Send Orders for Reprints to [email protected]

Current Biotechnology, 2014, 3, 45-59 45 Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Peter L. Bergquist*,1, Hugh W. Morgan2 and David Saul3

1Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, New South Wales 2109, Australia; and Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland 1142, New Zealand 2Department of Biological Sciences, Thermophile Research Unit, Waikato University, Hamilton 3240, New Zealand 3ZyGEM Corporation Ltd, Waikato Innovation Park, Hamilton 3216, New Zealand

Abstract: Enzymes from extreme thermophiles that grow above 70 °C have a number of attractions in industrial applications. They are often highly resistant to denaturing conditions and are stable at elevated temperatures and over a range of pH values. There has been a widespread search for micro-organisms producing novel enzymes and where found, most publications (and research grant applications) promise that their superior properties would be suited to particular industries that operate at elevated temperatures - for example, bleaching of kraft pulp in the pulp and paper industry. Yet examination of the academic and patent literature reveals few of these proteins adopted in industrial enzymology. Most employed successfully have been as laboratory reagents, particularly Thermus aquaticus DNA polymerase, which made the polymerase chain reaction possible and revolutionized gene manipulation at a laboratory level. Extremozymes marketed for the laboratory are lower volume / higher value products and have been cloned and expressed (usually) in Escherichia coli. This review examines the characterization and application of thermophilic enzymes for several activities that have been identified and (usually) produced in recombinant bacteria. DNA polymerases, glycosyl hydrolases, lipases and proteases from extreme thermophiles are described and evaluated for their potential and actual applications in biotechnology. Some of the barriers to widespread industrial acceptance are described. Emphasis is placed on a number of examples illustrated by the anaerobic extreme thermophiles Caldicellulosiruptor sp. and Dictyoglomus sp. with which the authors are familiar. A number of attractive enzymes are not scalable economically in Escherichia coli or the organism from which the gene has been isolated is an obligate anaerobe and enzyme yields from the native organism are low. Consequently, attention has turned to commonly used ‘cell factories’ to provide suitable yields of enzymes used as bulk chemicals. These 'factories' are usually fungi such as Saccharomyces cerevisiae and Trichoderma reesei or bacteria such as Bacillus sp. A number of challenges, such as codon usage incompatibility, must be overcome to achieve economic yields. Keywords: Cell factories, DNA polymerases, extremozymes, glycosyl hydrolases, industrial enzymology, proteinases, pulp bleaching.

INTRODUCTION small differences in amino acid sequence gave them remarkable comparative stabilities. The study of micro-organisms able to survive and multiply in extreme environments is a relatively recent Microorganisms are largely neglected in studies of phenomenon. Environments that were identified as having biodiversity yet are the most phylogenetically and extremes of temperature, salinity and pH, etc were physiologically diverse group on the planet. Figures vary, considered to be effectively sterile until the second half of but most microbiologists agree that we have probably the 20th century. The realization that these environments identified less than 1% of the total microbiota that exists in harboured a diverse assemblage of organisms was natural environments. For example, the total number of fascinating initially and there was curiosity and speculation bacterial species worldwide is conservatively estimated at as to how their macromolecular make-up was adapted to 13-50 million, yet to date, only 4,000 species have been these conditions. It was soon realised that these organisms described [1, 2]. Much of the problem has been caused by possessed proteins that were in many ways similar to those dependence on culture-based enrichment techniques that fail in the familiar more mesophilic microorganisms, but often to support most organisms [3]. The traditional techniques now have been augmented by a powerful array of DNA- based methods that have allowed the amplification of small

*Address correspondence to this author at the Department of Chemistry and subunit (SSU) rRNA and other genes directly from natural Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie environments. Uncultured microorganisms make up the University, Sydney, New South Wales 2109, Australia; major part of the Earth’s biological diversity. In many Tel: +61 2 9850 8614; Fax: +61 29850 8313; environments, >99% of the microorganisms cannot be Email: [email protected]

2211-551X/14 $58.00+.00 © 2014 Bentham Science Publishers 46 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. cultured by standard techniques and the uncultured fraction the notion that the active site is more flexible than the whole includes diverse organisms that are only distantly related to protein and loss of activity occurs before denaturation. In the cultured ones. Therefore, cultivation-independent methods classical model, the two rate constants for the processes are essential to understand the genetic diversity, population (ΔG*cat and Δ G*inact ) will lead to a temperature optimum structure and ecological roles of the majority of (Topt) that is dependent on the length of assay because microorganisms. Metagenomics, the culture-independent thermal denaturation is time dependent [12, 13]. A change in genomic analysis of an assemblage of microorganisms, has the assay duration will change the apparent optimum the potential to answer many fundamental questions in temperature. The equilibrium model introduces new thermal microbial biodiversity and ecology [4, 5] and allows the parameter Teq where the concentrations of the active and examination of the total genomic diversity of a sample and inactive enzyme are equal and allows prediction of the avoids the over-representation of fast growing members of temperature at which the enzyme activity is maximal (Topt, the microbial community. ref. [12]). Accordingly, they propose that successful screening for high(er) temperature enzymes will need to take The stabilities discovered from both enrichment and into account both thermal stability and activity (via T ), metagenomic studies were not limited solely to temperature eq unlike the classical model which suggests both activity and but to a range of conditions which led to denaturation of resistance to thermal denaturation can be achieved by their mesophilic counterparts. The ability of some enzymes such as lipases to show activity in organic solvents selecting for thermal activity alone. Similarly, the equilibrium model teaches that in enzyme engineering encouraged their use in bio-transformations in the studies using rational or directed mutation procedures, an biopharmaceutical industry to perform reactions with regio- increase in thermostability will not necessarily lead to and enantio-specificity. The general ‘green’ trend for enhanced activity at high temperatures as the temperature of industrial chemistry that became evident towards the end of T must be shifted also [13]. These considerations will have the last century and the realization that enzymatic processes eq could be carried out under mild catalytic conditions, an effect also on enzyme reactions in a reactor to give a product, as the output at given times and temperatures will stimulated the discovery of new extremophile organisms, vary between the two models. Given the general lack of and by inference, their enzymes. This shift occurred against a understanding of enzyme kinetics by the average molecular background of improving techniques to examine biologist engaged in biotechnological outcomes, it is microbiological diversity without needing traditional culture possible that resources are being wasted from the point of methods. As a result, a number of enzymes that showed characteristics that could aid their incorporation into enzyme identification to product formation as many researchers appear to be unfamiliar with, or ignorant of, both industrial processes were isolated and their characteristics the classical and equilibrium models [15]. published. Many authors extrapolated (or imagined) the industrial benefits of their particular enzyme from laboratory One area that has adopted an enzymatic step is the pulp bench experiments and every enzyme that had even the and paper industry in the bleaching of pulp, largely as the remotest possibility of a commercial use was proposed as result of R&D by Viikari and collaborators [17]. The new being industrially-relevant in published papers and research process had the advantage of requiring effectively no grant applications. Yet the reality is that only a few of these alteration to the current procedures and infrastructure at kraft enzymes have found a place in industrial processes, largely mills apart from the addition of a metering pump. Kraft pulp as a result of cost, plant design and a general reluctance to comes out of the cook at temperatures between 70 °C and change a system that works. 