Mining enzymes from extreme environments

Manuel Ferrer1, Olga Golyshina2,3, Ana Beloqui1 and Peter N Golyshin2,3

Current advances in metagenomics have revolutionized the research in elds of microbial ecology and biotechnology, enabling not only a glimpse into the uncultured microbial population and mechanistic understanding of possible biogeochemical cycles and lifestyles of extreme organisms but also the high-throughput discovery of new enzymes for industrial bioconversions. Nowadays, the genetic and enzymatic differences across the gradients from neutral and pristine to extreme and polluted environments are well documented. Yet, extremophilic organisms are possibly the least well understood because our ability to study and understand their metabolic potential has been hampered by our inability to isolate pure cultures. There are at least two obstacles for reaping the fruit of the microbial diversity of extremophiles: rst, in spite of the recent progress in development of new culturing techniques most extremophiles cannot be cultured using traditional culturing technologies; and second, the problem of the very low biomass densities often occurs under the conditions hostile for life, which often do not yield enough DNA and reduces the effectiveness of cloning.
Addresses 1 Division of Applied Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain 2 Division of Microbiology, HZI Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 3 Institute of Microbiology, Technical University of Braunschweig, Biozentrum, 38106 Braunschweig, Germany Corresponding author: Ferrer, Manuel (mferrer@icp.csic.es)

Current Opinion in Microbiology 2007, 10:207214 This review comes from a themed issue on Ecology and industrial microbiology Edited by Victor de Lorenzo and Margaret J McFall-Ngai Available online 4th June 2007 1369-5274/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.05.004

deep under the surface of the ocean, in salty environments and both in environments with high and low pH. The rst sequenced genome of an extremophile was that of Methanococcus jannaschii, a single-celled microbe living near hydrothermal vents, 2600 metres below sea level, where temperatures approach the boiling point of water and the pressure is sufcient to crush a common submarine [2]. Since then, the costs and accuracy of highthroughput sequencing were signicantly improved and we expect to have over 1000 draft genomes in the next couple of years [3], from which, approximately 600 should be prokaryotes and 460 should be eukaryotes [4]. Currently, about 4% of all sequencing projects deal with extremophiles that have industrial applications. Their genome assembly will be of practical importance, as we know that the genomes of extremophilic bacteria and archaea could be even more sophisticated than those from non-extremophiles: Thermotoga, Sulfolobus and Picrophilus species have 15004000 open reading frames (ORFs), comprising more complex biochemistries in terms of unique proteins, metabolites and enzymes (with up to 40% genes coding for hypothetical proteins). The genome sequencing data are currently being used to detect duplicate or missing genes within known pathways, to detect novel differences in metabolic capacities between two or more genomic datasets or to identied extreme-related genes; however, if the nal goal is the identication of extremozymes (the unique biocatalysts produced by extremophiles) we must also improve annotations through empirical work on specic candidates, because in silico prediction tools often fail to suggest the function for a large fraction of predicted proteins [5]. Extremozymes have developed molecular mechanisms of adaptation to extreme physico-chemical conditions [6]. As they are active and/or stable under extreme conditions (i.e. thermophilic, psychrophilic, acidophilic, alkalophilic, halophilic and others) they have a great practical importance for industrial applications [7,8]. In recent years, scientists have begun to mine extremophiles for information that might lead to new technologies, such as heat-resistant molecules for use in healthcare [9]. Although, the valuable enzymatic and genomic information of individual organisms represents a good starting point to dene the repertoire of extreme enzymes and protein structural elements, or folds, and to understand how protein structure relates to function, new and more efcient strategies are needed because the cultivation of microbes, especially those living in extreme environments, often presents a bottleneck [10]. Here, some key contributions are reviewed, current challenges are highlighted and the future of enzyme discovery
Current Opinion in Microbiology 2007, 10:207214

Introduction
Signicant progress in understanding the specic metabolic conversions of living cells has been achieved in the past decades. However, for extremophilic organisms this information is rather scarce. The extremophiles not only survive, but thrive in the harshest environments on earth previously thought to prohibit all forms of life. They are widely distributed organisms in nature and have started to be investigated intensively [1]. Among them, the thermophiles were the rst extremophiles to be discovered, but other extremophiles have been found living in ice,
www.sciencedirect.com

208 Ecology and industrial microbiology

from extreme organisms and their communities is speculated on, focussing on metagenomics.

Extremophile categorization: not all proteins from extreme environments are extremozymes
Extreme environments are physically isolated from other habitats on our planet due to their nature, and often have been for thousands of years. This has resulted in the selection of unusual organisms found there and probably has also prevented their dispersal. These environments are therefore expected to yield novel microbial diversity and unknown cellular gene products with interesting properties and new catalytic activities [9]. One should consider that enzymes from harsh environments can be categorized into two general classes. First, in some environments, for example those characterized by extreme temperature or pressure, the prevailing extreme environmental parameter extends into the intracellular milieu, and both intracellular and extracellular biochemical machineries are therefore exquisitely adapted to such conditions. However, in other environments, for instance those characterized by extremes of pH or salinity/osmolarity, or the presence of organic solvents or high levels of radiation, extremophiles invest considerable metabolic resources in order to exclude or compensate for the extreme parameter, and to create and maintain intracellular conditions more typical of non-extremophiles (mesophiles). Their cytoplasmic conditions are therefore distinct from those of the habitat, and intracellular enzymatic functions are not adapted, when compared to those of the extracellular enzymes, to the prevailing extreme environmental conditions (they are not expected to be extremozymes). The only known exception to this rule is the acidophilic ferrous-oxidizing archaeaon Ferroplasma acidiphilum, the intracellular enzymes of which have been reported to work in an extremely acidic pH [11,12]; recent evidence has shown that this unique biochemical machinery is directly correlated to the incorporation of iron into the protein molecules, thus acting as structural element or as a Lewis acid that decreases the pH optimum [13]. Therefore, these differences should be taken into consideration when trying to mine enzymes from extreme environments or microorganisms. For this reason, the focus here is on the most recent advances in those enzymes showing extremophilicity.

