug-resistant_Staphylococcus_aureus_and_Escherichia_coli Sibero, M.T., T.U. Bachtiarini, A.Trianto, A.H. Lupita, D.P. Sari, Y. Igarashi , E. Harunari , A.R. Sharma , O.K. Radjasa , and A. Sabdono. 2019. Characterization of a yellow pigmented coral-associated bacterium exhibiting anti-bacterial activity against multidrug resistant (MDR) organism. Egyptian Journal of Aquatic Research. https://www.sciencedirect.com/science/article/pii/S168742851830075X Cristianawati, O., A. Sabdaningsih, L.E. Becking, M.M. Khoeri, H. Nuryadi, A. Sabdono, A. Trianto, and O.K. Radjasa. 2019. Biological activity of sponge-associated fungi from Karimunjawa Islands, Indonesia against pathogenic Streptococcus pneumonia. Biodiversitas 20(8): 2143-2150. DOI: 10.13057/biodiv/d200807 Trianto, A., O.K. Radjasa, A. Sabdono, S.I. Muchlissin, R. Afriyanto, Sulistiowati, S.K. Radjasa, P. Crews, and E. McCauley. 2019. Exploration culturable bacterial symbionts of sponges from Ternate Islands, Indonesia. Biodiversitas 20(3):776-782. DOI: 10/13057/biodiv/d200323. Back to PEER Cycle 5 Grant Recipients