Microrganism prototype pipeline

Microrganism prototype pipeline

Discovery and exploitation of natural compounds from bacteria and fungi

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 secondary metabolites from marine bacteria and fungi. This prototype pipeline brings together the expertise and facilities of partners from the MIRRI and EMBRC research infrastructures to target and release the potential of these organisms from isolation through characterisation to end product.

Bacteria and fungi 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 prototype 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 are being targeted, characterized and prepared for scale up and production of active compounds.

 

Cultivation and culture collection

  • Improved targeted isolation and characterization of strains (substrate pulse experiment)
  • Biomass production
  • Fermentation and extraction scale up   

EMBRIC partners are enhancing and developing new tools for isolating the yet uncultured e.g. DSMZ substrate pulse on marine microbial communities to identify active cells and a biofilm tool for microbial isolation utilized by DSMZ has facilitated microbial isolation. Challenging cells with specific substrates not only can provide indication of what may be required for growth media but can help target organisms with specific properties. The expertise of the microbiologists across EMBRC and MIRRI can be harnessed to facilitate the creation of sufficient biomass from organisms hitherto difficult or impossible to cultivate.

Access Providers available for this pipeline element:

 

Determination of organism potential

Working with slow growing marine organisms can be laborious and expensive in time and resources so it is important that effort focusses on the organisms with potential. There are numerous methodologies that can be applied and the microorganism prototype pipeline explores and evaluates some of them for example:

  • Characterizing the metabolome; GC/MS and LC/MS(/MS) pipeline; correlating metabolism with gene expression data
  • Genome reading to determine biosynthetic gene clusters (BGCs)
  • Comparing approaches to unlocking natural product biosynthesis using:   
    • Chemical elicitors (rapid but untargeted)
    • Heterologous expression (cosmid and bac libaries) – slow but precise and affording opportunity for further manipulation
  • Liquid chromatography-tandem mass spectrometry (LC-MSMS), Proton (Hydrogen) nuclear magnetic resonance (HNMR), Carbon-13 nuclear magnetic resonance (CNMR), Heteronuclear Single Quantum Correlation (HSQC), Nuclear Overhauser Effect Spectroscopy (NOESY)  characterization

Metabarcoding technology can characterize the species compositions of mass samples of environmental DNA.

Access Providers available for this pipeline element:

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Extract production

  • Test efficiency of different extraction and characterization techniques
  • Selection of phenotypic assays; peptide arrays; mutant generation and sequencing; chemical probes
  • Develop specific purification procedures for each molecule - Extraction/isolation example natural products

Extraction method limits compounds available in the sample for assay – EMBRIC expertise can identify the appropriate stage of the organism’s life cycle or the triggers to express the target chemistry. Identifying the active component of the extract requires different technologies; the resources of RIs can be accessed to carry out process efficiently whilst utilizing their expertise e.g. EU-OPENSCREEN provides infrastructure for Chemical Biology and its translation to medicine, agriculture, bioindustries. HZI has protocols for the detection and identification of different metabolite classes: from small volatile compounds (e.g. short-chain fatty acids) to larger polar and nonpolar compounds of primary and secondary metabolism. Different state-of-the art routes to access, then heterologously express, identified biosynthetic gene clusters encoding novel chemistries are being explored and compared. Selected metabolites are purified by reversed phase and normal phase chromatography, structurally characterized by NMR and their biological profile is established in phenotypic assays (e.g. antibacterial, antifungal, cytotoxic).

Access Providers available for this pipeline element:

Purification of characterized compounds

  • Mode of action analysis for isolated and structurally characterized compounds
  • Characterize the metabolome by untargeted GC/MS and LC/MS
  • Correlate metabolism with gene expression data
  • Test efficiency of different extraction and characterization techniques
  • Test extracts and pure compounds in cellular bioassays
  • For bioactive compounds: Mode of action studies, peptides arrays, mutant generation and sequencing
  • Links to relevant data sets outside the partners to add value   

Compound identification in GC-MS analyses is carried out using NIST’11 spectral library; a GC-MS in-house library Golm Metabolome database is available for comparative purposes. For LC-MS/MS workflow, HZI uses freely-available databases, 610 in-house standards and a self-programmed MS2 spectra clustering algorithm.

