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Extremely thermophilic microorganisms have been sources of thermostable and thermoactive enzymes for over 30 years. However, information and insights gained from genome sequences, in conjunction with new tools for molecular genetics, have opened up exciting new possibilities for biotechnological opportunities based on extreme thermophiles that go beyond single-step biotransformations. Although the pace for discovering novel microorganisms has slowed over the past two decades, genome sequence data have provided clues to novel biomolecules and metabolic pathways, which can be mined for a range of new applications. Furthermore, recent advances in molecular genetics for extreme thermophiles have made metabolic engineering for high temperature applications a reality.
Microbial life in terrestrial hot springs, such as those present in Yellowstone National Park (USA), has been observed and studied in earnest since at least as early as the 1960s [1-3]. However, the discovery of microbial life in deep sea thermal vents and shallow marine seeps in volcanic regions of the world in the late 1970s and early 1980s [4-6] led to the realization that so the so called extreme thermophiles (Topt ≥ 70°C) were more phylogenetically, physiologically and geographically diverse than first thought. Many novel genera and species, both archaea and bacteria, were isolated and described, including several that became model microorganisms because of their interesting metabolic features and relative ease of cultivation in laboratory settings (see Table 1). Three archaea, Pyrococcus furiosus, a marine fermentative anaerobe (Topt 98-100°C) [5,7], Thermococcus kodakaerensis (Topt 85°C) also a marine fermentative anaerobe [8,9], and Sulfolobus solfataricus, a terrestrial heterotrophic acidophilic aerobe (Topt 80°C, pHopt 3.5) [10,11], and one bacterium, Thermotoga maritima, a marine fermentative anaerobic bacterium (Topt 80°C) [6,12,13], became the focus of most fundamental and biotechnological studies addressing life at high temperatures. In fact, much of what is known about extreme thermophile physiology and enzymology to date has been based on the study of these “model” microorganisms (sub-classified as hyperthermophiles because of their Topt ≥ 80°C). Recently, as discussed below, genetic systems have been established for the three archaea, further enhancing their value as model systems.
Difficulties in isolating extreme thermophiles because of the harsh and often inaccessible environments from which they come, and subsequently cultivating these microorganisms in laboratory settings, initially presented significant challenges to their study and, consequently, to associated biotechnological applications. In the early 1990s, however, successful attempts to clone and express genes from hyperthermophiles in mesophilic recombinant hosts (e.g., Escherchia coli) facilitated efforts to produce specific enzymes for characterization and application [14,15]. Furthermore, since 1995, when the genome sequence of the hyperthermophile Methano(caldo)coccus jannaschii was reported , related efforts for many high temperature microorganisms have enabled and accelerated projects to not only identify promising biocatalysts for single-step biotransformations but also to discover metabolic pathways, cellular features, and biological phenomena that are relevant to biotechnology. Now, armed with virtually unlimited access to genome sequence data, and aided by molecular genetics and ‘omics’ tools, the prospects for biotechnology at elevated temperatures have never been more promising, and poised to go beyond single-step biocatalysts (i.e., the use of a single enzyme for a single biotransformation).
For the most part, the isolation of many currently known and studied extremely thermophilic microorganisms happened primarily in the late 1970s through the early ‘90s [17,18], although isolation of several Sulfolobus species was reported in the early 1970s . Description of new isolates continues to appear in the literature, but most often reside within genera of previously studied extreme thermophiles. As such, discovery of significantly different extremely thermophilic genera and species is becoming rare. Coupled to this is the fact that the criteria for designating a new isolate as “novel” have become more stringent, given the availability of genome sequences and related quantitative measures for differentiating among microorganisms. Thus, new reports of truly novel extreme thermophiles based on more than marginal differences in 16S rRNA phylogeny and subtle variations in growth physiology are infrequent.