diversity. These approaches provide little information on enzyme diversity but are very important for an estimation of the total number of prokaryotes on Earth, predicted to be about 46 1030 cells, which represents around 108 separate genospecies [10,14]. Recent analysis of few seawaterbased microbial habitats revealed the above gures are actually an underestimate of the overall microbial diversity [15]. Another point is that among the currently known 52 bacterial phyla, half of these lack a single cultivated representative and only few thousands are represented in microbial cultures (among them, 4% being extremophiles) [15,16]. 16S rRNA gene phylogenies of variety of microbial communities analysed these days suggest more new taxa are out there. Recent data suggests that the community composition and diversity can shift greatly in human-perturbed areas, as well as within small vertical and horizontal distances, possibly in response to changes in the extreme physical and chemical conditions [17,18,19].

Protein engineering versus metagenomics

Over 3.3 million non-redundant protein sequences (up to 40% being hypothetical), have so far been predicted and deposited in electronic databases; from this number only 8% correspond to extremophiles. In the past two years, 45 extremozymes have been identied from pure cultures isolated from different extreme environments [2051,52 57] (see details in Table 1). These include thermostable, cold-active, acidophilic, alkalophilic, barophilic and halophilic hydrolases (i.e. galactosidases, cellulases, amylases, esterases, lipases, mannases, arabinofuranosidases, chitinases, xylanases, phosphotriesterase and b-lactamase), lyases, oxidoreductases and laccases, to mention just a few. Although, many of these enzymes have been proven to be useful for chemical synthesis, one still cannot use these for the formation of desired linkage as they still might exhibit some unpredictable features with unnatural substrates and often exhibit low operational stability. To overcome these problems, protein activation and stabilization are traditionally used. For example, using protein engineering and chemical modication screening it is currently possible to create articial extremozymes with altered protein topology, thermal stability and tolerance to organic solvents [22,58]. Additionally, combinatorial immobilization techniques are providing effective methods for optimizing operational performance, in addition to aiding the recovery and re-use of biocatalysts. However, these strategies have a few hindrances, such as the requirement for highly efcient high-throughput screening assays, the large numbers of possible mutant variants and the problems of selection of the best enzyme for the next mutagenesis/panning round, because the accumulation of subtle changes at genetic level do not always lead to the best enzymatic tness. A new chapter in extremozyme discovery is also about to be written as we move from culture-dependent techniques to metagenomics. This approach uses the harvesting
www.sciencedirect.com

Estimates of microbial diversity in extreme and non-extreme microbial communities

The microbial diversity on our planet is mostly comprised by the microorganisms, in terms of both absolute numbers of cells and of the biomass, and the numbers of species. Different culture-independent technologies, both indirect (e.g. DNA-ngerprinting analyses, 16S rRNA gene libraries analyses, quantitative PCR and serial analysis of sequence tags) and direct (enumeration through uorescence in situ hybridisation [FISH] and its variants), have been used to access the immense reservoir of microbial
Current Opinion in Microbiology 2007, 10:207214

Mining enzymes from extreme environments Ferrer et al. 209

Table 1 Extremozymes discovered by cultivation-dependent and independent approaches in the period 20052006 (the extreme environments from which these extremozymes were isolated are shown).
Enzyme pHopt Oligosaccharide-degrading enzymes Trehalase 6.5 Trehalose-6-phosphate synthase 6.07.0 and trehalose-6-phosphate phosphatase b-Galactosidase 7.0 b-Galactosidase 6.0 b-Galactosidase 6.8 b-Galactosidase 4.0 b-Glycosidase 5.05.5 b-1,3-Glucanase 9.0 2 a-Glucosidases 2.54.0 a-Glucosidase 2.53.0 Mannase 10.0 Alpha-L-arabinofuranosidase 7.0 Cellulase Cellulase Cellulase Endoglucanase 9 Endoglucanases a-Amylase a-Amylase Xylanase Feather-degrading enzyme Family 18 chitinase Cyclodextrinase Ester hydrolases and proteases Cholesterol esterase Esterase 12 Esterases Esterase Esterase Esterase 5 Esterases Benzoyl tartrate esterase Phosphotriesterase Lipase TEM-b-lactamase Protease Miscellaneous Homoserine transsuccinylase Oligopeptide-binding protein DNA-binding protein Gellan lyase Isocitrate dehydrogenase L-Threonine dehydrogenase Dihydrofolate reductase 3 Sulfur oxygenase reductases Protein-disulde oxidoreductase Sulte oxidase Catalase Polyphenol oxidase Laccase Ferritin Sec traslocase (ATPase) DnaK protein Nitrate reductase Malate dehydrogenase 6.07.0 5.59.0 5.6 7.0 3.011.0 6.0 7.0 6.0 8.810.3 4.55.0 5.59.0 12.0 10.5 7.011.0 8.0 1.5 6.0 8.09.0 7.5 8.0 8.0 8.0 6.08.8 8.0 7.0 8.0 10.0 7.38.8 8.0 11.0 7.5 3.59.5 5.0 7.0 8.0 7.5 7.5 4.09.0 Properties Topt (8C) 88 100 Other Rhodothermus marinus Thermus thermophilus RQ-1 Shallow marine hot springs Hot springs [20] [21] Source Environment Refs