Access Providers available for this pipeline element:

 

Legal framework

There is extensive legislation concerning the safe handling, use and distribution of micro-organisms at the national, regional and international levels. Micro-organisms of hazard groups 2, 3 and 4 are hazardous substances and as such fall under the EU Biological Agents Directive and are dangerous goods as defined by the International Air Transport Association (IATA) Dangerous Goods Regulations where requirements for their packaging are defined.  The potential mis-use of microorganisms has introduced control measures in place for biosecurity to control access to dangerous pathogens.  Most recently the Nagoya Protocol is being implemented by nations to ensure benefit sharing occurs as a result of accessing genetic resources. The legislation and supporting documents are often difficult to find and understand.  EMBRIC offers advice and interpretation to help the implementation of such legislation. Examples are:

  • Health and Safety
  • Classification of Microorganisms on the Basis of Hazard
  • Quarantine regulations
  • Postal Regulations and Safety
  • Packaging
  • Regulations governing distribution of cultures
  • Legislation on the Proliferation, Distribution and Misuse of Dangerous Pathogens         
  • Export Licensing Measures
  • Convention on Biological Diversity and the Nagoya Protocol        
  • Ownership of Intellectual Property Rights (IPR)  
  • Safety information provided to the recipient of microorganisms

Access Providers available for this pipeline element:


Possible research workflows with this discovery pipeline

Example 1

We are a small biotech company creating a small molecule library for large pharmaceutical companies to access and screen for activities with potential as marketable products, do you have access to microorganisms with novel properties that would be relevant for inclusion in our libraries? The EMBRIC partners may have organisms with relevant chemistry in their ex situ collections or the microorganism prototype pipeline could be used to target organisms from marine samples that have the range of chemistry of interest. EU-OPENSCREEN has created its own library of characterized compounds that can be screened.

Example 2

Company X has a chemical a target and are in the process of isolating the active compound themselves through their own in-house screens. EMBRIC is asked to help provide as many potential candidates as possible and particularly freshly isolated strains with novel attributes. Targeting these as ‘de novo’, in the vast oceans, is somewhat difficult but usually begins with a metagenomics approach. The EMBRIC partners can support targeted isolation programmes; it is possible to target and “hunt” genes in nature; these can be cloned and expressed. New discoveries and technologies are appearing swiftly and it should be possible to target and isolate whole organisms with specific properties: e.g. with recent innovations such as the iChip. Additionally, mBRCs with their networks of collectors, isolators and depositors can support researcher-targeted isolation programmes.

Example 3

A new company wishes to develop their own specific route or pipeline to discovery. In this specific scenario, EMBRIC partners provide links to where the microorganisms may be sourced providing extracts or compounds. ELIXIR and some of the EMBRIC cluster partners can provide tools and software for combined metabolite profile and genome analysis. The technologies and practices at this stage of the pipeline are institutional (some are outlined above) but many appear in the scientific literature, on which experienced practitioners in EMBRIC can advise, and to which they can provide references. In addition, EU-OPENSCREEN provide compound libraries and storage with individual mBRCs and institutional compound libraries providing additional services.

 

Contact person for questions and inquiries

David Smith
CAB International (CABI)
Tel:        +44 (0)1491 829114
Email:   d.smith@cabi.org
www.cabi.org

 

Relevant publications

Trindade, M., van Zyl, L., Navarro-Fernández, J. & Abd Elrazak, A. (2015). Targeted Metagenomics as a Tool to Tap Into Marine Natural Product Diversity for the Discovery and Production of Drug Candidates. Frontiers in Microbiology http://dx.doi.org/10.3389/fmicb.2015.00890.
Clin Microbiol Infect 2012; 18: 1185–119310.1111/1469-0691.12023:
http://www.ncbi.nlm.nih.gov/pubmed/23033984  http://onlinelibrary.wiley.com/doi/10.1111/ele.12162/abstract
Rhode et al. 2013. http://ijs.sgmjournals.org/content/63/Pt_7/2374.long
Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products” Natural Product Reports; DOI: 10.1039/C5NP00111
Anne Osbourn, Rebecca Goss and Guy T. Carter (Eds) (2014). Natural Products: Discourse, Diversity, and Design. Wiley-Blackwell; 552 pages; ISBN: 978-1-118-29806-0; http://eu.wiley.com/WileyCDA/WileyTitle/productCd-1118298063.html

 


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