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D 6.1 EMBRIC Showcases 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 b EMBRIC showcases: prototype pipelines from the microorganism to product discovery (Revised 2019) HORIZON 2020 - INFRADEV Implementation and operation of cross-cutting services and solutions for clusters of ESFRI 1 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 (Revised 2019) Project start date: June 2015 (48 months) Submission due date: May 2019 Actual submission date: Apr 2019 Work Package: WP 6 Microbial pipeline from environment to active compounds Lead Beneficiary: CABI [Partner 15] Version: 1.0 Authors: SMITH David [CABI Partner 15] GOSS Rebecca [USTAN 10] OVERMANN Jörg [DSMZ Partner 24] BRÖNSTRUP Mark [HZI Partner 18] PASCUAL Javier [DSMZ Partner 24] BAJERSKI Felizitas [DSMZ Partner 24] HENSLER Michael [HZI Partner 18] WANG Yunpeng [USTAN Partner 10] ABRAHAM Emily [USTAN Partner 10] FIORINI Federica [HZI Partner 18] Project funded by the European Union’s Horizon 2020 research and innovation programme (2015-2019) Dissemination Level PU Public X 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 Services 2 Abstract Deliverable D6.1b replaces Deliverable 6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery with the specific goal to refine technologies used but more specifically deliver results of the microbial discovery pipeline. The objective of work package 6 Microbial pipeline from environment to active compounds was to develop coherent chains of high-quality services for access to biological, analytical and data resources and deploying common underpinning technologies and practices for useful secondary metabolites 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. Deliverable D3.1 Map of centres of expertise and best practices from EMBRIC work package 3 provides a comprehensive coverage of the cluster capabilities. Work package 6 demonstrated how the discovery pipelines can utilize the partner RIs and specifically how some of the services can be linked to produce specific end products. EMBRIC has established an overarching and operational structure to facilitate the integration of the multidisciplinary value chains of services. The partner RIs are committed to retain such activities as legacy to the EMBRIC project. 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. Population genomics has provided us with a picture of what might be there and an idea of the chemistry they may perform. EMBRIC’s microorganism pipeline aims to unlock this potential. Utilizing the specialist expertise and facilities of key laboratories, organisms that are difficult to cultivate or have yet to be grown were targeted, characterised and prepared for scale up and production of active compounds. DSMZ undertook to make difficult-to-culture bacteria amenable to subsequent natural compound analyses. Optimal growth conditions for some of these organisms were determined to enable strains with novel properties to be grown. Following de-replication, DSMZ isolated 264 species of slow growing bacteria from sea water, sediment and sponges of the Pacific Ocean representing the phyla Actinobacteria, Bacteroidetes, Proteobacteria and Rhodothermaeota. The most interesting were selected for cosmid library production. Two Arcobacter strains are described as novel species in the genus and those exemplary strains 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. 9 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 were fine-tuned user fully sequenced samples were put through the microbial pipeline and demonstrated that interesting compounds could be discovered. CABI focused on the regulatory environment addressing issues around access to genetic resources and access and benefit sharing in particular. The microorganism 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, a total of 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. 3 Contents Abstract ................................................................................................................................ 3 1. Introduction .................................................................................................................... 6 1.1 Microorganism prototype pipeline ................................................................................ 6 Cultivation and culture collection .................................................................................... 7 Determination of organism potential ............................................................................... 7 Extract production .......................................................................................................... 8 Purification of characterised compounds........................................................................ 8 Legal framework ............................................................................................................ 8 2. Protocols for isolation, selection, growth of strains and extract supply ......................... 10 2.1. Sample collection and processing ............................................................................ 10 2.2. Cultivation strategies ............................................................................................. 10 2.2.1 Single dilution high-throughput in liquid media. ................................................... 10 2.2.2 Direct plating method .......................................................................................... 11 2.2.3 Growth in biofilms ............................................................................................... 11 2.2.4 Chemotaxis chambers ........................................................................................ 11 2.3. Taxonomic affiliation of isolates ................................................................................ 12 2.4. Maintenance procedures .......................................................................................... 13 2.5. Fermentation of bacteria for natural product production ............................................ 13 3. The genomic characterisation, cosmid library production and analysis ........................ 14 3.1 Gene Clusters Prediction ........................................................................................... 14 3.2 Genomic DNA Preparation ........................................................................................ 15 3.3 Vector DNA Preparation ............................................................................................ 15 3.4 Construction of Cosmid Library ................................................................................. 16 3.5 Transformation of Gene Clusters and Secondary Metabolite Heterologous Expression ........................................................................................................................................ 17 4. Analysis of extracts ...................................................................................................... 20 4.1 Analysis of extracts at USTAN ................................................................................... 20 4.1.1 Secondary metabolites production at Ustan ........................................................ 20 4.1.2 Secondary metabolites extraction ....................................................................... 20
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