1015 15 520 010 85 70 60 60 55 100 40 40 100 95 4060 50 50 1520 60 95 75 45 55 60 50 50 95 4867 030 100 20 50 70 90 90 70 20 100 53 7580 100 60 010 60 92 120 74 1530 1525 1040

Fe-protein Fe-protein SDS-tolerant Urea-tolerant Organic solvent tolerant Organic solvent tolerant Organic solvent tolerant First conversion: esterase-lipase Fe-protein Tolerant to 4M salts and 40 MPa Urea-tolerant New copper ligands

Arthrobacter sp. SB Arthrobacter sp. C2-2 Planococcus sp. L4 Guehomyces pullulans Alicyclobacillus acidocaldarius Nocardiopsis sp. strain F96 Ferroplasma acidiphilum Ferroplasma acidiphilum Bacillus sp. strain JAMB-750 Thermotoga maritima MSB8 Pseudoalteromonas sp. DY3 Soil metagenome Pyrocuccus horikoshii Pyrococcus horikoshii Cow rumen metagenome Haloarcula hispanica Haloarcula sp. strain S-1 Environmental DNA library Bacillus pseudormus FA30-01 Rhodothermus marinus Cow rumen metagenome Burkholderia cepacia strain ST-200 Deep-sea sediment metagenome Cow rumen metagenome Cow rumen metagenome Ferroplasma acidiphilum Mud and sediment-rich water metagenome Deep-sea metagenome Rhodotorula mucilaginosa Sulfolobus solfataricus Pseudoalteromonas haloplanktis Cool-seep sediment metagenome Natrinema sp. J7 Thermotoga maritima Aeropyrum pernix Deinococcus radiopugnas Geobacillus stearothermophilus 98 Colwellia psychrerythraea Pyrococcus horikoshii Alkaliphilic and halotolerant bacillus strain Gold-bearing metagenome Pyrococcus horikoshii Thermus thermophilus Vibrio salmonicida Cow rumen metagenome Thermus thermophilus Pyrococcus furiosus Thermotoga maritima Shewanella sp. Ac10 Koliella antarctica Flavobacterium frigidimaris KUC-1

Antarctic sea water Antarctic sea water Supercial saline soils Antarctic sea water Acidic creek Soil sample Acid mine drainage, acidic pools Acid mine drainage, acidic pools Alkaliphilic environments Geothermally heated marine sediments Deep-sea sediments Soil Hydrothermal uid Hydrothermal uid Animal microora Hypersaline waters Brine pool Flat sediments Poultry farm soil Shallow marine hot springs Animal microora Soil Deep-sea sediments Animal microora Animal microora Acid mine drainage, acidic pools Mud sediments Deep-sea water Sewage treatment plant Acidic hot spring Antarctic sea water Cool-seep sediments Salt mine Geothermally heated marine sediments Hot springs Radiated samples Hot water samples Arctic marine sediments Hydrothermal uid Alkaliphilic and halophilic samples Gold-bearing concentrates Hydrothermal uid Hot springs Fish (cod) microora Animal microora Hot springs Hot springs Geothermally heated marine sediments Antarctic sea water Antarctic sea water Antarctic soil

[22] [23] [52] [53] [26] [27] [11] [12] [28] [29] [30] [61] [31] [32] [62] [33] [34] [63] [35] [36] [59] [37] [64] [65] [65] [11] [66] [67] [55] [39] [56] [68] [41] [42] [43] [44] [45] [46] [47] [48] [69] [49] [50] [51] [70] [52] [53] [54] [55] [56] [51]