100 °C and pH~13 and would be expected to be a target for The biotechnological advantages of thermophilic and an alkalophilic, thermophilic xylanase. Yet, in general, mesophilic enzymes are used after some cooling and pH hyperthermophilic enzymes have been listed in many adjustment of the pulp. Why is this? In the amounts used, the reviews over the past two decades (for example, refs. [6-11]) enzyme must be regarded as a bulk chemical and cost is an but the temperature stability and high T of α-amylase from opt important factor. Furthermore, alkalophilic xylanases from Bacillus species appears to be one of the few enzymes used thermophiles have proved to be elusive and even the sole in large-scale industrial applications-in this case, starch degradation. enzyme available in commercial production must be used in kinetic excess, thereby increasing costs. In addition, the price of pulp follows a cyclic pattern and there is a tendency to PHYSICO-CHEMICAL ISSUES RELATED TO eliminate enzyme-aided bleaching on price downturns. THERMOSTABILITY Further, academic researchers making claims of industrial applicability for xylanases they have isolated have seldom Recent results and the proposal of a new model for the effect of temperature on enzyme activity may have a performed bleaching tests on the insoluble industrial substrates. Worse, many studies fail to be cognizant of the considerable influence on biotechnological applications of common rules of enzyme kinetics and have assayed enzymes enzymes from extreme thermophiles in areas ranging from under substrate-limited conditions (see Gibbs et al. [18, 19]). biodiversity screening, enzyme engineering and enzyme production in a bioreactor [12-16]. The effect of temperature Although there are some thermophilic xylanases that on enzyme activity has been interpreted as increased have been characterized thoroughly, their production from temperature gives increased activity - but results in a loss of their native organisms is not economically viable because the activity as a result of enzyme denaturation (the classical parent organisms are obligate anaerobes. There have been model). The model of Daniel and collaborators introduces an unexpected barriers to bulk heterologous production in inactive non-denatured form of the enzyme as an common industrial hosts (reviewed in Nevalainen et al. [20]; intermediate (the equilibrium model). Their results support Nevalainen and Peterson [21]; and updated in this review). Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 47

In contrast to the demonstration of potential, actual with the same induction characteristics in the same application of enzymes from hyperthermophiles in industrial recombinant host gave an increased yield but the amount of situations depends on perceived suitability, protein costs and enzyme was not additive with gene copy number. Many of minimal alteration to current processes. Industrial enzymes the problems apparent with heterologous enzyme production such as proteases, glycosyl hydrolases and lipases, etc by Trichoderma reesei have been reviewed comprehensively usually are produced by filamentous fungi or Gram-positive by Nevalainen and Peterson [21]. Bacteria as these microorganisms secrete enzymes directly The lesson learned is that it is not simply a case of into the fermentation medium, leading to cost savings in shifting a gene coding for an enzyme into a standard ‘cell downstream processing. The selection of the microorganism factory’ but substantial optimization and genetic alteration for use as a ‘cell factory’ usually requires it to have GRAS may be required. It is likely that a number of issues will have status (Generally Regarded as Safe) and include to be resolved before economically-viable yields are Trichoderma reesei, Kluveromyces lactis, various achieved. Issues relating to low levels of expression that will Aspergillus species and the bacterium Bacillus subtilis need resolution may be summarized as: Is the yield the result (reviewed in refs. [22-24]). These expression strains have of codon usage? Is protein folding involved? Does been optimised for overproduction of their own or related glycosylation have an effect? Is proteolytic processing enzyme(s) of interest but heterologous enzymes from other appropriate? Is degradation of product by the host’s families and orders frequently are expressed poorly and both proteases a significant factor? As well, there exist the many the strain and the gene may need modification as well as factors involved with cell physiology and fermentation scale- optimization of the culture conditions and the physiology of up that need to be taken into account [20, 21]. the host. Poor yield in common recombinant hosts has deterred These difficulties are particularly intrusive for enzymes widespread adoption by industry of thermophilic enzymes derived from hyperthermophiles. Growth conditions are such as glycosyl hydrolases and proteases in industrial usually anaerobic and the yields are low. In most cases there processes for which they might be considered ideal on an a are no, or only rudimentary, genetic systems available. priori basis. In contrast, thermophilic DNA polymerases and Cloning and expression in E. coli usually will provide to a lesser extent, proteases, have found widespread sufficient enzyme for proof of principle confirmation but is applications in the life sciences and diagnostics markets. The not cost-effective for production. We have investigated the targeted customers use microgram rather than kilogram expression of one hyperthermophilic enzyme in detail in quantities of these enzymes in their procedures and so Trichoderma reesei with the objective of bulk production for existing expression hosts (usually E. coli) provide adequate industrial purposes. The enzyme, a β-1,4 xylanase from the amounts of enzymes for commercial production. Typically, obligately anaerobic bacterium Dictyoglomus thermophilum the enzymes are used in gene cloning and mutation, pathway Rt46. B1 was produced in E. coli, showed an outstanding synthesis, synthetic biology and pathogen diagnostic areas apparent T performance, was thermostable (half-life 24 opt that are not as cost-sensitive as industrial enzymes. hours at 85 °C) and showed superior bleaching ability on kraft pulp compared to commercial products [25]. In this review we shall outline the isolation, and uses of Dictyoglomus genes have a high A-T content and some selected enzymes from extreme thermophiles. Many of Trichoderma genes a high G:C content. Consequently, it was these enzymes show outstanding properties and have been necessary to resynthesise the gene with Trichoderma codon pivotal in the explosion of knowledge in contemporary preferences before expression was achieved [26]. The yield genetics. A number of these enzymes that can be regarded as was still low compared to a similar family 11 xylanase laboratory reagents have made major contributions to cloned into Trichoderma from the fungus Humicola [27]. engineering new organisms, for example, in agricultural and Alterations to the gene cassette and the provision of KEX- biofuels areas. Others have potential that will be realized like cleavage sites adjacent to the recombinant protein only when yields can be raised, costs reduced and the new provided only marginal improvements in yield [28]. approaches are embraced by commercial plant engineers and Subsequent studies showed that Trichoderma reesei Rut-C30 managers. Some aspects regarding the problems of scale up suffered from secretion stress effects even with the in heterologous hosts have been mentioned above. expression of homologous proteins as shown by physiological changes in the fungal hyphae and the up- PROTEASES regulation of genes associated with the unfolded protein response (UPR) such as pdi1 and the ER-associated Proteases have played an important role on the degradation response (ERAD) such as sec61 [29]. Another processing of natural products for many centuries. They are study showed that the gene for a T. reesei Rut-C30 aspartyl probably still the most widely used class of industrial protease was up-regulated nearly 10-fold when recombinants enzyme with diverse applications in the detergent and food were expressing the heterologous xylanase and this effect industries (reviewed in [33]). Although proteases are used was presumed to contribute towards lowering the yield of the widely in detergent formulations none of the enzymes have enzyme [30]. been isolated from thermophiles. Accordingly, they are not discussed here. Some yield improvement was reported when the hyperthermophilic xylanase gene was expressed from An advantage of thermophily in proteases is that above alternative promoters but there was no correlation between 65 - 70 °C the substrate of the enzyme, usually proteins from copy number (gene dosage) and the amount of xylanase mesophilic organisms, denatures and becomes more activity measured [31]. Miyauchi et al. [32] have shown that amenable to hydrolysis. This effect results in a marked the xylanase gene expressed from three different promoters increase in specific activity above this point in the 48 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. temperature range. In addition, proteins normally recalcitrant buffers compatible with the PCR and inhibitory substrates to hydrolysis denature and become susceptible to hydroysis. (tannins and dyes) remain insoluble. These facts make EA1 proteinase efficacious for releasing trace DNA from difficult Proteases are one class of hydrolase that have found forensic substrates such blood stains and samples presented novel application as a low volume/high value laboratory reagent in nucleic acid isolation. The most commonly used on dyed fabrics and plant material [40]. enzyme for this purpose is Proteinase K, a serine proteinase originally isolated from the fungus Engyodontium album GLYCOSYL HYDROLASES FROM CALDICELLU- (formerly Tritirachium album; [34]). A thermophilic serine LOSIRUPTOR SP. proteinase from Thermus Rt4.1A was cloned and the Members of the genus Caldicellulosiruptor are anaerobic recombinant product demonstrated to be able to recover PCR-ready DNA from blood [35] and generally to be thermophiles with a Topt above 70 °C able to hydrolyze a broad spectrum of plant polysaccharides as sole carbon and superior to Proteinase K in a number of applications - energy source. Species distribution exhibits wide geographic including chromosomal DNA preparation in agarose plugs spread with isolates characterized from hot pools in New for pulsed field gel electrophoresis [36, 37]. Such was the Zealand [41], the USA [42], Iceland [43] and Russia [44]. temperature-activity profile of the enzyme that tissue could Species exhibit a spectrum of activity on crystalline cellulose be digested at 75 °C (just below the melting temperature of the agarose plug) and on lowering the temperature, residual from weakly active (for example Ca. hydrothermalis, Ca. owensensis) to strongly cellulolytic (Ca. saccharolyticus, activity was minimal, thereby allowing cleavage of the DNA Ca. bescii). using mesophilic restriction endonucleases. Typically, the alternative procedure using Proteinase K required 2-3 days Their ability to degrade crystalline cellulose, substrates washing the agarose blocks to remove the enzyme in order to like untreated switchgrass and to ferment a wide range of prevent hydrolysis of the nucleases. With Rt4.1A, this step pentose and hexose sugars holds attraction for the conversion could be dropped entirely. Fung and Fung [38] also of biomass to either feed chemicals or as a platform demonstrated Rt4.1A proteinase's usefulness in the organism for production of bioethanol. Not surprisingly, preparation of stable mRNA from mammalian cells for their cellulolytic and hemicelluloytic enzymes have attracted cDNA production and for a few years the enzyme was considerable attention. Since cellulosomes are absent in commercialised under the name PreTaq™ but it was species of Caldicellulosiruptor, they provide a contrast to the marketed poorly and soon forgotten. cellulolytic system of Clostridium thermocellum that uses a cell-bound cellulosomal enzyme complex to deconstruct The exceptionally high thermal stability of Rt4.1A plant polysaccharides. The unique feature of cellulose proteinase was an advantage for tissue digestion, but it was utilization in Caldicellulosiruptor is the production of large also a limitation in that the enzyme was difficult to extracellular modular multi-domain proteins containing two inactivate. This characteristic meant that residual proteinase different functional enzyme activities, interlinked usually by became a problem for high temperature processes such as the polymerase chain reaction (PCR). Ideally, a heat-kill step cellulose binding domains and proline-threonine-rich linker regions (which are thought to provide flexibility to the should be no greater than 95 °C so that a thermal cycler can overall complex). The functional enzymes are arranged in be used and boiling avoided. With Rt4.1A proteinase, this multiple combinations; for example xylanase with inactivation could be achieved only by adding EGTA - a mannanase, xylanase with endoglucanase, endoglucanase chelating agent that is undesirable in most downstream with mannanase etc, and some examples include nucleic acid manipulation steps. debranching enzymes [45]. From genome sequence Alternative proteinases were sought that fitted all the alignments, genes for these multi-domain enzymes are criteria desirable for nucleic acid extraction: (i) optimally aggregated into discrete domains in the genome and each active above 70 °C, (ii) heat inactivated at 85 - 95 °C (iii) domain contains the genes for several multi-domain active in buffer compatible with most downstream enzymes. There is diversity in the ability of the different applications (notably the Polymerase Chain Reaction) and species in the genus to deconstruct plant polymers, (iv) aggressively hydrolytic without the addition of particularly crystalline cellulose, with some species being detergents. The enzyme selected was a neutral strongly cellulolytic (for example Ca. bescii, Ca. metalloproteinase secreted by Bacillus sp. strain EA1. This saccharolyticus) whereas others are weak or inactive. These proteinase (termed EA1 proteinase) differed from its nearest properties are reflected in the composition of multi-domain homologue from Bacillus caldolyticus by just one amino glycosyl hydrolases found in the respective genomes, where acid, but this substitution more than doubled the half life of weakly cellulolytic species are either lacking the required the enzyme at 85 °C (115 minutes compared to 45 minutes; glycosyl hydrolases (particularly family GH 48) or have [39]) making it the most thermostable member of the truncated versions of these enzymes. Features of these thermolysin family of proteinases (EC 3.4.24.27). multidomain glycosyl hydrolases, together with the ability of A recombinant version of the proteinase is marketed as Caldicellulosiruptor to co-ferment hexose and pentose prepGEM™ by ZyGEM Corporation (New Zealand) and is sugars simultaneously, have attracted attention to these the basis for a number of kits designed for both RNA and species as likely candidates for a consolidated bioprocessing DNA extraction. Given the high specific activity, the enzyme system (CBP) for conversion of plant biomass [46, 47]. can be used at 50-fold lower concentrations than Proteinase However, end-products of fermentation do not include K, making supply simple and cost efficient. Furthermore, the ethanol and substantial re-direction of intracellular energy mild, neutral buffer and with thermophily replacing the need metabolism will be required by genetic transformation to for detergents, means that nucleic acids are extracted in address this deficiency. Alternatively, the glycosyl hydrolase Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 49 genes themselves are viewed as likely candidates for transfer complexes, but also exist on a broader scale in the genome. to alternative hosts that contain an ethanologenic capacity. Gene clusters of multi-domain glycosyl hydrolase enzymes in Ca. saccharolyticus and strain Tok7B.1 have quite The complete genomes of eight Caldicellulosiruptor different configurations and content [50] and this species have now been annotated and comparative genomics undertaken, reflecting the interest shown in the capacity of observation extends to other species with wider geographical distributions. More extensive genomic comparison of other these species to deconstruct plant polysaccharides [48, 49]. It species/strains might allow a consensus on the core content is likely that the ability to hydrolyze crystalline cellulose is of GH enzymes required for plant polysaccharide an ancient trait which has been lost more recently in those deconstruction. It may reveal also the optimal configuration members of the genus which have a low growth on cellulose, of GH enzyme combinations in the multi-domain while other species have retained and enhanced this capacity, probably through gene duplication and modification [45, 50], configuration along with the optimal number and type of CBM and PT domains [49]. Cellulolytic capability is and similar hypotheses have also been proposed for the associated with GH families 5 to 9, 12, 44, 45, 48, and 61; proliferation of cellulosomal enzymes [51]. This those in the genus Caldicellulosiruptor encompass GH recombination or gene shuffling within the multi-domain families 5, 9, 44 and 48 often in multiple copies and paired construction could lead to an unlimited number of possible with a variety of other enzyme functions, eg xylanase, enzyme pairings as the microorganism seeks a selective growth advantage on a recalcitrant substrate [50]. mannanase, debranching enzymes. The presence or absence of the GH family 48 enzyme appears to be the defining The Carbohydrate-Active Enzymes Database (CAZy characteristic of strongly cellulolytic speciesbut the multiple database; www.cazy.org/; ref. [52]) provides a description of copies of the GH family 5, 9 and 44 cellulases also present the families of structurally-related catalytic and suggests that synergy of enzyme action is at least as carbohydrate-binding modules of proteins that catalase the important in the ability to degrade these complex substrates hydrolysis, synthesis or modifications of carbohydrates. It [45]. contains families of glycosyl hydrolases that hydrolyse or Cloning and expression of many genes for multi-domain rearrange glycosidic bonds. In the strongly cellulolytic enzymes has now been accomplished such as ManA [54], members of the genus, the glycoside hydrolase genes for GH CelA [55], CelB [56], xylanase [57] and many others [49]. families 9 and 48 (those GH families associated with ability The CelA enzyme remains one of the largest multi-domain to degrade crystalline cellulose) are aggregated at specific loci on the chromosome, along with GH families associated cellulases reported with 1,751 amino acids. The protein contains an N-terminal family 9 GH and a C-terminal family with mannan, xylan and side chain-degrading activities. The 48 GH linked by two type 3 CBM and Tp domains. different enzyme functions in the multi-domain enzymes are Truncated versions of these enzymes have been produced linked by carbohydrate binding modules (CBM) which are with altered thermostability and enzyme activity. characterized by a planar array of polar and aromatic Interestingly, truncated expression products of a cloned celE residues that are likely to be involved in binding to cellulose (and hemicellulose) and are important in ensuring close domain from Caldicellulosiruptor strainTok7B.1 exhibited unusual results when used in a stone-washing assay to contact between substrate and enzymes. In the genus remove cellulose strands from denim material. Products Caldicellulosiruptor, the