INMARE

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Oceans of enzyme possibilities

INMARE sampling. © Carla CCR de Carvalho

The world’s oceans are full of interesting microorganisms, yet their wider potential has not yet been fully explored. The INMARE project brought together researchers from several different disciplines to discover new enzymes and bioactives from marine environments, which will help chemical and pharmaceutical companies work more effectively, as Professor Peter Golyshin explains The world’s oceans are home to an abundance of marine life that is uniquely adapted to living in sometimes very harsh environments. This is particularly true of marine microorganisms. Enzymes from microorganisms adapted to living in these extreme environments hold great potential for biotechnology. Significant parts of the oceans are inhabited by psychrophilic microbes for example, which are well adapted to low temperatures, while many other ‘extremophiles’ live in extreme conditions in marine environments. “For example, there are thermophiles that live at high temperatures, in hot vents. Other organisms prefer alkaline conditions, or elevated salt concentrations, there are also organisms that thrive under conditions of multiple extremes,” outlines Peter Golyshin, Professor of Biology at Bangor University in the UK. Professor Golyshin and his colleagues in the INMARE project set out to streamline the enzyme discovery process. “Many people have invested a lot of time into improving enzymes, yet often it doesn’t lead anywhere. In this project, our aim was to screen enzymes from microbes that have already evolved to operate in extreme environments,” he explains. These extreme environments hold a great deal of scientific interest, as they can provide important insights into the origins of life, in addition there is also a strong applied dimension to the project’s work. Chemicals are often synthesised in harsh conditions for example, so companies need enzymes that can cope. “For industry, it is important to have enzymes that can operate under the conditions of industrial processes,” stresses 34

Professor Golyshin. In the pharmaceutical and chemical industries, many substrates have very low solubility, so a reaction needs to be set up accordingly. “The reaction must be set up in the right conditions, e.g. with high pressure, temperature or high concentration of solvents, to enable solubility of substrates,” explains Professor Golyshin. “Microbes that thrive in similar environments may be capable of producing enzymes to do these reactions. That’s the rationale behind exploring these environments - we hope to find some microbes and their enzymes that have these corresponding features.”

“In MAMBA we gathered very large sets of genomic and metagenomic resources, which we screened for enzymatic activities. In INMARE we are carrying on with that work, but we also have some new sampling sites and new ways to identify enzymes and metabolites,” says Professor Golyshin. Samples have been gathered from many different environments. “For example, our Norwegian colleagues are taking samples of hydrothermal fields (‘black smokers’) in the northern part of the mid-Atlantic ridge. Microbial DNA is then extracted to establish genomic libraries, which are screened for

“There may be millions of different microbial species, most of which are not amenable to cultivation in the laboratory, and therefore their enzymes and metabolites remain very difficult for us to access,” explains Professor Golyshin. A technique called ‘metagenomics analysis’ helps researchers to gain access to industrially relevant enzymes from as yet uncultured microorganisms. “Essentially, we harvest DNA from the environment, then we perform shotgun DNA sequencing to identify which genes are present in the sample. Then we identify the relevant ones and express them in different microbial hosts, like yeasts, pseudomonads or bacilli, for example,” continues Professor Golyshin. “We also establish gene libraries, then screen those for the defined activity that we are interested in. In this way, we can discover genuinely new enzymes.” This could include substrate-promiscuous enzymes capable of catalysing different reactions, which could help chemical and pharmaceutical companies work more efficiently. Researchers in the project have established a large collection of esterases, which have been analysed against a similarly large quantity of ester substrates, and Professor Golyshin says the results so far are very interesting. “We’ve found that a large proportion of these enzymes can take not just one or two substrates, but many more. Many

of these enzymes can do different things and work with different reactions. We have some enzymes that catalyse reactions with 76 substrates,” he outlines. Some commonalities in the structure of the enzymes have also been found, which holds important implications in terms of the project’s wider agenda. “We are now able to predict promiscuity just by looking at the sequence of the protein. This is one of the most important discoveries we have made in the project,” continues Professor Golyshin.

Commercial sector The project’s industrial partners play an important role in guiding this research and ensuring it is relevant to commercial needs, which is an important part of the wider agenda. While it is of course difficult to forecast the outcome of this kind of research, Professor Golyshin is keen to translate the project’s work into commercial development. “We try to deliver enzymes or compounds to the commercial sector that could be used in the future,” he stresses. One

Sampling equipment being deployed from a research vessel off the coast of Messina, Italy. © CNR (Consiglio Nazionale delle Ricerche)