www.sciencedirect.com

Current Opinion in Microbiology 2007, 10:207214

210 Ecology and industrial microbiology

of DNA from an environmental sample (or from an enrichment), its archiving in the metagenomic libraries in appropriate hosts, and subsequent screening of these libraries for a gene of interest [10]. Metagenomics constitutes a challenging research domain with great potential for both fundamental and industrial applications that range from the understanding of microbial adaptation and evolution to the discovery of new enzymes for their direct use [14,59,60]. Following this technology, extreme environments such as deep sea water, the Arctic, goldbearing ores, rumen, worm guts and so on, have been explored during the past two years in order to isolate about 36 new extremozymes [61,62,63,64,65,66,67,68,69,70] (see examples in Table 1). Their analysis has revealed some interesting facts. For example, through a metagenomic approach, a novel esterase (O.16) with one of the highest levels of structural and functional complexity known to date has been isolated and characterized from the deep sea hypersaline anoxic basins (DHABs) of the Eastern Mediterranean Sea [67]. This enzyme has been shown to be the rst hydrolase to possess three catalytic triads composed of Ser, His and Asp with the active-site serine embedded in consensus sequence motifs: Gly-XSer-X-Gly or Gly-Asp-Ser-Leu (X denotes any amino acid). Therefore, one can suggest that if this enzyme is a proxy of other enzymes and other metabolic activities in the DHABs, then considerable new microbial diversity and novel biological activities and mechanisms might still be discovered in these fascinating habitats. Further, the combination of metagenomics and enzyme evolution has successfully been applied to the discovery of the rst lipase acting in the conversion of the sn-2 positions of triglycerides [65]. This unusual and rare conversion might open new opportunities for the creation of nutritional lipids containing essential fatty acids. Recently, it has also been reported that the retrieval from a metagenome expression library of a bovine rumen, an anaerobic environment, of a new polyphenol oxidase with laccase activity [70]. This laccase is unusual in two respects. Firstly, it lacks any sequence relatedness to the known laccases but shares some with hypothetical proteins (DUF152) which are conserved in bacteria and which comprise about 750 database entries. The study has thus revealed the function of the proteins of this family; in addition to the protein from the metagenome, two proteins from Bacteroides and Escherichia coli were characterized suggesting that DUF152 is a new laccase family. Secondly, the characterised protein exhibits much higher activity and substrate afnities than previously described laccases. These were just recent examples of the potential of metagenome technology when applied to extreme environments for giving new biomolecules (other good examples are shown in Table 1). Although exhibiting interesting properties, the number of extremozymes discovered by metagenome mining is still rather low, especially considering the potential of this technology for nding new activities. In this respect
Current Opinion in Microbiology 2007, 10:207214

one should also be aware of certain difculties that are related to efcient DNA and RNA extraction, cloning and gene expression in appropriate hosts. Over the past two years few alternatives were explored to overcome these limitations.

Extreme environments as a source of metagenome libraries: challenges and bottlenecks

One of the main challenges for mining enzymes from extreme environments is not only to develop a robust screening or selection system to select for specic extremozymes, but also to overcome the problem of the low biomass yields and very low cell numbers that hinder high yields of DNA for cloning [17]. To solve this problem, new methods are required to establish libraries for metagenome analysis of low cell-density extreme environments that can further be subjected to high-throughput screening. Recent examples, which can be applied to samples from harsh environments, are based on the combination of environmental whole genome amplication (WGA) with library construction for metagenome analysis of contaminated low cell-density environments. Using multiple displacement amplication (MDA) with f29 DNA polymerase, it was amplied up to 24 ng of sufcient quality DNA from an initial amount of 0.4 ng, which is equivalent to the DNA from approximately 105 cells [17,71]. Even though this technique is biased, yields rather short DNA fragments and often produces articial sequences, this method can be considered as being able to deal with rare and very dilute DNA samples for consequent PCR typing. Although the common E. coli expression systems have been predicted to be able to successfully express up to 40% of genes with sequences that are available in the public databases [72], and other alternative hosts for library construction and screening with different expression capabilities are currently under development (Bacillus subtilis, Pseudomonas putida, Bacillus species [Firmicutes], Streptomyces lividans [Actinobacteria] or Rhizobium leguminosarum [Alphaproteobacteria]) [73], many decits still remain in the expression of, for instance, metagenomes derived from archaea-dominated communities. This makes the choice of a suitable heterologous expression host crucial. To overcome these limitations, many future efforts should be focussed on the generation of metagenome libraries based on a broader host range vectors, thus making it possible to clone in extremophilic organisms such as Thermus thermophilus or Sulfolobus sulfataricus, and consequently this should help to optimize the gene expression. This technology should be developed in parallel with improving direct and fast systems for identication of extremozymes. Recently, a screening system using a unique direct selection technique, a technology that enables the identication of thermostable mutants in T. thermo thodes (Evry, France; philus has been developed by Biome
www.sciencedirect.com

Mining enzymes from extreme environments Ferrer et al. 211

http://www.biomethodes.com). This system relies on a thermophilic microorganism, enabling plate selection of thermostable mutants after overnight cultivation, being potentially applicable in metagenome library screening for enzymes with improved thermal activity and/or stability. The approaches based on the above technologies could ultimately become common in future metagenome screening trials and will shorten the way to directly retrieve enzymes of interest. Hosts for psychrophilic gene cloning and expression are less understood. However, the recent discovery of a single protein (Cpn60) isolated from and Antarctic bacterium, Oleispira antarctica, that radically expands the low temperature growth limit of E. coli [74], might enable future development for metagenome library construction and screening at temperatures as low as 10 8C. In fact, a collection of vector derivatives ArcticExpress, containing cpn genes have been established and developed into a potent tool to produce stable recombinant proteins from psychrophilic organisms (technology is patented and currently used by Stratagene Ltd to produce gene expression cell lines; http://www.stratagene.com). Finally, recently it has been reported that systematic screening of signal peptides should be of practical importance when expressing DNA fragments of metagenome extreme origin where new protein secretion pathways could be found [75]. Finally, it should be noted that high-throughput sequencing which is becoming even more advanced with the broader use of new techniques [76] has revolutionized the study of microbial community metagenomes [77]. There are certain limitations and difculties in the use of high-throughput sequencing and gene prediction for extremozyme discovery not only because of difculties with genome assembly and other challenges in interpretation of large datasets but also because our inability to predict in silico the extremophilic features in many known annotated protein families. It would therefore require a combination of thorough annotation and wetlab work in order to reveal the structuralfunctional characteristics of the new enzymes. Therefore, a function-based screen for a particular conversion seems to be the best option for looking for new enzymes and corresponding reactions. For this reason, the identication of functional motifs in extremozymes by sequence comparison and the use of these motifs for PCR detection or gene probing in DNA libraries might not necessarily lead to the discovery of unusual extremozymes with novel structural and functional features. More importantly, the search for genes and/or enzymes could be based on easy-to-use colorimetric assays that can be handled in automated facilities; for that, sensitive highthroughput screening methods to test vast numbers of clones for enzymes for specic applications have to be established. Whatever the case, it is important to mention that both the functional-based and sequence-based approaches for discovered enzymes should rely not only
www.sciencedirect.com