most common CBM is of the containing a family 9 GH together with two CBMs showed family 3 type, which is implicated with activity on low activity in the assay, whereas truncated versions where crystalline cellulose substrates. The number of CBM3 either one or both CBMs were absent had increased activity modules in a multi-domain enzyme varies from one to three depending on the species and the enzyme type. The ratio of [58]. Thus, the presence of the CBMs seemed to antagonize the ability of the enzyme to function, contrary to CBMs to GHs correlates with ability to deconstruct plant expectations. Synergism between enzymes in the same polysaccharides and as expected, this ratio is high in species multi-domain complex and between complexes is assumed of the genus Caldicellulosiruptor. and sometimes reported [59, 60]. The gene celB from Ca. saccharolyticus was the first multi-domain enzyme to be cloned and expressed in E. coli Full characterization of many of these expressed enzymes have been reported with a view to their incorporation into [53]. CelB was shown to contain an N-terminal exo- industrial processes, particularly pulp bleaching [61]. In glucanase activity and a C-terminal endo-glucanase activity general, major difficulties are the requirement for the separated by regions with high proline and threonine content enzyme to meet existing process parameters, and the need to (PT boxes). In homologues of this enzyme in other species, produce enzymes at a volume and cost which is currently there is variation in the number of CBM domains, with two being present in Ca. obsidiansis and three in uneconomic. Transcriptomes and proteomes of different species of Caldocellulosiruptor on different plant substrates Caldicellulosiruptor strain Tok 7B.1. The latter is notable in will be helpful to indicate the enzymes that are most likely to that it is also an isolate from the same thermal region in New be important in deconstruction. Initial studies have been Zealand as the type species and indicates the possibility of undertaken on Ca. saccharolyticus grown on xylan and diversity even in geographically-similar environments and xyloglucans, of Ca. bescii grown on filter paper and xylan this observation is important for gene discovery - a small geographical area may have several variants of a type. One [62-64] and a proteomic analysis of seven Caldicellulosiruptor species grown on Avicel [49]. hypothesis concerning the architecture of this genetic region Generally, these studies were consistent with expectations in is that recombination after gene duplication led to the that largely cellulolytic enzymes were up-regulated on diversity of modular enzymes. Differences between species cellulose substrates and xylanolytic or hemicellulolytic are manifest not only in the architecture of the multi-domain enzymes up-regulated on hemicellulose substrates, but few 50 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. enzymes were detected that were unique to a particular Although many of their kinetic properties are exceptional, substrate. The transcriptome varied with time of growth, they have had to be cloned and expressed in standard indicating adaptation to changing concentration or as binding fermentation strains as hosts and their low G:C content has sites on the substrate became saturated and the enzymes were required significant genetic engineering skills to provide released to the supernatant. suitable recombinants for expression. Some of the main applications have required inexpensive enzymes in bulk (for A recent discovery is the role of S-layer-linked enzymes example, pulp bleaching in paper manufacture) and they are in both substrate hydrolysis and binding the cell close to the a replacement for well-established but mesophilic enzymes polysaccharide substrate via the cell wall S-layer. A modular currently used in the industry. The major applications have family GH5 endoglucanase/xylanase with a type 28 CBM involved glycosyl hydrolases, but new uses in value-added characterised from Ca. saccharolyticus was shown to have the unique property of being multifunctional against C5 and products involving drug precursor transformations have been reported recently. C6 polysaccharides, including microcrystalline cellulose, while only possessing one catalytic module [65]. Furthermore, the presence of the CBM28 was critical for PULP BLEACHING adherence to Avicel and activity on insoluble substrates [65] There has been significant interest in xylanases with and the enzyme possessed S-layer-linked (SLH) domains which were responsible for attaching the cell to the substrate. alkaline pH optima and stability under alkaline conditions in the bleaching of paper pulp. This interest primarily has been Other SLH proteins unique to Ca. saccharolyticus have been due to the proven utility of xylanases in kraft pulp bleaching described which are larger than the multi-domain enzymes, where the pulp comes out of the kraft cook at high bind tightly to Avicel but with barely detectable endo- temperature and at an alkaline pH [17]. Several research glucanase activity [65]. groups have prospected for novel organisms producing Genome comparisons of the different Caldicellulosi- xylanases in natural and man-made alkaline environments ruptor species indicates that the multi-domain gene clusters (for example, refs. [69-71]). A number of groups have are flanked by mobile elements or partial mobile elements attempted directed evolution of xylanases to increase the pH suggesting these are areas of either lateral gene transfer or optimum of known xylanases (for example, refs [19, 72, transformation [64]. This arrangement could facilitate the 73]). The objective of these studies has been to better match transfer of multiple blocks of enzyme functions to more the enzyme to industrial conditions encountered in pulp suitable expression hosts. The degree of heterogeneity that bleaching and to reduce reliance on chemicals in the exists in the genes that constitutes these gene clusters, and bleaching process. the diverse combinations of different GH family enzymes in Mathrani et al. [74] filed a patent describing the multi-domain enzymes hints at a continuing quest to find Dictyoglomus B1 and its xylanase(s). This strain had low the optimum combination for rapid plant polysaccharide cellulase activity and was isolated initially from a pulp and deconstruction. Since conserved regions abound in the multi- domain enzymes, the propensity for directed gene shuffling paper mill. They could not claim priority for the organism because of a prior publication by Patel et al. [75]. Their to produce new combinations would seem to be high. The claims regarding pH range and temperature range were development of the improved pyrF transformation system attributed to the presence of more than one enzyme but may [66] for clostridial organisms, would allow testing such have resulted from substrate limitation under the conditions constructions either in a Caldicellulosiruptor host or other given of the enzyme reactions as shown in their examples. expression system and the development of tailored combinations for a particular substrate. The potential for The Dictyoglomus strain B1 xylanase was produced under anaerobic conditions in a continuous fermentor using the gene expression in the native species has been increased individual parameters determined from batch culture trials substantially by the recent demonstration of the necessity to and 73 °C, pH8. A dilution rate of 0.112h-1 gave optimal methylate the transforming DNA with an N4-methylase xylanase activity in the supernatant [76]. The enzyme cloned from the genome of Ca. bescii to obtain preparation isolated from the culture supernatant was transformants [67]. probably a mixture of Family 10 and 11 enzymes as deduced from the amount of free xylose determined by HPLC after ENZYMES FROM DICTYOGLOMUS pulp treatment. The preparation gave a brightness increase of 2 ISO units using peroxide-bleached pine pulp [77]). Gibbs The phylum Dictyoglomi consist a single genus, et al. [78] cloned the xynA gene from a λZap library of D. Dictyoglomus, with two type strains and several related pure thermophilum Rt46.B1 and expressed it in E. coli using an culture isolates. All isolates are thermophilic, anaerobic, Gram-type negative rods. A major distinguishing phenotypic over-expression plasmid vector. The apparent optimal temperature at pH6.5 was 85 °C (at the time, the most feature is the formation of spherical bodies in late stationary thermophilic xylanase reported) and had half-life at that phase of growth, the function of which is not understood. temperature of 24 hours. The partially-purified enzyme was Most isolates are fermentative using a range of simple able to release colour from unbleached Pinus radiata pulp carbohydrates, but some isolates are able to grow on and broke down soluble xylan into xylobiose and xylotriose. crystalline cellulose and chemolithotrophy using carbon monoxide as energy source has been reported for one pure An account of the cloning and expression of xynA and xynB genes from D. thermophilum Rt46.B1 was published by culture [68]. Morris et al. [27, 79] who demonstrated that both XynA and There have been relatively few applications for XynB released reducing sugars from Pinus radiata kraft Dictyoglomus enzymes as a result of a number of factors. pulp. The xynB gene coded for XynB, a family 11 xylanase Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 51 that was found to contain a catalytic domain and a xylan- XynB from Dictyoglomus thermophilum Rt46.B.1 was binding domain. A more thermostable derivative, XynB6, anticipated to have greater commercial potential if its was constructed and over-expressed in E. coli and the performance