Nonetheless, interesting isolates continue to be reported. In the last several years, several new fermentative anaerobes have been isolated (Table 2). For example, Acidilobus saccharovorans [20,21], a terrestrial thermoacidophiic crenarchaeon (Topt 80-85°C, pHopt 3.5-4) contributes to closure of the anaerobic carbon cycle in terrestrial hot springs by complete oxidation of organic compounds (acetate, ethanol, and lactate). Also, Pyrococcus yayanosii is one of only a handful of microbes (and the only Pyrococcus species) shown to be an obligate piezophile (Topt 98°C, Popt 52 MPa); growth was not observed at atmospheric pressure, but rather between 20 and 120 MPa .
One promising approach to the isolation of novel microorganisms from high temperature environments is to consider mixed cultures and consortia. For example, a three-species archaeal consortium, capable of deconstructing crystalline cellulose, was recently described . This consortium, with members related to archaea from the genera Ignisphaera, Thermofilum, and Pyrobaculum, was able to partially dissolve filter paper after incubating for 30 days. In other cases, inter-species interactions are critical for isolation of new extreme thermophiles. Along these lines, the discovery of nanoarchaea was reported, occurring as parasitic partners with the extremely thermophilic archaeon, Ignicoccus hospitalis . Evidence from genome sequence data of lateral gene transfer between Nanoarchaeum equitans and I. hospitalis was linked to how these two microorganisms adapted to growth by sulfur-H2 respiration coupled to inorganic carbon and nitrogen fixation. As DNA sequencing rates increase and costs continue to decrease, metagenomes from thermal environments will be examined for hints to unusual physiologies , as well as clues to new enzymes and metabolic pathways, the components of which can be produced recombinantly (perhaps, in extremely thermophilic hosts) for further analysis.
As mentioned above, access to genome sequence information for extreme thermophiles (which has facilitated functional genomics, proteomics and other ‘omics-based’ approaches) has enabled rapid advances in our understanding of these microbes’ physiology over the past 15 years, despite the lack of genetic systems. In some cases, genome sequence information has been used to ask global questions about how thermophilic proteins fold and function at high temperatures . Along these lines, lower levels of structural disorder and functional simplification determined at the level of individual genes and proteins, as well as of whole genomes, was proposed as the basis for prokaryotic thermophily . Genome sequence information led to the provocative proposal that high temperature bacteria and archaea have lower spontaneous mutation rates than mesophiles . The relationship between mesophiles and thermophiles has been given new perspective through comparative genomics. For example, reverse gyrase, an enzyme involved in thermophilic transcriptional processes to deal with uncoiling DNA, was once thought to be a defining feature of extreme thermophiles [29,30]. However, the genome sequence of Nautilla profoundicola (Topt 40-45°C) encodes the gene for this enzyme, likely acquired through lateral gene transfer within the submarine hydrothermal environment that it inhabits . The phylogenetic lines between thermophily and mesophily may be more blurred than expected. Mesophilic members of the Order Thermotogales (or “mesotogas”) were recently identified , as well as related species that have very broad growth temperature ranges , raising questions of about the thermal direction of microbial evolution. It is also clear that extreme thermophiles from the same genus can have differences in their genome sequences that map to subtle but significant differences in their growth physiology . Finally, examination of the P. furiosus and S. solfataricus proteomes, with respect to metal content, revealed a far more extensive set of metals implicated in protein structure and function, including “non-biological” metals, such as uranium and vanadium . Since similar results were found for Escherchia coli, it seems that the microbial world employs more of the periodic table for biological function than previously thought.
In certain cases, genome sequences of extreme thermophiles have revealed physiological insights not previously known, despite years of microbiological study. The genome sequence of Metallosphaera sedula, an extremely thermoacidophilic archaeon (Topt 73°C, pHopt 2.0) , originally isolated and studied for its ability to mobilize metals from sulfidic ores , confirmed the presence of a novel CO2 fixation pathway (3-Hydroxypropionate/4-Hydroxybutyrate cycle) . Consequently, this archaeon possesses a much more versatile growth physiology than previously thought . In fact, M. sedula bioenergetics can be fueled by CO2/H2 autotrophy, heterotrophy, and metal/sulfur oxidation, separately or in combination (mixotrophy) .