on appropriate agar-based colorimetric assays that combine many substrates onto a single plate, but also on liquid high-throughput screening facilities. In this case, future advances should focus on designing cell-free systems with a double aim: to increase substrate bioavailability and to reduce inhibition and cross reactivity of cell components. Finally, in order to facilitate the discovery of unknown extreme genes, the should be further efforts towards creating new high resolution molecule arrays capable of binding with functional-like properties of specicity and afnity; these can be used on capture arrays in a similar fashion to antibodies and might have advantages for enzyme mining.

Conclusions
This review is an attempt to draw the readers attention to the ongoing research on extremophilic organisms and their communities as promising sources for enzymes with unusual properties. Environmental genomics helps us to understand the mechanisms and functioning of microbial communities and ecosystems in extreme environments and the biology of individual constituents of these communities, and it sheds light on the unknown microbial diversity and provides a census of the genes and proteins that could be useful for practical applications. Activitybased discovery in extreme environments of novel enzymes that are not related to any previously known proteins with the same function, or that exhibit unique structural features, or belong to the families of proteins with unknown and/or unclear function and thus dene their function, marks the importance of these environments for enzyme mining through a metagenomic approach. This approach is extremely important for the development of several new products and to provide the tools for the resolution of conversions which are not amenable to the existing enzymes or chemical synthesis. Obviously, to be prepared for these challenges for enzymatic/chemical conversions we have to establish new screening techniques for corresponding reactions and, nally, put more emphasis on the experimental functional and structural characterisation of these new proteins.

Update
It has recently been reported about the generalities and specializations in adaptation to extreme conditions by the thermoacidophilic enzymes from the unicellular red algae Galdieria sulphuraria (Cyanidiaceae), as one example of a eukaryotic extremophile [78]. Also, it has been shown that the gradients of physicochemical factors inuence both the growth and survival of life and enzyme biochemistries in deep-sea environments [79].

Acknowledgements
This research was supported by Spanish and European Community Projects 200680I126, BIO2006-11738, EVK3-2000-00042 BIODEEP, EVK3-2002-00077 COMMODE and MERG-CT-2004-505242 BIOMELI and MetaGenoMik Project supported by the Federal Ministery for Science and Education (BMBF). AB thanks the Spanish a for a FPU fellowship. Ministerio de Ciencia y Tecnolog Current Opinion in Microbiology 2007, 10:207214

212 Ecology and industrial microbiology

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Javaux EJ: Extreme life on Earth past, present and possibly  beyond. Res Microbiol 2006, 157:37-48. An interesting overview of the impact of microbial diversity on the Earths environments and possible implications for astrobiological research. 2. Bult CJ, White O, Olsen GJ, Zhou L, Fleischmann RD, Sutton GG, Blake JA, FitzGerald LM, Clayton RA, Gocayne JD et al.: Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii. Science 1996, 273:1058-1073.

