Brazilian Journal of Microbiology (2007) 38:398-405 ISSN 1517-8382 EXPLORING THE BIOTECHNOLOGIAL APPLICATIONS IN THE ARCHAEAL DOMAIN Alquéres, S.M.C.1; Almeida, R.V.2; Clementino, M.M.3; Vieira, R.P.1; Almeida, W.I.1; Cardoso, A.M.1*; Martins, O.B.1 1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 3Instituto Nacional de Controle da Qualidade em Saúde, Fundação Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil. Submitted: March 25, 2007; Returned to authors for corrections: July 16, 2007; Approved: July 29, 2007. MINI-REVIEW ABSTRACT Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. The extremophilic nature of many archaea has stimulated intense efforts to understand the physiological adaptations for living in extreme environments. Their unusual properties make them a potentially valuable resource in the development of novel biotechnological processes and industrial applications as new pharmaceuticals, cosmetics, nutritional supplements, molecular probes, enzymes, and fine chemicals. In the present mini-review, we show and discuss some exclusive characteristics of Archaea domain and the current knowledge about the biotechnological uses of the archaeal enzymes. The topics are: archaeal characteristics, phylogenetic division, biotechnological applications, isolation and cultivation of new microbes, achievements in genomics, and metagenomic. Key words: Archaea, Biotechnology, Extremozymes, Genomic, Molecular Phylogeny. Archaea domain For 15 years after their recognition, the Archaea were generally In the late 1970s, Carl Woese and his colleagues at the known only as inhabiting hostile environments (27). Within the University of Illinois studied relationships among prokaryotes past two decades, the use of molecular techniques, including and proposed that life should be divided into three domains: PCR-based amplification of 16S rRNA genes, has allowed a Archaea, Bacteria, and Eucarya. They found that there were culture-independent assessment of microbial diversity, indicating two distinctly different prokaryotic groups. Those “bacteria” a wide distribution of mostly uncultured archaea in normal that lived at high temperatures or produced methane were habitats, such as ocean and lake waters and soil (10). Nowadays clustered together as a group far away from the usual bacteria the use of 16S rDNA clone libraries to map the diversity of (Fig. 1) and the eukaryotes (51). uncultivated prokaryotes from natural populations has provided Woese argued that Archaea, Bacteria, and Eucarya each a revolutionary advance for interpreting microbial evolutionary represent a primary line of descent that diverged early from an relationships. These molecular surveys have produced more than ancestral progenote with poorly developed genetic machinery. 20,000 archaeal 16S rRNA gene sequences from environmental This hypothesis is reflected in the name Archaea, from the Greek studies, extending the known groups and increasing the number archae, meaning ancient. Later he treated these groups formally of novel lineages. In Brazil, our group study the archaeal diversity as domains, each consisting of several kingdoms. This separation in tropical environments and show that the detection of a and organization of tree of life have become very popular, although substantial number of uncultured phylotypes suggests that this the idea of the progenote itself is not generally supported (50). region harbors a pool of novel archaeaplankton taxa (11,47). *Corresponding Author. Mailing address: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde - Bloco D, subsolo, sala 5. 21941-590 - Rio de Janeiro – Brasil. Tel.: +55 21 25626751 - Fax: +55 21 22708647. E-mail: [email protected] 398 Biotechnological applications in the archaeal domain Archaeal characteristics Several features set the Archaea apart, for example, archaea have a single cell membrane containing a peptidoglycan-like wall which is different in bacteria. Furthermore, both eubacterias and eukaryotes have membranes composed mainly of glycerol- ester lipids, whereas archaea have membranes composed of glycerol-ether lipids. These differences may be an adaptation to extreme environments (7). Archaeal organisms also have flagella that are notably different in composition and development from the flagella of bacteria. Individual archaea cells range from 0.1 to over 15 µm in diameter, and some form aggregates or filaments up to 200 µm in length. They occur in various shapes, such as spherical, rod-shaped, spiral, lobed, or rectangular, and they also exhibit a variety of different types of metabolism. Archaeal and bacterial metabolic