Biotechnology of Marine Extremophiles

J. Querellou
Ifremer, Centre de Brest
Laboratory of Microbiology and Biotechnology of Extremophiles
BP 70, 29280 Plouzané
France

Extended abstract

“The sea is regarded as the origin of life on Earth and the oceans include the largest habitats on Earth hosting the most ancient forms of life. Over billions of years, marine microbes have moulded the global climate and structured the atmosphere. Knowledge of the biochemical processes that adapted, diversified and evolved in very different and extreme environments is the basis for discoveries in biotechnology” (ESF Marine Board, Position Paper 5, 2002). This statement reflects the current opinion of most of the scientific community in Europe. The fields of biotechnology that could benefit from mining the extremophiles are very broad and cover the search for new bioactive compounds for industrial, agricultural, environmental, pharmaceutical and medical uses. However, this potential remains to a large extent unexplored and, in respect of the drugs available on the market, only 30% have been developed from natural products and so far less than 10% have been isolated from marine organisms.

Extremophilic Archaea and Bacteria thrive in various extreme environments on our planet. They include thermophiles and hyperthermophiles colonising coastal hot springs, shallow and deep-sea hydrothermal vents, or psychrophiles thriving in deep ocean trenches, abyssal plains, polar oceanic regions and continental margin cold seeps. Extremophiles include also halophiles, thermoacidophiles and thermoalkaliphiles, named according to the properties of their respective environments. In addition, exploration of subterranean environments below the sea floor, through ODP, has revealed the existence of an important microbial biomass, most of it still uncultured and uncharacterised (Pedersen, 2000).

The potential impact of marine microbial resources on the biotechnology industry is mainly dependent upon biodiversity exploration and genomics dedicated programmes as well as specifically targeted biotechnological projects. Moreover, access to and preservation of the marine extreme environments is a major issue.

1. Besides microorganisms already available in public collections, exploration of extremophiles in the various extreme marine environments (hydrothermal vents, cold seeps, subterranean environments), either with cultural methods or total DNA molecular based approaches, is the most promising way to increase the probability of finding novel bioactive compounds.
2. Access to sampling. The huge potential of marine extremophiles relies on access to sampling. The legal property is defined according to international regulations, and is therefore granted for EU member states in their EEZ. In international waters however, the property is in fact attributed to those able to sample the deep-sea, in fact a very small number of countries. Conservation of marine extreme environments has not been an issue up to very recently and this could change in the near future.
3. Since the very first complete genome sequencing of Haemophilus influenzae in 1995, genomic approaches have been successfully introduced in analyses of microbial genomes for gene identification and function assignment. Genomics and proteomics are therefore needed to insure the growth of databases, to discover novel genes and gene products through data mining. Expression libraries, constructed from total DNA extracted from crude samples, provide a short cut to gene repertoire without culture-based limitations.
4. Specifically targeted biotechnological programmes are needed in order to characterise the properties of the novel bioactive compounds and assess their potential for development. When, as very often observed, the properties of the candidate molecule do not fit completely with the industrial requirements, engineering of the molecule is needed.

Exploration.
Exploration of microbial biodiversity in these environments has opened a new era for microbiologists already blessed by the establishment of Archaea as a separate domain (Woese and Fox, 1977) and the successive introduction of molecular biology and genomics as basic and powerful tools. Besides the classic approach of strain isolation through culture, a new window has been opened during the last decade with the use of molecular markers (16S rRNA mainly) to characterise the microbial biodiversity of various ecological niches (Amann et al., 1996). One of the main conclusions is the growing emergence of clusters of “environmental clones” with no representative in culture, illustrating the “count-plate anomaly”. The number of extremophilic species described has increased exponentially during the last two decades and encompasses Archaea, Bacteria and more recently some associated archaeal and bacterial viruses. Since the cultured strains already available could account for less than 1% of the total number of species in marine extreme environments, ecogenomics, based on the complete sequencing of clones containing cosmids or bacterial artificial chromosomes with inserts from extremophiles, considerably extend the probability of finding new genes and their products suitable for the industry. Traditional and direct approaches to the finding of novel products are however still effective. For example, sampling of deep-sea hydrothermal vents microorganisms colonising selective substrates and subsequent cultures on corresponding selective media have led to the discovery of new thermostable esterases/lipases, proteases (keratinases) and xylanases.