the underexplored rare biosphere. Proc Natl Acad Sci USA 2006, 103:12115-12120. Authors reported the remarkable diversity of microbial communities of deep seawater and hydrothermal vents which turned out to be two orders of magnitude more complex than expected, or previously estimated for any microbial environment. This is a landmark publication suggesting the current gures on microbial diversity of even such simple and dilute environments are rather incorrect and underestimate the overall biological diversity of the biosphere. 16. Ginolhac A, Jarrin C, Gillet B, Robe P, Pujic P, Tuphile K, ` re G, Simonet P, Nalin R: Bertrand H, Vogel TM, Pierrie Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol 2004, 70:5522-5527. a JA, Podar M, Chen W, 17. Abulencia CB, Wyborski DL, Garc Chang SH, Chang HW, Watson D, Brodie EL, Hazen TC, Keller M: Environmental whole-genome amplication to access microbial populations in contaminated sediments. Appl Environ Microbiol 2006, 72:3291-3301. 18. Reed AJ, Lutz RA, Vetriani C: Vertical distribution and diversity  of bacteria and archaea in sulphide and methane-rich cold seep sediments located at the base of the Florida Escarpment. Extremophiles 2006, 10:199-211. A study that examined the bacterial and archaeal communities by using molecular phylogenetic analysis at three vertical zones of cold seep sediments of the Florida Escarpment. 19. Prokofeva MI, Kublanov IV, Nercessian O, Tourova TP,  Kolganova TV, Lebedinsky AV, Bonch-Osmolovskaya EA, Spring S, Jeanthon C: Cultivated anaerobic acidiphilic/ acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats. Extremophiles 2005, 9:437-448. Fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats by different acidophilic or acidotolerant thermophilic prokaryotes. 20. Jorge CD, Sampaio MM, Hreggvidsson GO, Kristjanson JK, Santos H: A highly thermostable trehalase from the thermophilic bacterium Rhodothermus marinus. Extremophiles 2007, 11:115-122. 21. Silva Z, Alarico S, da Costa MS: Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Extremophiles 2005, 9:29-36. 22. Coker JA, Brenchley JE: Protein engineering of a cold-active beta-galactosidase from Arthrobacter sp. SB to increase lactose hydrolysis reveals new sites affecting low temperature. Extremophiles 2006, 10:515-524. 23. Skalova T, Dohnalek J, Spiwok V, Lipovova P, Vondrackova E, Petrokova H, Duskova J, Strnad H, Kralova B, Hasek J: Coldactive beta-galactosidase from Arthrobacter sp C2-2 forms compact 660 kDa hexamers: crystal structure at 1.9 A resultion. J Mol Biol 2005, 353:282-294. 24. Lauro BD, Rossi M, Moracci M: Characterization of a betaglycosidase from the thermophilic bacterium Alicyclobacillus acidocardarius. Extremophiles 2006, 10:301-310. 25. Masuda S, Endo K, Koizumi N, Hayami T, Fukazawa T, Yatsunami R, Fukui T, Nakamura S: Molecular identication of a novel beta-1,3-glucanse from alkaliphilic Nocardiopsis sp. strain F96. Extremophiles 2006, 10:251-255. 26. Hatada Y, Takeda N, Hirasawa K, Ohta Y, Usami R, Yoshida Y, Grant WD, Ito S, Horikoshi K: Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme. Extremophiles 2005, 9:497-500. 27. Miyazaki K: Hyperthermophilic alpha-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression and characterization of the recombinant protein. Extremophiles 2005, 9:399-406. 28. Zeng R, Xiong P, Wen J: Characterization and gene cloning of a cold-active cellulase from a deep-sea psychrophilic bacterium Pseudoalteromonas sp. DY3. Extremophiles 2006, 10:79-82. www.sciencedirect.com

3. 

Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crecy-Legard V, Diaz N, Disz T, Edwards R et al.: The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 2005, 33:5691-5702. The new approach for the annotation of genomes, where experts annotate single subsystems over the complete collection of genomes, rather than having an annotation expert attempt to annotate all of the genes in a single genome. 4. ller R: The impact of bacterial genomics on Bode HB, Mu natural product research. Angew Chem Int Ed Engl 2005, 44:6828-6846. Ye Y, Osterman A, Overbeek R, Godzik A: Automatic detection of subsystem/pathway variants in genome analysis. Bioinformatics 2005, 21:i478-i486.

5.

6. 

Tehei M, Zaccai G: Adaptation to extreme environments: macromolecular dynamics in complex systems. Biochim Biophys Acta 2005, 1724:404-410. A solid review focussing on the molecular mechanisms of adaptation to extreme physico-chemical conditions, and in particular, providing tools to quantify the macromolecular adaptations to extreme temperatures. 7. Egorova K, Antranikian G: Industrial relevance of thermophilic Archaea. Curr Opin Microbiol 2005, 8:649-655.

8. 

Podar M, Reysenbach A-L: New opportunities revealed by technological explorations of extremophiles. Curr Opin Biotechnol 2006, 17:250-255. An interesting summary on genomics and metagenomics data emphasizing a few points on the importance of extremophiles for biotechnology.

9. 

Antranikian G, Vorgias CE, Bertoldo C: Extreme environments as a resource for microorganisms and novel biocatalysts. Adv Biochem Eng Biotechnol 2005, 96:219-262. Here the authors provide a thorough overview of the physiology, metabolism, enzymology and genetic properties of the extremophilic microorganisms. 10. Green BD, Keller M: Capturing the uncultivated majority. Curr Opin Biotechnol 2006, 17:236-240.

11. Golyshina OV, Golyshin PN, Timmis KN, Ferrer M: The pH  optimum anomaly of intracellular enzymes of Ferroplasma acidiphilum. Environ Microbiol 2006, 8:416-425. The study that discovered the cytosolic enzymes as acidic extremozymes. 12. Ferrer M, Golyshina OV, Plou FJ, Timmis KN, Golyshin PN: A novel alpha-glucosidase from the acidophilic archaeon Ferroplasma acidiphilum strain Y with high transglycosylation activity and an unusual catalytic nucleophile. Biochem J 2005, 91:269-276. 13. Ferrer M, Golyshina OV, Beloqui A, Golyshin PN, Timmis KN: The  cellular machinery of Ferroplasma acidiphilum is iron-proteindominated. Nature 2007, 445:91-94. The rst organism to possess an iron-protein dominated enzyme repertoire. 14. Deutschbauer AM, Chivian D, Arkin AP: Genomics for environmental microbiology. Curr Opin Biotechnol 2006, 17:229-235. 15. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR,  Arrieta JM, Herndl GJ: Microbial diversity in the deep sea and Current Opinion in Microbiology 2007, 10:207214