Genome sequencing has also brought renewed interest to extreme thermophiles that had been isolated 20 years ago. For example, the genome sequences of Caldicellulosiruptor saccharolyticus [40,41] and Caldicellulosiruptor bescii  (formerly Anaerocellum thermophilum) shed new light on microbial mechanisms [43,44] and enzymology [45-47] of lignocellulose deconstruction. Furthermore, comparative genomics of eight Caldicellulosiruptor species revealed key determinants of lignocellulose degradation, based on the core and pan genomes of this genus .
As mentioned above, studies of extreme thermophiles, and hence biotechnological applications, have been hampered to a certain extent by the limited availability of tools for genetic manipulation and metabolic engineering. Selection of thermostable markers, high temperature solid media, need for anaerobic conditions, and lack of defined media are among the challenges faced. Despite these obstacles, in recent years new molecular genetics systems have been developed for several extreme thermophiles, as well as being refined and expanded in cases where these had previously existed [49,50].
Initial success with molecular genetics in extreme thermophiles was achieved with the extremely thermoacidophilic archaeon S. solfataricus. The fact that this archaeon grows aerobically and can be cultivated on solid media no doubt contributed to progress in this regard. Most genetic manipulation in this species has utilized lacS mutants [51,52]. DNA is introduced to the cell via electroporation and growth on lactose (which requires lacS complementation) is used to select for successful transformation. This strategy has been used as the basis to generate deletion mutants for the study of copper response [53,54], toxin-antitoxin pairs , and antimicrobial proteins . In addition, virus-based vectors encoding genes under the control arabinose- and heatinducible promoters have been used to over-express proteins in S. solfataricus [57,58], and reporter systems for monitoring gene expression have been developed based on β-galactosidase (lacS)  and β-glucuronidase (gusB) .
In recent years, success with molecular genetics has gone beyond Sulfolobus species. Development of genetic techniques for euryarchaeal Thermococcales has been facilitated by isolation of naturally competent strains of T. kodakaraensis and P. furiosus [60,61]. Manipulation of both species initially hinged upon pyrF, the gene encoding orotidine-5ʹ-monophosphate (OMP) decarboxylase; 5-fluoroorotic acid (5-FOA) can be used to select for pyrF mutants, because cells with the functional gene are sensitive to this compound. On the other hand, media lacking uracil can be used to select for complementation of pyrF, as the gene is required for uracil biosynthesis. Once pyrF mutants of P. furiosus and T. kodakaraensis were generated [60,62], methods were developed for performing selections in complex media [63,64], over-expressing proteins, secreting proteins , and generating “markerless” deletions that allow multiple manipulations [62,66].
These tools have facilitated more targeted investigation of fermentative H2 metabolism, a defining feature of Pyrococcus and Thermococcus species. One problem, central to understanding hydrogenase function, is producing sufficient amounts of active forms of the enzyme to study and evaluate. This issue was addressed for soluble hydrogenase I (SHI) from P. furiosus, which was recombinantly produced in E. coli by using an anaerobically-driven promoter native to this bacterium . Furthermore, an engineered hydrogenase, consisting of two of the four native subunits, was overexpressed heterologously in the native host, P. furiosus, and found to utilize electrons directly from pyruvate ferredoxin oxidoreductase without the involvement of an intermediate electron carrier (NADPH or ferredoxin) . There are biotechnological implications of this work. Hydrogen production is normally growth-associated, but an electron carrier-independent hydrogenase might partially decouple these processes, resulting in higher yields of hydrogen.
Directed gene knockouts have been pivotal in probing metabolic mechanisms related to H2 generation in P. furiosus  and T. kodakaerensis [70,71]. Deletion of surR, which encodes a transcriptional regulator, revealed that this gene is required for expression of the membrane-bound hydrogenase , the primary H2-evolving enzyme . Deletion of cytosolic hydrogenases limited growth of the microbe, but increased specific H2 production rates, suggesting that re-oxidation of H2 by these enzymes is an important energy conservation mechanism in this species [70,71]. Deletion of the membrane-bound oxidoreductase complex, which is required for sulfide production [69,70], also resulted in increased specific hydrogen production [69-71].