Access to samples and marine extremophiles
Access to hypersaline ponds and seas as well as shallow hot springs is usually easy and does not require heavy equipment. On the contrary, sampling of deep-sea continental margin cold seeps and hydrothermal vents is dependent upon specialised equipment such as vessels, man-operated submarines or ROVs. European countries possess all the basic equipment to fulfil that purpose, but increasing costs make integration necessary. Development of new samplers is also required in order to remain competitive with the US and Japan.

Genomics applied to marine microorganisms.
Since microorganisms constitute the basis of any marine ecosystem whether they belong to photosynthetic, chemosynthetic or heterotrophic groups, their contribution to the functioning of marine ecosystems is of enormous importance. They are heavily involved in the recycling of major elements on a global scale. Knowledge of the genome sequences of key species is a prerequisite to study microbial responses to environmental changes. It is also the basis of gene mining for new potential biotech applications. Marine microbe complete genome projects account for approximately 15% of the total genome projects and European contribution has been limited to one third of this work, the main effort being carried out by US consortia. This critically emphasises the need of a significant increase in means related to marine genomics in Europe. Comparative genomics can be used to compare enzyme inventories of different extremophiles and complete already available biochemical data (hyperthermostable proteases for example (Ward et al., 2002)) or to unravel discrepancies between experimental and genomic data, possibly suggesting new metabolic pathways. It significantly contributes to the identification of new genes and functions as demonstrated by the re-annotation of Pyrococcus abyssi (Cohen et al., 2003). The rapidly expanding proteomic technologies are also expected to increase the list of proteins and enzymes involved in functional multiprotein complexes in DNA replication, transcription, translation and various metabolisms.

Microbial marine biotechnology: brief view of leading fields.
The main fields of interest are enzymes and biopolymers.
Enzymes from hyperthermophiles have been a major target of biotech programmes in the last decade (e.g. EU FP4 and FP5 “Extremophiles as cell factories”). Thermostable proteases, lipases, esterases, starch and xylan degrading enzymes have been actively sought and in many cases found in bacterial and archaeal hyperthermophilic marine microorganisms (Bertoldo and Antranikian, 2002). In some cases, the substrate specificity and the yield do not completely fit the industrial requirements and enzymes have to undergo an additional protein engineering step (e.g. DNA shuffling). The best known commercial success of thermostable enzymes is the Taq DNA polymerase, obtained from Thermus aquaticus (Yellowstone hot spring). Marine Thermococcales have been an important source of high fidelity thermostable DNA polymerases (Pfu, Vent, Pab, etc.) (Hamilton et al., 2001), accounting for 30% of total sales. In addition, the high structural conservation and complementation of DNA replication proteins between euryarchaeal Pyrococcus and human make hyperthermophilic archaea a model of choice to study eukaryotic DNA replication (Henneke et al., 2000).

Microbial exopolysaccharides (EPS), especially those produced by some mesophilic Vibrios and Alteromonas strains isolated from deep-sea hydrothermal vents, display interesting properties currently under evaluation for therapeutic uses. The main fields concerned are tissue regeneration and cardiovascular diseases (antithrombotic/proangiogenic effects). Characterisation and preliminary results on anticoagulant activities of the EPS showed that native EPS were deprived of effects whereas sulphated derivatives were active (Guezennec, 2002).

References.

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Acknowledgements: We thank the Brittany Regional Council and EU (Repbiotech programme) for their financial support.