Mining enzymes from extreme environments Ferrer et al. 213

29. Kang HJ, Vegat K, Fukada H, Ishikawa K: Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii. Extremopiles 2006, 11:251-256. 30. Kashima Y, Mori K, Fukada H, Ishikawa K: Analysis of the function of a hyperthermophilic endoglucase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose. Extremophiles 2005, 9:37-43. 31. Hutcheon GW, Vasisht N, Bolhuis A: Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 2005, 9:487-495. 32. Fukushima T, Muzuki T, Echigo A, Inoue A, Usami R: Organic solvent tolerance of halophilic alpha-amylase from a haloarchaeon, Haloarcula sp. strain S-1. Extremophiles 2005, 9:85-89. 33. Kojima M, Kanai M, Tominaga M, Kitazume S, Inoube A, Horikoshi K: Isolation and characterization of a featherdegrading enzyme from Bacillus pseudormus FA30-01. Extremophiles 2006, 10:229-235. 34. Hobel CF, Hreggvidsson GO, Marteinsson VT, Bahrani-Mougeot F, Einarsson JM, Kristjansson JK: Cloning, expression and characterization of a highly thermostable family 18 chitinase from Rhocothermus marinus. Extremophiles 2005, 9:53-64. 35. Takeda Y, Aono R, Doukyu N: Purication, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200. Extremophiles 2006, 10:269-277. 36. Merone L, Mandrich L, Rossi M, Manco G: A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties. Extremophiles 2005, 9:297-305. 37. Shi W, Tang XF, Huang Y, Gan F, Tang B, Shen P: An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. 17: gene cloning, expression and characterization. Extremophiles 2006, 10:599-606. 38. Goudarzi M, Born TL: Purication and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase. Extremophiles 2006, 10:469-478. 39. Palmieri G, Casbarra A, Fiume I, Catara G, Capasso A, Marino G, Onesti S, Rossi M: Identication of the rst archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix. Extremophiles 2006, 10:393-402. 40. Filipkowski P, Koziatek M, Kur J: A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans. Extremophiles 2006, 10:607-614. 41. Derekova A, Sjoholm C, Mandeva R, Michailova L, Kambourova M: Biosynthesis of a thermostable gellan lyase by newly isolated and characterized strain of Geobacillus stearothermophilus 98. Extremophiles 2006, 10:321-326. 42. Maki S, Yoneta M, Takada Y: Two isocitrate dehydrogenases from a psychrophilic bacterium, Colwellia psychrerythraea. Extremophiles 2006, 10:237-249. 43. Shimizu Y, Sakuraba H, Kawakami R, Goda S, Kawarabayasi Y, Ohsima T: L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization. Extremophiles 2005, 9:317-324. 44. Redecke L, Brehm MA, Bredehorst R: Cloning and characterization of dihydrofolate reductase from a facultative alkaliphilic and halotolerant bacillus strain. Extremophiles 2007, 11:75-83. 45. Kuroita T, Kanno T, Kawai A, Kawakami B, Oka M, Endo Y, Tozawa Y: Functional similarities of a thermostable protein-disulde oxidoreductase identied in the archaeon Pyrococcus horikoshii to bacterial DsbA enzyme. Extremophiles 2007, 11:85-94. 46. Di Salle A, DErrico G, La Cara F, Cannio R, Rossi M: A novel thermostable sulite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli. Extremophiles 2006, 10:587-598. www.sciencedirect.com

47. Lorentzen MS, Moe E, Jouve HM, Willansen NP: Cold-adapted features of Vibrio salmonicida catalase: characterization and comparison with the mesophilic counterpart from Proteus mirabilis. Extremophiles 2006, 10:427-440. 48. Miyazaki K: A hyperthermophilic laccase from Thermus thermophilus HB27. Extremophiles 2005, 9:415-425. 49. Tatur J, Hagedoorn PL, Overeijnder ML, Hagen WR: A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus. Extremophiles 2006, 10:139-148. 50. Pretz MG, Remigy H, Swaving J, Albers SV, Garrido VG, Chami M, Engel A, Driessen AJ: Functional and structural characterization of the minimal Sec translocase of the hyperthermophile Thermotoga maritima. Extremophiles 2005, 9:307-316. 51. Yoshimune K, Galkin A, Kulakova L, Yoshimura T, Esaki N: Coldactive DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold-temperatures. Extremophiles 2005, 9:145-150. 52. Hu JM, Li H, Cao LX, Wu PC, Zhang CT, Sang SL, Zhang XY, Chen MJ, Lu JQ, Liu YH: Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychrophilic and halotolerant Planococcus sp. L4. J Agric Food Chem 2007, 55:2217-2224. 53. Nakagawa T, Ikehata R, Uchino M, Miyaji T, Takano K, Tomizuka N: Cold-active acid beta-galactosidase activity of isolated psychrophilic basidiomycetous. Microbiol Res 2006, 161:75-79. 54. di Rigano VM, Vona V, Lobosco O, Carillo P, Lunn JE, Carfagna S, Esposito S, Caiazzo M, Rigano C: Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana. Plant Cell Environ 2006, 29:1400-14009. 55. Zimmer C, Platz T, Cadez N, Giffhorn F, Kohring GW: A cold active (2S,3R)-(-)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa. Appl Microbiol Biotechnol 2006, 73:132-140. 56. Luo Y, Zheng Y, Jiang Z, Ma Y, Wei D: A novel psychrophilic lipase from Pseudomonas uorescens with unique property in chiral resolution and biodiesel production via transesterication. Appl Microbiol Biotechnol 2006, 73:349-355. 57. Oikawa T, Yamamoto N, Shimoke K, Uesato S, Ikeuchi T, Fukioka T: Purication, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel Antarctic psychrotolerant, Flavobacterium frigidimarinis KUC-1. Biosci Biotechnol Biochem 2005, 69:2146-2154. 58. Siddiqui KS, Cavicchioli R: Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modication with oxidized polysaccharides. Extremophiles 2005, 9:471-476. nez-Abarca F, Golyshin PN: Genome and 59. Ferrer M, Mart metagenome minining for novel catalysts. Curr Opin Biotechnol 2005, 16:588-593. 60. Lorenz P, Eck J: Metagenomics and industrial applications. Nat  Rev Microbiol 2005, 3:510-516. A thorough review describing examples of gene expression in different host for metagenome library construction as well as for understanding the potential of this technology. 61. Voget S, Steele HL, Streit WR: Characterization of a metagenome-derived halotolerant cellulose. J Biotechnol 2006, 126:26-36. 62. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A et al.: Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microora. Environ Microbiol 2005, 7:1996-2010. A thorough example of gene mining from animal microora. In this paper, few dozen of hydrolase genes were retrieved from bovine rumen metagenome, few of which showed extremophilicity. Other studies have examined extreme bacterial and archaeal communities of animalhost Current Opinion in Microbiology 2007, 10:207214