under alkaline conditions could be improved. A recombinant enzyme was examined for its ability to bleach gene shuffling procedure was developed in an attempt to use Eucalyptus kraft-oxygen-treated pulp in Elemental and Total directed evolution to achieve a better performance at alkaline Chlorine-Free procedures, proving superior to XynA and two pH (Degenerate Oligonucleotide Gene Shuffling, DOGS commercial xylanase preparations in trials [27, 79, 80]. At [85,86]). An extensive examination of a library generated by the lowest chemical charge, pulp treated with the xylanase family shuffling did not allow detection of any progeny with had a brightness 6-8% ISO greater than the brightness of the improved pH characteristics. This result was explained later untreated reference pulp (reviewed in Bergquist et al. [28, when it was shown that the measurements of alkaline pH 81]). activity of the other enzymes in the shuffling were artefactual [19]. Gene shuffling experiments between D. It was realized that it was not economically feasible to thermophilum xynB and Bacillus agaradhaerens badX genes prepare the enzyme in E. coli and considerable effort was demonstrated that alkaline activity was determined by a expended on optimizing XynB expression in the high- number of changed amino acids distributed throughout the secreting fungus Trichoderma reesei Rut-C30. The native shuffled sequence and was not the result of a single mutated gene was not expressed by the fungus until the codon usage was modified to resemble that of Trichoderma [26]. Early site. experiments and yields of D. thermophilum XynB and construction of the shuttle vector for producing sufficient OTHER APPLICATIONS OF DICTYOGLOMUS plasmid DNA in E. coli for transformation into Trichoderma, GLYCOSYL HYDROLASES where the DNA is integrated into a chromosome, were The gene for the β-mannanase from D. thermophilum reviewed by Bergquist et al. [82]. Accordingly, the vector was larger than typical bacterial vectors and had provision Rt46.B1, ManA, was isolated from a λZap library as for the xylanase genes from this organism. It was cloned into a heat- for Trichoderma regulatory and termination sites. The xynB inducible over-expression vector and the enzyme was gene was fused to the catalytic core of the Trichoderma cbh1 produced in E. coli. The purified enzyme had an apparent gene. Several promoters were trialled to allow expression temperature optimum of 80 °C and a pH optimum of 5.0 and secretion of the enzyme into the culture supernatant of under the conditions used. It was particularly active on locust Trichoderma [28, 82]. It has been shown recently that expression is improved by having the multiple copies of the bean gum and examination of the products of digestion suggested that ManA only acted on substituted mannans gene under the control of other inducible promoters [31]. [87]. This type of enzyme may have applications in oil Around the same time, Walsh and Bergquist [83] recovery techniques [88, 89]. expressed the xynA gene in Kluyveromyces lactis. This yeast Nielsen et al. [90] used cattle manure as a substrate for a secretes few native proteins and does not hyperglycosylate heterologous enzymes. A high copy number plasmid vector two-step anaerobic fermentation at a biogas plant in Denmark. The lignocellulosic fibers are incompletely was modified to include the signal sequence of the K. lactis digested in the manure. Caldicellulosiruptor lactoaceticus killer toxin and a strong, inducible promoter. The xynA gene and Dictyoglomus strain B4a were tested for their ability to was ligated as an in-frame fusion into the vector and it was increase the yield of methane. Strain B4a only uses xylan transformed into K. lactis strain CBS1065. The yield and xylose as a substrate for growth but it gave lower yields obtained of approximately 90% pure XynA was greater than that produced in E. coli and showed activity in pulp of methane than Caldicellulosiruptor that has enzymes for the hydrolysis of a greater variety of polysaccharides. bleaching but was expressed at a lower level than commercially viable. GENERAL GENOMICS There have been a number of reports on the activity of various xylanases at alkaline pH’s. Process conditions in A thermostable amylase gene was cloned from a HindIII kraft pulp bleaching generally favour an enzyme that is library from D. thermophilum H-6-12 and expressed in E. active at high pH values. The activities of several glycosyl coli by Fukusumi et al. [91]. No applications were described hydrolase family 11 xylanases claimed to be active under and the gene (amyA) was not annotated in the full genome alkaline conditions were re-determined under optimal sequence of the thermophile when it became available. The conditions and found to have optima in the pH 5-6 range protein was significantly larger than the other two amylases [19]. Only one enzyme tested, BadX from Bacillus subsequently cloned from D. thermophilum H-6-12 but did agaradhaens, was shown to have an alkaline pH optimum. not appear to be a precursor for either AmyB or AmyC. A Significant activity at pH values higher than 8 appears often further paper reported on the cloning of two more amylases to be the result of excess enzyme added to the reaction from this organism [92]. It was suggested that the enzymes mixtures so that substrate is limiting. The different nature of may be useful in starch degradation but no tests were laboratory and industrial substrates needs to be taken into reported apart from a TLC analysis of the saccharides consideration in designing assay conditions. In some cases, produced on hydrolysis of starch that showed differences significant differences were observed in pH profiles between the three enzymes. The authors noted the unusually generated using a small molecule substrate when compared low G:C content of the DNA of the genes encoding these to those generated using xylan [19, 84]. Nearly all microbial enzymes [92]. The D. thermophilum H-6-12 genome has xylanases characterized exhibit acidic or neutral pH optima been sequenced and only two putative amylases have been [19]. recorded with AmyB classified as belonging to family 57 52 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. and AmyC to family 13 (hapmap.expasy.org/proteomes/ statements about its possible use in pulp bleaching but no DICT6.html). demonstration of its applicability. The equivalent enzyme expressed in E. coli that had some N-terminal amino acids Mathrani, and Ahring [93] described the isolation and and the carbohydrate binding module deleted previously had characterization of a strictly xylan-degrading Dictyoglomus from a man-made, thermophilic anaerobic environment in a been shown to give a good bleaching performance by Morris et al. [27]. pulp mill. Their largely taxonomic description drew attention to the fact that the G:C content of the genomic DNA was low In an attempt to reduce pre-treatment of lignocellulose, but was higher than the type species, D. thermophilum with D. thermophilum Rt46.B1 xynA and xynB genes were the implication that it was a distinct species. They described synthesized with codon optimization for plants and were thermophilic and alkalophilic xylanases from several expressed constitutively from the CaMV 35S promoter for Dictyoglomus isolates including an Icelandic culture B4, transient expression in tobacco and stable expression in type strain D. thermophilum and Dictyoglomus B1 Arabidopsis. The biochemical characteristics of both (described earlier). Crude enzymes from culture supernatants enzymes were retained and active enzyme could be assayed were used. The pH assays most probably were substrate- from dried stem tissue [99]. It was proposed by the authors limited as they showed very broad peaks and they were all that as the temperature of plant growth was well below the reported as % of maximal activity rather than kcat/Km. The apparent temperature optima of the enzymes, they could be temperature assays similarly appeared to be substrate activated after harvest and would contribute to biomass limited. They made the claim of being the first to report breakdown under relatively mild conditions. alkaliphilic, thermophilic xylanases, but the results may have Sunna et al. [100] characterized a linker peptide with been due to substrate limitation [19]. high affinity towards materials containing silica. A McCarthy et al. [94] reported on the crystal structure of recombinant form of D. thermophilum XynB was fused to the β-1, 4-xylanase from Dictyoglomus thermophilum the specific C-terminal linker sequence and bound to zeolite, Rt46B.1. The structure was solved at 1.8Å resolution and retaining enzyme activity. These experiments indicated that a showed that the enzyme had a single domain fold fusion protein plus matrix could provide a method for characteristic of family 11 xylanases composed of a jelly roll enzyme concentration from dilute supernatant solutions after of two twisted β-sheets that create a deep substrate-binding fermentation or an immobilized enzyme system for re-use in cleft containing the two catalytic residues Glu90 and industrial processes. Although the system appeared to be Glu180. It is one of the most thermostable xylanases for scalable, no large volume trials have been reported. which the structure has been solved and has a slightly Glycosyl hydrolase genes from D. turgidum were extended C-terminus in comparison to mesophilic isolated by Brumm et al. [101, 102] after library production equivalents. This structure allowed the modeling of mutants in a plasmid vector and by direct cloning from genomic created by directed evolution. An X-ray structure of a DNA based on whole sequence annotation. They identified xylanase from a Streptomyces species with similarities