genes share common evolutionary aspects (6). However, the transcriptional and translational machinary of Archaea is much more similar to Eucarya than Bacteria (28). For instance, archaean translation uses eukaryotic initiation and elongation factors, and their transcription involves TATA-binding proteins and TFIIB (14). The study of protein synthesis in Archaea led to a much deeper understanding of this process not only in the Archaea, but in all domains of life (8,33,52). Many archaeans are extremophiles, some live at very high temperatures, often above 100ºC, as found in geysers and submarine black smokers. Others are found in very cold habitats, highly saline, acidic, or alkaline water. They are able to live in the anoxic mud of marshes and at the bottom of the ocean, even thrive in petroleum deposits deep underground. However, many archaeans are mesophiles, and have been found in environments like marine plankton (5), sewage, and soil (36). Futhermore, many Figure 1. Carl Woese view at the procariotic tree of life. We methanogenic archaea are found in the digestive tracts of animals reproduced in silico Woese’s experiment by phylogenetic such as ruminants, termites, and humans (15,42). relationships of 16S rDNA sequences from Bacteria and Archaea. We used thirty-five eubacterial and four methanogenic Phylogenetic division archaeal sequences from GenBank database. Phylogenetic and The Archaea domain is divided on the basis of 16S rRNA molecular evolutionary analyses were conducted using MEGA gene sequences into four phylogenetically distinct phylum: (Kumar et al. 2001). Euryarchaeota, Crenarchaeota, Korarchaeota and Nanoarchaeota (Fig. 2). Cultivated crenarchaeotes presents a thermoacidophilic Crenarchaeota, which comprise an important number of phenotype. The term “cren” means spring or fount and expresses uncultured groups from marine plankton, freshwater, and soil the resemblance of this phenotype to the ancestor of the samples (13,33,39) domain Archaea. Most cultured representatives of the The Euryarchaeota phylum, the term “eury” means broad or Crenarchaeota are extreme thermophiles or hyperthermophiles. wide, contains organisms that are highly diverse in their A broad variety of metabolic pathways is evident. Aerobically physiology, morphology and natural habitats. During the last growing chemolithotrophs gain energy by the oxidation of decade, numerous reclassifications within the Euryarchaeota various sulfur compounds, molecular hydrogen or ferrous iron. have been carried out, mainly based on the results of 16S rRNA Anaerobic chemolithotrophs reduce sulfur, thiosulfate or sequence comparisons. Five major groups are known within produce nitrate, hydrogen sulfide or ammonia. Organotrophic this kingdom: the obligate anaerobic methanogens, the extreme growth occurs on complex organic substrates, sugars, amino halophiles, the hyperthermophilic sulfate reducers, the acids or polymers such as starch and cellulose. However, Thermoplasma group, and finally, the Thermococcus- cultivated species appear to represent a minority in Pyrococcus group (28, 35). 399 Cardoso, A.M. et al. Biotechnological Applications The extremophilic nature of many Archaea has stimulated intense efforts to understand the physiological adaptations for living in extremes environments and to probe the potential biotechnological applications of their stable cellular components. This is particularly true of their enzymes (called extremozymes), which are able to remain catalytically active under extremes of temperature, salinity, pH and pressure. Many interesting enzymes have been isolated from extremophilic microbes (41). Specific archaeal metabolites have also been purified and characterized and some of them have potential industrial uses (Table 1). However, several technical difficulties have prevented the large-scale industrial application of enzymes and special metabolites from extremophilic sources, the most important being the availability of these compounds (40). Genes encoding several enzymes from extremophiles have been cloned in mesophilic hosts, with the objective of overproducing the enzyme and altering its properties to suit commercial applications. Escherichia coli, Bacillus subtilis and yeasts have been Figure 2. Phylogenetic tree of 16S rRNA genes from archaea with used successfully as mesophilic hosts for several archaeal genomes representing the four archaeal groups (Nanoarchaeota, genes. Genetic engineering techniques are valuable tools for Korarchaeota, Crenarchaeota, Euryarchaeota). The numbers at the creating novel biocatalysts that can improve bioprocesses