214 Ecology and industrial microbiology

habitats. On this topic, the rst barophile was found in the gut of an amphipod and few studies have found that gut environments often present extreme habitats. For example, stomachs are regions of low pH and insect hindguts can be regions of high (>12) pH. Thus, one might expect that these environments would present many opportunities for the discovery of enzymes adapted to extreme conditions. 63. Lee MH, Lee CH, Oh TK, Song JK, Yoon JH: Isolation and characterization of a novel lipase from a metagenomic library of tidal at sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol 2006, 72:7406-7409. 64. Park HJ, Jeon JH, Kang SG, Lee JH, Lee SA, Kim HK: Functional expression and refolding of a new alkaline esterase, EM28L8 from deep-sea sediment meagenome. Protein Expr Purif 2007, 52:340-347. 65. Reyes-Duarte D, Polaina J, Lopez-Cortes N, Alcalde M, Plou FJ,  Elborough K, Ballesteros A, Timmis KN, Golyshin PN, Ferrer M: n of a carboxylesterase into a triacylglycerol Conversio lipase by a random mutation. Angew Chem Int Ed Engl 2005, 44:7553-7557. First example of the conversion by protein engineering of a true carboxylesterase mined from cow rumen metagenome into a true triacylglycerol lipase without modication of the shape and active site. 66. Rhee JK, Ahn DG, Kim YG, Oh JW: New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol 2005, 71:817-825. 67. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN,  Martins Dos Santos VA, Yakimov MM, Timmis KN, Golyshin PN: Novel microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin. Chem Biol 2005, 12:895-904. First enzymatic analysis of a metagenome library constructed from DHABs of Mediterranean Sea. The study demonstrated the structuralfunctional changes of the enzymes along the changes in ionic strength, oxygen concentration and hydrostatic pressure. 68. Song JS, Jeon JH, Lee JH, Jeong SH, Jeong BC, Kim SJ, Lee JH, Lee SH: Molecular characterization of TEM-type b-lactamases identied in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea). J Microbiol 2005, 43:172-178. 69. Chen ZW, Liu Y-Y, Wu J-F, She Q, Jiang C-Y, Liu S-J: Novel bacterial sulphur oxygenase reductases from bioreactors

treating gold-bearing concentrates. Appl Microbiol Biotechnol 2007, 74:688-698. 70. Beloqui A, Pita M, Polaina J, Martinez-Arias A, Golyshina OV,  Zumarraga M, Yakimov MM, Garcia-Arellano H, Alcalde M, Fernandez VM et al.: Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis and phylogenetic relationships. J Biol Chem 2006, 281:22933-22942. First discovered and fully characterized polyphenol oxidase with laccaselike activity by metagenome approach. The study has revealed the function of a large family of conserved hypothetical bacterial proteins. 71. Yokouchi H, Fukuoka Y, Mukoyama D, Calugay R, Takeyama H, Matsunaga T: Whole-metagenome amplication of a microbial community associated with scleractinian coral by multiple displacement amplication using w29 polymerase. Environ Microbiol 2006, 8:1155-1163. 72. Gabor EM, Alkema WB, Janssen DB: Quantifying the accessibility of the metagenome by random expression cloning techniques. Environ Microbiol 2004, 6:879-886. 73. Wexler M, Bond PL, Richardson DJ, Johnston AWB: A wide range-host metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase. Environ Microbiol 2005, 7:1917-1926. 74. Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN: Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol 2003, 21:1266-1267. 75. Brockermeier U, Casters M, Freudl R, Jockwer A, Noll T, Eggert T: Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol 2006, 362:393-402. 76. Metzker ML: Emerging technologies in DNA sequencing. Genome Res 2005, 15:1767-1776. 77. DeLong EF: Microbial community genomics in the ocean. Nat Rev Microbiol 2005, 3:459-469. 78. Weber AP, Horst RJ, Barbier GG, Oesterhelt C: Metabolism and metabolomics of eukaryotes living under extreme conditions. Int Rev Cytol 2007, 256:1-34. 79. Lauro FM, Bartlett DH: Prokaryotic lifestyles in deep sea habitats. Extremophiles 2007, doi: 10.1007/s00792-006-0059-5.

Current Opinion in Microbiology 2007, 10:207214

www.sciencedirect.com