To date, the largest impact of enzymes from extreme thermophiles have had on science and technology relates to their use in catalyzing the polymerase chain reaction (PCR) . Beyond that, the first thoughts for biotechnological applications for enzymes from these extremophiles turned to thermostable and thermoactive “drop in” replacements for industrial enzymes already in use (e.g., proteases, amylases, glucose isomerases). Several insightful reviews have appeared over the past 25 years that examine scientific and biotechnological aspects of biocatalysis at elevated temperatures (for a recent excellent review see ). One of the opportunities afforded by biocatalysts that function at temperatures approaching and exceeding 100°C is the exploitation of the intrinsic features that underlie their unprecedented thermostability and thermoactivity. In this sense, the key strategic questions to consider are whether the biocatalytic process requires high temperatures, and, as a consequence, if high temperatures confer any strategic advantage.
The structural stability of enzymes from extreme thermophiles makes them attractive candidates for protein engineering, based on the premise that these are less susceptible to undesired consequences from genetic manipulations. The extensive protein engineering of β-glycosidases from S. solfataricus to catalyze chemo-enzymatic synthesis of oligosaccharides serves as an excellent example in this regard . In another case, cofactor specificity of an alcohol dehydrogenase from P. furiosus could be modified by site-directed mutations in the co-factor binding pocket . Building on the theme of enzymes from extreme thermophiles as robust protein engineering targets, the transcription factor Sso7d from S. solfataricus was used as a scaffold for creating binding partners to a variety of biomolecules . The thermostability of these enzymes was also a key factor in examining the effect of microwaves on biocatalysis. Under certain conditions, the biocatalytic rates of enzymes from P. furiosus, S. solfataricus and T. maritima could be enhanced under microwave irradiation, a prospect not possible for mesophilic enzymes whose stability was significantly impacted by microwaves .
Key to many enzyme applications is the capability to immobilize the biocatalyst for stabilization or for a specific process strategy (e.g., re-use or localization). Enzymes from extreme thermophiles present some opportunities, as well as challenges, when it comes to immobilization . For example, entrapment in a porous gel as a means of immobilization, commonly employed for mesophilic enzymes, is problematic because of the thermolability of the matrix. Alternatively, carbohydrate binding domains from extreme thermophiles can be employed in fusions with extremely thermophilic enzymes for immobilization, as was demonstrated with the chitin-binding domain from a P. furiosus chitinase and the xylose isomerase from Thermotoga neapolitana .
Not surprisingly, enzymes from extreme thermophiles have been examined closely for applications related to the emergence of biofuels. It has been argued that the deconstruction of lignocellulose is best done at elevated temperatures, either because thermal factors facilitate this process, or because biofuels bioprocessing already involves thermal steps for pretreatment . To this point, computational studies suggest that thermal contributions to enzyme plasticity and molecular motion at high temperatures play a role in enhancing cellulose-binding domain and catalytic domain synergy in cellulose . The genomes of species within the extremely thermophilic genus Caldicellulosiruptor encode a host of multi-domain glycoside hydrolases that contribute to the breakdown of crystalline cellulose and hemicellulose [43,48]. Recent work has looked at the contributions of the various domains within these enzymes to complex carbohydrate hydrolysis [46,81] and the potential role of certain multi-domain glycoside hydrolases, which also use S-layer homology domains to anchor to the cell envelope . The high temperature, cellulose-degrading consortium of archaea, described above, also gave rise to the discovery of a novel hyperthermophilic cellulase, a multi-domain enzyme exhibiting optimal activity at 109°C .
Biocatalysis based on whole cells has also been the objective of efforts with extreme thermophiles. For example, the degradation of toxic pollutants has been demonstrated; S. solfataricus 98/2 could utilize phenol for growth in a fed-batch bioreactor . The recovery of base, precious and strategic metals through whole cell bio-oxidation processes has been a long-term goal for extreme thermoacidophiles and recent efforts have focused on identifying process bottlenecks and improved processing strategies. It is becoming clear, not surprisingly, that mixed cultures will be the most effective approach to biohydrometallurgy and could be a way to overcome problems with surface passivation by jarosite and elemental sulfur by-products .