We harvest DNA from the environment, then we perform shotgun DNA sequencing to identify which genes are present in the sample. Then we identify the relevant ones and express them in different microbial hosts, like yeasts, pseudomonads or bacilli. Sampling sites This work builds on the earlier MAMBA project, in which researchers gathered and stored large numbers of genetic resources from microorganisms in different marine environments, including deep hypersaline anoxic basins in the Eastern Mediterranean Sea, epibionts of marine invertebrates, or petroleum-depleting marine microbial communities. The project brings together 24 multi-disciplinary partners from across Europe, providing a solid foundation to identify interesting enzymes, characterise them and find ways to apply them in industry.

activities. Alternatively the DNA can be directly sequenced and then analysed for genes for enzyme candidates to be cloned and characterised,” outlines Professor Golyshin. “We also have other partners who take samples from sea floor brine lakes in the Mediterranean. There are also further under-explored sites, including those with shallow hot vents.” These environments are home to some unique microbes, Professor Golyshin and his colleagues sought to harness their wider potential. However, it’s very difficult to disentangle the activity of certain microbes and identify industrially relevant enzymes.

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INMARE Industrial Applications of Marine Enzymes: Innovative screening and expression platforms to discover and use the functional protein diversity from the sea

Project Objectives

The main objectives of INMARE were to: 1. Streamline and significantly shorten the pipelines of marine enzyme and bioactive compound discovery for industrial applications; 2. Develop marine enzyme collections with a high proportion of promising enzymeallrounders; 3. Identify new lead products and deliver prototypes for new biocatalytic processes based on marine microbial enzyme resources for targeted production of fine chemicals, drugs and materials for use in environmental clean-up applications.

Project Funding

INMARE was funded by the European Union’s Horizon 2020 research and innovation programme, grant agreement No. 634486.

Project Partners

There are 24 INMARE Project Partners Please see website for full details: http://www.inmare-h2020.eu/participants.php.en

Contact Details

Professor Peter Golyshin, INMARE Coordinator School of Natural Sciences Bangor University, Gwynedd, LL57 2UW T: +01248 383 629 E: p.golyshin@bangor.ac.uk W: http://www.inmare-h2020.eu/ Ferrer M, Martínez-Martínez M, Bargiela R, Streit WR, Golyshina OV & Golyshin PN. Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. Microb Biotechnol. 2016, 9(1), 22-34. DOI:10.1111/1751-7915.12309. All INMARE publications can be found here: http://www.inmare-h2020.eu/publications.php.en

Professor Peter Golyshin

area in which these enzymes could be applied is the pharmaceutical industry, which could benefit from a more streamlined development process. “Currently it takes a long time to bring a new discovery from the laboratory to the market, as it has to go through pre-clinical and clinical trials. A lot of money and time is required,” points out Professor Golyshin. “One of our partners is developing anti-tumour drugs, and through the work of the project they have succeeded in finding a new compound, which they have already patented.” A further important dimension of the project’s work is the possibility of using new marine enzymes in environmental clean-up operations. Petroleum constituents can cause a lot of damage when they leak into marine environments. “Petroleum constituents dissolve the membranes of microbial cells,” explains Professor Golyshin. Certain microbes can help clean up marine environments which have been exposed to these types of pollutants. “There are microbes that can degrade petroleum hydrocarbons. They are naturally present in the sea, but normally they are found in very low numbers. We’ve found that when we take a sample of sea-water and add some petroleum, the number of these bacteria increase - they comprise up to 90 percent of the total microbial community. These bacteria are naturally associated with algae, or unicellular eukaryotes, which photosynthesise and produce fats, lipids, and other molecules similar to petroleum components,” continues Professor Golyshin. These bacteria can degrade petroleum effectively in the right conditions, when they have access to oxygen and nitrogen, while researchers are also working on enzymes capable of degrading plastic. This is a prominent issue at the moment, with concern rising over

Image of a 5 litre fermenter. © Carla CCR de Carvalho

the amount of plastic in the world’s oceans and its ecological impact, and Professor Golyshin says a lot of attention is being paid to this area. “The EU and different national funding agencies are looking to improve our basic knowledge, aiming to build a toolbox to degrade plastic,” he outlines. This will remain an important part of Professor Golyshin’s agenda in future, along with building on the progress that has been made over the course of the INMARE project and moving towards commercial applications. “A spin-off company has already been established, and a number of patents have been filed, which was a major part of the initial project application. The project has recently concluded, and we have produced more than 100 peer-reviewed papers and book chapters, 4 patents and one startup company.” It would seem that INMARE’s ambitious voyage of discovery has certainly been a successful one. INMARE Scientist Professor Michail Yakimov, Institute for Coastal Marine Environment (IAMC), CNR. © CNR (Consiglio Nazionale delle Ricerche)

Peter Golyshin is Professor of Biology at Bangor University, a role in which he investigate the environmental genomics of microorganisms. He performed the first omics-based studies in marine obligate hydrocarbonoclastic bacteria, that play a key role in marine oil degradation worldwide. He pioneered activity-based metagenomic enzyme discovery in cow rumen, earthworm gut and deep-sea environments.

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