to 12 novel glycosyl hydrolases by shot-gun and direct PCR- XynB was reported by Wouters et al. [95] and was placed in amplification techniques. This microorganism has had its the same subclass as Rt46.B1 XynB since it was claimed to genomic DNA completely sequenced and annotated, be active in alkaline conditions. They noted that the 3 allowing correlation between in silico and isolation results. cysteines in Rt46.B.1 do not form disulfide bridges and that The most outstanding feature was the paucity of cellulase XynB has a longer C-terminus that may contribute to its genes and an alternative pathway for cellulose digestion for thermostabiity. Detailed methods for the isolation, this strain was suggested. Two of the enzymes have been purification and assay of XynA (Family 10) and XynB commercialized for research purposes (Lucigen Corpn., (Family 11) from D. thermophilum Rt46.B1 were published Middleton, WI, USA). by Bergquist et al. [96]. A GH3 β-glucosidase was isolated apparently by direct The influence of temperature on the fluorescence cloning from the D. turgidum sequenced genome. The intensity and enzyme activity of the fusion protein of GFP enzyme produced in E. coli was characterized and found to and D. thermophilum Rt46.B1 xylanase B was examined by be a monomer of 86kDal molecular mass. It was able to Zhang et al. [97]. They fused GFP to the N-terminus with a digest isoflavones from spent coffee grounds, particularly linker to maintain flexibility between the two proteins to converting diazdin to diazdein. It was suggested that the assist folding and found that the refolding properties of the enzyme may be useful for adding value to a product largely gfp portion of the fusion were more temperature-dependent used in compost since isoflavone aglycones have established than that of the XynB portion. They suggested that gfp therapeutical applications and are superior pharmaceutically fusion proteins could aid in the understanding of the folding to isoflavone glycosides [103]. properties of proteins. D-lyxose is a pharmaceutical precursor for antitumour Zhang et al. [98] reported on the expression and and immunostimulatory agents and can be produced from D- characterization of the Dictyoglomus thermophilum Rt46B.1 xylulose by D-lyxose isomerase. The gene was isolated from xylanase xynB gene in Bacillus subtilis. Their results were D. turgidum by similar methods employed by the authors for essentially a confirmation of the work of Morris et al. [27] the β-glucosidase from the same organism [104]. The but the enzyme was expressed in a different bacterial host. enzyme had a higher apparent optimal temperature, They mistakenly attributed the origin of the strain to the deep thermostability and productivity compared to the mesophilic Atlantic Trench but it was isolated from a geothermal pool in homologs that have been evaluated and may be useful in the Rotorua region of New Zealand. The enzyme was secreted into the supernatant by the host and there were Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 53 biochemical transformations involved with therapeutic agent indistinguishable from Taq DNA polymerase with respect to production. error frequencies and the expected 5’-3’ exonuclease activity. It appears that the original selection of T. aquaticus Oh and colleagues (see, for example, [105, 106, 108- for DNA polymerase production was a fortuitous but far- 110]) have isolated a series of genes identified as present in the genomes of Dictyoglomus turgidum and reaching event. Caldocellulosiruptor saccharolyticus from mining of the Holliger and colleagues in a series of papers described completed genomic sequences. They have characterized a L- the use of Taq DNA polymerase in directed evolution with fucose isomerase that catalyzes the isomerization of L-fucose the key initial advance being the directed evolution of the to L-fuculose and D-arabinose to D-ribulose [105]. A similar polymerase itself by compartmentalised self-replication. This gene has been cloned, expressed and characterised from Ca. technique is essentially emulsion PCR where the polymerase saccharolyticus [106] but the D. turgidum enzyme had gene delivered in its E. coli expression host is amplified in superior kinetics and a higher apparent optimal temperature. individual compartments formed by water-in-oil emulsions Neither paper suggested applications for the enzymes but and each initial gene copy is separated from other gene they could be used in the preparation of rare sugars. copies in their own compartments. After three rounds of Similarly, L-rhamnose isomerases have been isolated and compartmentalized replication they isolated a mutant characterized from D. turgidum and Ca. saccharolyticus polymerase with thermostability increased more than ten- [107, 108]. This type of enzyme has a potential application fold compared to wild-type Taq DNA polymerase and in the enzymatic production of the strawberry flavor agent in another that was more than 130-fold resistant to the inhibitor the food industry and in the production of other rare sugars. heparin [112]. A subsequent publication [113] described the The thermophilic enzymes showed superior kinetic behavior extension of the polymerase substrate specificity so that 3’- compared to their mesophilic counterparts. Another enzyme mismatches and a basic sites were by-passed by mutant that has been produced from the D. turgidum genome was polymerases derived by compartmentalized self-replication characterized as a cellobiose-2-epimerase, with activity on β- and were incorporated into the PCR products, unlike the 1,4-and α-1,4-gluco-oligosaccharides and which has situation with the wild-type enzyme. potential for the production of rare oligosaccharides [109]. A Other publications have described mutant Taq DNA β-glucosidase from D. turgidum was shown to have polymerases that are able to incorporate unnatural bases hydrolytic activity on β-linked sugar residues but not on α- efficiently into the PCR product, such as hydrophobic base linked ones. It was proposed that the enzyme could be used analogues, using compartmentalized self-replication (Loakes to provide rare gingeno sides with pharmacological effects et al. [114]; reviewed in Loakes and Holliger [115]) and [110]. In all of these papers, there is laboratory Leconte et al. [116] using phage display, showing that demonstration of the activity and merits of these ‘boutique’ polymerases could be evolved to allow expansion of the enzymes but as yet, no demonstration of acceptance or use genetic code (see also Leconte et al. [116, 117]). by industry although the potential is evident. The compartmentalization self-replication approach was BACTERIAL DNA POLYMERASES extended further by first including a ‘molecular breeding’ step [118] using Thermus aquaticus, T. thermophilus and T. This section is not intended as a review of the flavus DNA polymerase genes to provide a pool of polymerase chain reaction (PCR) or of the applications of recombinant genes for compartmentalized self-replication in Taq DNA polymerase and its use in enzyme engineering, the presence of primers with multiple mismatches gene expression and synthetic biology in general. This representative of lesions in ancient DNA. The selected material has been covered adequately in undergraduate chimeras outperformed the original Taq DNA polymerase textbooks and the general literature. However, we comment and allowed the amplification of genomic DNA from cave on some lesser-known applications of the enzyme and note bears ~47,000 and 60,000 years old [119]. This technique its role in directed evolution and as a mutagen. Taq DNA was extended even further into the isolation of mutant polymerase is almost exclusively a laboratory reagent and polymerases with increased resistance to environmental can be produced economically on a small scale, both as a inhibitors such as bone dust, humic acid, peat extract and tar. recombinant and non-recombinant product. It has a major A different set of Thermus DNA polymerase genes were use in gene construction for expression in heterologous used in the breeding program apart from T. aquaticus and T. vectors and, by manipulation of the synthetic conditions and thermophilus to generate the novel chimeric polymerases reagents, can be used for library formation in directed [120]. While these ‘tailored’ polymerases have widespread evolution. It plays a central role in Synthetic Biology and utility in a wide range of fields they must be considered as Systems Biology research. being limited to academic and laboratory applications. Some may be qualified for applications such as forensics but they Gibbs et al. [111] re-examined the phylogeny of the are not expected to be a major or bulk product in the Thermus species that have been described and showed they commercial market. Many will be particularly useful in gene could be separated into 8 distinct clades on the basis of 16S and genetic pathway construction and systems biology but it rRNA gene sequence analysis. Twenty-two DNA is unlikely that they will form a major commercial industry. polymerase genes were cloned and sequenced and eight enzymes representing each of the clades were purified to From an early stage it was observed that Taq DNA homogeneity and characterized enzymologically. Each of the polymerase had measurable reverse transcriptase (RT) enzymes was able to perform PCR but none were as activity [121] and Myers and Gelfand [122] reported that the thermostable as the original polymerase from T. aquaticus as RT activity of the polymerase from T. thermophilus (Tth shown by half-life measurements. Otherwise, they were polymerase) was 100-fold greater than that shown by Taq. 54 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al.