Until very recently, metabolic engineering involving enzyme and pathways from extreme thermophiles were carried out in mesophilic hosts. For example, the archaeal isoprenoid ether lipid biosynthesis pathway was reconstructed in E. coli to produce digeranylgeranylglyceryl phosphate (DGGGP) . The strategic use of temperature for bioprocessing was demonstrated by cloning a hyperthermophilic α-amlyase from T. maritima into transgenic sweet potato . Below 40°C, the normal growth temperature range of the plant, no significant amylase activity could be detected nor was plant growth and development impacted. But upon switching to 80°C, the hyperthermophilic enzyme was activated such that the plant storage root was rapidly hydrolyzed. This created a mechanism for first producing the sweet potato starch then using a thermal switch to convert the starch into a fermentable sugar for biofuels applications.
Of course, the next frontier is metabolic engineering at high temperatures. In fact, the rapid progress with molecular genetics for extreme thermophiles has given rise to the prospect of using these microorganisms as recombinant hosts for metabolic engineering. The three archaea shown in Table 1 offer the most near-term prospects in this regard. For example, S. solfatarcius P2 differs from S. solfataricus 98/2 in that the latter strain is less able grow on surfaces. However, the insertion of two genes from P2 into 98/2, encoding α-mannosidase and β-galactosidase, enabled 98/2 to mimic P2 by attaching to glass and forming static biofilms . The use of thermally-driven gene regulation has been elegantly demonstrated using P. furiosus as a recombinant host. When microorganisms are employed to generate a desired product, the production pathway often competes with the microbe’s natural biosynthesis pathways for key intermediates or cofactors. In this situation, one might envision an ideal two-stage process in which biomass is generated during the first stage with minimal product formation, while cellular activity ceases and the desired product is generated during the second stage (see Figure 1). The recent proof-of-concept work of Basen et al.  uses temperature as the switch to halt cell growth and initiate product formation. Lactate dehydrogenase from Caldicellulosiruptor bescii (Topt = 78°C) was cloned into P. furiosus (Topt = 98°C) under the control of a P. furiosus “cold shock” promoter which is turned on at 70-75°C. In this situation P. furiosus can be cultured at 98°C until it reaches a high cell density, at which point it can be transferred to 72°C, resulting in expression of the heterologous lactate dehydrogenase and formation of lactate (a product that P. furiosus is unable to produce naturally).
In the near future, we will likely see the first demonstrations of high temperature strains used as hosts for biotechnological applications. For example, in biofuels production, the ultimate goal is to create metabolically-engineered extreme thermophiles that breakdown lignocellulose and convert fermentable sugars to liquid biofuels (socalled “consolidated bioprocessing”). At high temperatures, there is the possibility that biofuels can be recovered directly through direct evaporation and distillation (see Figure 2). Other strategic uses of high temperatures will likely emerge as tools for molecular genetics in extreme thermophiles become more firmly established.
Although discovered more than 40 years ago, in many ways extremely thermophilic microorganisms are just at the beginning when it comes to biotechnological applications. Virtually any enzyme that is identified in a mesophile has a homologous version in an extreme thermophile, typically with significantly higher levels of thermostabilty, if not thermoactivity. As genetic systems for extreme thermophiles become more widely used and more tractable, the challenge will be to exploit elevated temperatures to improve upon existing bioprocessing strategies, or even better, make possible novel multi-step, biotransformations. Ideas along these lines have already been proposed , and it is only a matter of time before these process concepts are demonstrated and put into practice.
The authors acknowledge support from the U.S. Department of Energy ARPA-E Program (DE-AR0000081), U.S. Department of Energy GTL Program (DG-FG02-08ER64687), U.S. National Institute of Health (R01GM90209), and the U.S. Defense Threat Reduction Agency (HDTRA1-09-1-0030). AD Frock acknowledges support from an U.S. National Institutes of Health Biotechnology Traineeship (T32 GM008776-06).
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