Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) Grant Agreement Number: 654008 EMBRIC European Marine Biological Research Infrastructure Cluster to promote the Blue Bioeconomy Horizon 2020 – the Framework Programme for Research and Innovation (2014-2020), H2020-INFRADEV-1-2014-1 Start Date of Project: 01.06.2015 Duration: 48 Months Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery (M36) HORIZON 2020 - INFRADEV Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 1 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) Implementation and operation of cross-cutting services and solutions for clusters of ESFRI Grant agreement no.: 654008 Project acronym: EMBRIC Project website: www.embric.eu Project full title: European Marine Biological Research Infrastructure cluster to promote the Bioeconomy Project start date: June 2015 (48 months) Submission due date : May 2018 Actual submission date: May 2018 Work Package: WP 6 Microbial pipeline from environment to active compounds Lead Beneficiary: CABI Version: 9.0 Authors: SMITH David GOSS Rebecca OVERMANN Jörg BRÖNSTRUP Mark PASCUAL Javier BAJERSKI Felizitas HENSLER Michael WANG Yunpeng ABRAHAM Emily Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 2 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) Project funded by the European Union’s Horizon 2020 research and innovation programme (2015-2019) Dissemination Level PU Public PP Restricted to other programme participants (including the Commission Services) RE Restricted to a group specified by the consortium (including the Commission Services) CO Confidential, only for members of the consortium (including the Commission X Services Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 3 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) Abstract The objective is to develop coherent chains of high quality services for access to biological, analytical and data resources and deploying common underpinning technologies and practices for the route to useful compounds from marine bacteria. This prototype pipeline brings together the expertise and facilities of partners from the MIRRI, EMBRC and EUOPENSCREEN research infrastructures to target and release the potential of microorganisms from isolation, through characterisation, to end product. Bacteria are key components of the marine environment, performing a wide range of biogeochemical and ecological functions yet we know very little about them. It is estimated that we have seen less than 1 percent in culture and the vast potential remains locked away. Molecular ecology has provided us with a picture of what might be there and an idea of the chemistry they may perform. The pipeline aimed to unlock this potential, utilizing the specialist expertise and facilities of consortium laboratories, organisms difficult to cultivate or have yet to be grown were targeted, characterised and prepared for scale up and production of active compounds. DSMZ undertook the task to make difficult-to-culture bacterial strains amenable to subsequent natural compound analyses. Optimal growth conditions for some of these organisms were determined and as a result bacterial strains with novel properties were isolated and grown. Following de-replicating of potential clones DSMZ isolated 264 species of slow growing bacteria from seawater, sediment and sponges of the Pacific Ocean representing the phyla Actinobacteria, Bacteroidetes, Proteobacteria and Rhodothermaeota. The most interesting of these will be selected for cosmid library production two Arcobacter species were fermented and organic extracts supplied to HZI for analysis. USTAN had the role of improving the efficiency and effectiveness of a harmonised natural product discovery pipeline. Cosmid libraries were established for test strains and these put through heterologous platforms to determine the presence of compounds. Nine gene clusters and a series of compounds were detected. Improvements to the process were made using elicitors and promoters and some difficult cyclic peptides were characterised. HZI improved the efficiency of different extraction and characterization techniques for use on different types of organisms and as a result isolates producing interesting compounds were discovered. Once the pipeline through the partner infrastructures was fine-tuned, fully sequenced samples were put through the pipeline and it was demonstrated that interesting compounds could be discovered. CABI focused on the regulatory environment addressing issues around access to genetic resources. The prototype pipeline resulted in a flexible system to access cross research Infrastructure expertise and facilities to meet specific user defined demand of the research community. In one single experiment to develop the EMBRIC microbial prototype pipeline, 264 rarely isolated species of bacteria were made available for study of their bioactive compounds. This demonstrates that coordinated and targeted isolation programmes engaging research teams from the many partners in the Research Infrastructures orchestrated for specific bioindustry needs could yield many thousands of candidate organisms. The potential for discovery of new interesting bioactive compounds is thus increased exponentially. Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 4 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) Contents Abstract 4 1. Introduction 7 1.1 Microorganism prototype pipeline 7 2. Protocols for isolation, selection, growth of strains and extract supply 11 2.1. Sample collection and processing 11 2.3. Taxonomic affiliation of isolates 14 2.4. Maintenance procedures 14 2.5. Fermentation of bacteria for natural product production 15 3. The genomic characterisation, cosmid library production and analysis 16 3.1 Gene Clusters Prediction 16 3.2 Genomic DNA Preparation 16 3.3 Vector DNA Preparation 17 3.4 Construction of Cosmid Library 18 3.5 Transformation of Gene Clusters and Secondary Metabolite Heterologous Expression 19 4. Analysis of extracts 22 4.1 Analysis of extracts at USTAN 22 4.2 Analysis of extracts at HZI 23 5. Regulatory Environment 26 5.1 Regulatory Guidance and Community Best Practices 28 5.2 Regulations impacting on collection, handling, use and distribution of organisms 31 5.3 Health and Safety 32 5.4 Microorganisms as hazardous substances 33 5.5 Classification of Microorganisms on the Basis of Hazard 33 5.6 Quarantine regulations 35 5.7 Postal Regulations and Safety 35 5.8 Packaging 36 5.9 Regulations governing distribution of cultures 36 5.10 Control of Distribution of Dangerous Organisms 36 5.11 Biological Weapons Convention 37 5.12 Export Licensing Measures 37 5.13 Control procedures: UKNCC control of Dangerous Pathogens 39 5.14 Convention on Biological Diversity 40 5.15 Ownership of Intellectual Property Rights (IPR) 40 Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 5 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) 5.16 Safety information provided to the recipient of microorganisms 41 5.17 Areas Beyond National Jurisdiction (ABNJ) 42 6. Outputs from the microorganism prototype pipeline 45 6.1 Strains collected from the Pacific Ocean by DSMZ 45 6.2 Cosmid libraries and biosynthetic gene clusters at USTAN 45 6.3 Characterisation of extracts at HZI 61 6.4 Significance of outputs 64 7. EMBRIC pathways to discovery 66 8. Summary 75 9. References and bibliography 77 Deliverable D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery Page 6 of 85 Deliverable D6.1 EMBRIC (Grant Agreement No. 654008) 1. Introduction The European Marine Biological Research Infrastructure Cluster (EMBRIC) is designed to accelerate the pace of scientific discovery and innovation from marine Bio-Resources. EMBRIC aims to promote new applications derived from marine organisms in fields such as drug discovery, novel foods and food ingredients, aquaculture selective breeding, bioremediation, cosmetics and bioenergy (http://www.embric.eu/). Researchers design their own experiments to lead to discovery of novel properties or products but such programmes can be enhanced by using the facilities available through European Research Infrastructures. EMBRIC addresses the steps to discovery from selection and isolation of the marine microorganisms to the characterisation and isolation of bioactive compounds. EMBRIC tests the different routes that can be followed and demonstrates the advantages of engaging with the most appropriate technologies available. Additionally, there are numerous areas where policy, regulations or legislation impact on the isolation, use and distribution of organisms which include health and safety, quarantine regulations, shipping, packaging, governing distribution of cultures including dangerous pathogens, export licensing measures, ownership of Intellectual Property Rights (IPR) and more. Compliance with this regulatory environment may be complicated and ever changing but it is not new. Individuals and their Institutions must address this but in many cases community best practices or common approaches have helped them. For example culture collections through the European Culture Collections’ Organisation (ECCO) and the World Federation for Culture Collections (WFCC) have supported their members in compliance