Reverse transcription bythermophilic polymerases would recombinant genetic constructions largely as a result of their have the advantage over mesophilic retroviral RT’s proof-reading ability and are frequently found in DNA commonly in use for cDNA synthesis as higher incubation polymerase mixtures where low error frequencies are temperatures could be used to destabilize RNA secondary required. Several Thermococcus enzymes have been structures that limit mesophilic RT’s as well as making commercialized as laboratory reagents (for example, Vent possible single-tube reactions for the synthesis of mRNA to DNA polymerase, New England Biolabs and KOD cDNA [122-124]. One of the disadvantages of using the RT polymerase, Toyobo Life Sciences). A number of activity of Tth was the requirement for Mn2+rather than Mg2+ incremental improvements have been made that are as the divalent cation in the reaction, resulting in higher error described in patent applications (for example, refs. [134- frequencies during cDNA synthesis [125], requiring its 136]). Archaeal DNA polymerases have been reviewed in removal, as well as leading to lower yields [126]. the wider context of thermophilic DNA polymerases by Perler et al. [137]. The cloning of archaeal polymerase genes Shandilya et al. [127] surveyed a number of diverse and their expression in E. coli may be complicated by the thermophilic bacteria for DNA polymerases that copied presence of inteins within the gene. In one example, no RNA efficiently at elevated temperatures in the presence of 2+ inteins were found in the DNA polymerase from Mg . Three out of nine DNA polymerases cloned and Thermococcus sp. 9°N-7 [138] but others have introduced expressed from the thermophiles showed outstanding RT activity as judged by their ratios of DNA polymerase/RT- restriction sites for reassembly of individually amplified fragments or primer extension to generate inteinless genes specific activity. Comparison of the DNA polymerase genes [139-141]. Griffiths et al. [142] could not isolate for Bacillus EA1, Clostridium stercorarium and recombinant colonies containing T. zilligii and Caldibacillus CompA2 showed six unique residues that were Thermococcus GT DNA polymerases and developed a not present in a Clostridium thermocellum DNA polymerase technique that predicted intein splice sites and constructed that has minimal RT activity. Unfortunately, modeling of the effect of these residues on the structure of Taq polymerase primers that allowed in-frame ligation of PCR products that eliminated the inteins. The resulting recombinant T. zilligii and their individual and combined effects on RT activity polymerase had a low error frequency equivalent to the (Gibbs MD, Bergquist PL. Unpublished, 2007) were not KOD1 enzyme and was able to generate PCR products at pursued. low template concentrations. Although the archaeal DNA Others have approached the RT activity of DNA polymerases are of considerable interest from the point of polymerases in different ways. A randomized approach using view of molecular mechanisms of replication, they have had error-prone PCR and an N-terminal deleted form of Taq a predominantly boutique role as laboratory reagents polymerase was used by Sauer and Marx [128] to generate a ensuring fidelity in PCR reactions, usually in association small library. They isolated two mutants that were able to with DNA polymerases from bacteria. reverse transcribe RNA in a sequence-dependent manner. Schönbrunner et al. [129] observed that many of the LIPASES problems of performing ‘long’ RT/PCR have been resolved by using a mixture of enzymes and employed a genetic Lipases (triacylglycerol hydolases) are enzymes capable fusion constructed from a Thermus isolate and Thermotoga of hydrolyzing triglycerides at the interface between the maritima DNA polymerases. Mutations were introduced by insoluble substrate and water. They are important rational design and a number of recombinant polymerases biocatalysts for a number of bio-transformations in synthetic were identified with 3’-5’ proofreading ability that were organic chemistry. Many show high levels of chemio-, regio- capable of single enzyme RT/PCR. Directed evolution was and enantio-selectivity and tolerate organic solvents. Their performed by Ong et al. [130] using Taq DNA polymerase catalysis of synthetic reactions with a broad spectrum of in a modified compartmentalized self-replicating system that substrates makes them suitable for a number of allowed diversification of a short region of the polymerase biotechnological applications. Microbial lipases and their gene (a ‘patch’) to be replicated. This system allowed the applications have been reviewed by Hasan et al. [143] isolation of a mutant able to incorporate ribonucleotide and although most examples describe mesophilic enzymes. deoxyribonucleotide triphosphates into the PCR product. Thermostability is a desirable attribute for The enzyme had essentially a wild-type sequence with four biotechnological applications of lipases in both hydrolytic localized mutations. A recent paper from the same group but and synthetic catalyses with increased solubility being a using the archaeal DNA polymerase from Thermococcus factor in the former case and resistance to denaturation in gorgonarius demonstrated that a single mutation allowing organic solvents in the latter. Initial papers on thermostable trans-lesion synthesis across an abasic site together with a ‘steric-gate’ mutation at the active site transformed the lipases described enzymes from Bacilli and emphasized applications [144-149]. Lipases from extreme thermophiles Thermococcus polymerase into an RNA polymerase; have been isolated only recently from culturable allowing them to claim that the adaptive path from a DNA to microorganisms [150] and from metagenomic DNA from and RNA polymerase was a short one [131]. high temperature enrichments [151]. Most interest on lipases has focused on synthetic applications in organic solvents, ARCHAEAL DNA POLYMERASES with, for example, the lipase from Thermoanaerobacter thermohydrosulfuricus highly S-stereospecific towards esters Archaeal DNA polymerases have been less prominent in of secondary alcohols in reactions carried out at 70 °C. research applications apart from extensive studies on their Sunna et al. [152] showed that the lipase from Geobacillus manner of replicating archaeal chromosomes [132, 133]. The Tp10.A.1 showed high enantioselectivity on racemic ester polymerases have found utility in applications involving Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 55 substrates and Hutchins et al. [153] extended this ability to and discussed. Knowledge of fermentation techniques lipases from Bacillus thermoleovorans and Geobacillus coupled with an understanding of the physiology of the strains OK4.A1 and Mk1.A1. Other applications are in organism are emphasized as necessary requirements for the detergent manufacture, the oils and fats and the flavours and enhanced production of heterologous proteins. aromas sections of the food industry and the treatment of Is there a role for a Synthetic Biology approach to tailor pitch in the pulp and paper industry [143, 154]. Salameh and microorganisms and their enzymatic pathways to industrial Wiegel [155] have reviewed the possible applications of processes? This approach is encountered frequently in the lipases from extreme thermophiles but note the continued recent literature. The past 25 years has seen an increased use of mesophilic enzymes in these industries. appreciation of the microbial biodiversity of extreme Temperature-stable lipases may have more mundane but environments. The search for hyperthermophilic microorgan- equally important uses. The heat stable lipases from the isms has revealed numerous Bacteria and Archaea able to Bacilli that were produced initially for applications in bio- grow above 70 °C and many of their enzymes have been transformations were tested against oils discharged into isolated and studied for their academic interest and possible waste waters (olive oil, palm oil, palm fruit oil and canola industrial uses. The genes for these enzymes reflect selective oil). The lipase from Geobacillus Tp10.A.1 produced in E. pressures in their evolution over substantial periods of time coli was significantly superior to the enzymes expressed and many researchers have considered them to be suitable from the genes from the seven other Bacilli in its ability to starting points for incremental improvement to fulfill roles hydrolyse the oils to triacyl glycerols for disposal(Te’o VSJ, that they may not have encountered in their evolution. The Bergquist PL. Unpublished, 2011). The lipase could have a emergence of systems biology and synthetic biology has role in bioremediation if it could be produced in bulk at an raised the question as to whether it may be better to design economic level. and build cells with customized novel functions rather than alter small areas of an existing genome by mutation or SOME FINAL ISSUES acquisition of heterologous genes. Essentially, the emphasis here is to put the engineering back into genetic engineering A survey of the published literature shows that there is by developing standardized parts (biobricks) for the considerable potential for extreme thermophiles and their construction of novel genomes, for example, see Gingko enzymes in a number of areas of biotechnology and a BioWorks, www.gingkobioworks.com. Their objective is to number of proof of concept experiments have been reported. apply engineering methodologies to the development of new The reality is that up to the present, there has been very organisms using standard components that operate limited uptake in areas that require enzymes as bulk predictably. Ginkgo researchers use a growing collection of chemicals. The main use of extremozymes has been in what re-usable genetic parts and host strains to customize are essentially niche areas involving DNA manipulation or organisms for particular needs. Similar themes are evident in specialized boutique areas that do not require bulk, such as the establishment of synthetic biology centres at MIT fragrances and flavor components. The situation may change (www.synbio.mit.edu), Harvard (www.wyss.harvard.edu) in the future as a result of the world-wide emphasis on bio- and the University of California at Berkeley energy and there will be a choice of continued screening and (www.biofab.org) and others. Recent publications have isolation of enzymes and pathways as contrasted with demonstrated that the ‘biobricks’ can be incorporated into wholesale construction of pathways and organisms using the redesigned pathways, replacing natural parts with synthetic developing techniques of synthetic biology that are arousing ones (see, for example, refs. [156-158]). The development of considerable interest. Irrespective of the route chosen, some the ‘Gibson Assembly’ [159-162] achieved through the use basic facts of cell physiology and enzymology will have to of Taq DNA polymerase to synthesize the overlapping PCR be accommodated to produce biocatalysts in large amounts products has allowed the construction of large DNA for industrial use at a suitable price. In this brief concluding assemblies for genomic mutation and pathway reassembly section, we have chosen to outline areas that we consider to where multiple genes for enzymes and their regulatory be important when viewed against the published literature factors are required. rather than provide a recapitulation of material that has been While the construction of improved single genes and summarized already. pathways may be imminent by way of synthetic biology, Most of the applications of Dictyoglomus and other there are some physico-chemical barriers that may be enzymes from hyperthermophiles that have been suggested difficult to overcome. For example, consider the complex require the proteins to be produced cheaply as bulk chemical situation that pertains in trying to change the pH optimum of products. Transfer of the genes into the usual ‘cell factory’ a family 11 xylanase using data from Bacillus circulans as strains normally used for bulk production has turned out to an example [163, 164]. Two glutamic acid residues are be difficult for the expression of genes from other sources involved at the active site. Glu172 has a dual role in (heterologous proteins). This area has been reviewed by catalysis: at the beginning of catalysis it acts as a general Nevalainen et al. and Nevalainen and Peterson [20, 21] who acid by donating a proton to the glycosidic oxygen. Hence it provide a commentary on the difficulties in getting must be protonated at near neutral pH which requires an expression of heterologous proteins in filamentous fungi, extremely elevated pKa for Glu172 of around 6.8, compared using D. thermophilum XynB as a model gene product. to the approximately 4.0 for free glutamic acid. Later in Alterations of the vector sequence to provide suitable catalysis, Glu172 has to act as a general base and remove a processing sites, tags for the secretion of the protein and the proton from water, which requires a completely different problem up-regulation of proteases attendant on the pKa. What is believed to happen is that the enzyme changes expression of the heterologous gene have been recognized shape during catalysis, thereby placing the Glu172 in a 56 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. different hydrophobic and ionic environment which lowers [18] Gibbs MD, Reeves RA, Choudhary PR, et al. Alteration of the pH its pK to around 6.0, thus favouring the acquisition of a optimum of a family 11 xylanase, XynB6 of Dictyoglomus a thermophilum. N Biotechnol 2010; 27: 803-6. proton. The enzyme then changes shape again and puts [19] Gibbs MD, Reeves RA, Choudhary PR, et al. The activity of Glu172 back in the 'starting' position where its pKa is back at family 11 xylanases at alkaline pH. N Biotechnol 2010; 27: 795- 6.8 [165]. These results suggest that to change the pH 802. optimum of a hyperthermophilic xylanase such as XynB, [20] Nevalainen KMH, Te’o VSJ, Bergquist PL. Heterologous protein will require quite elaborate structural changes to the enzyme expression in filamentous fungi. Trends Biotechnol 2005; 468-74. [21] Nevalainen H, Peterson R. Heterologous production of proteins in that might not be compatible with high temperature stability. Trichoderma. In: Biotechnology and Biology of Trichoderma; In addition, acid residues with very high pKa's like Glu172 Gupta VK, Ed. Elsevier Science B. V. Amsterdam 2013. In Press. are inherently unstable and to increase the pKa any further [22] Ward OP. Production of recombinant proteins by filamentous may destabilize the enzyme. Will the new Gene Engineers be fungi. Biotechnol Adv 2012; 30: 1119-39. [23] Schmallmey M, Singh A, Ward OP. Developments in the use of able to accommodate these requirements for enzyme activity Bacillus species for industrial production. Can J Microbiol 2004; in a biobrick? 50: 1-17. [24] Van Dijl JM, Hecker M. Bacillus subtilis: From soil bacterium to super-secreting cell factory. Microb Cell Fact 2013; 12: 3-6. CONFLICT OF INTEREST [26] De Faria FP, Te'o VSJ, Bergquist PL, et al. Expression and processing of a major xylanase (XYN2) from the thermophilic The authors are founding scientists and shareholders in fungus Humicola grisea var. thermoidea in Trichoderma reesei. ZyGEM Corpn. Ltd. Lett Appl Microbiol 2002; 34: 119-23. [26] Te’o VSJ, Cziferszky AE, Bergquist PL, et al. Codon optimisation of the thermophile xylanase gene xynB from Dictyoglomus ACKNOWLEDGEMENTS thermophilum for expression in Trichoderma reesei. FEMS Microbiol Lett 2000; 190: 13-9. Declared none. [27] Morris DD, Gibbs MD, Chin CW, et al. Cloning of the xynB gene from Dictyoglomus thermophilum strain Rt46B.1 and action of the gene-product on kraft pulp. Appl Environ Microbiol 1998; 64: REFERENCES 1759-65. [28] Bergquist P, Te’o V, Gibbs M, et al. Recombinant bleaching [1] Woese CR. Microbiology in transition. Proc Nat Acad Sci 1994; enzymes from thermophiles expressed in fungal hosts. In: 91: 1601-3. Applications of Enzymes to Lignocellulosics; Mansfield SD, [2] Pace NR. A molecular view of microbial diversity and the Saddler JD, Eds. Amer Chem Soc Symp Series 2003; 855: 435-45. biosphere. Science 1997; 276: 734-40. [29] Kautto L, Grinyer J, Paulsen I, et al. Stress effects caused by the [3] Staley JT, Knonopka A. Measurement of the in situ activities of expression of a mutant cellobiohydrolase I and proteasome non photosynthetic microorganisms in aquatic and terrestrial inhibition in Trichoderma reesei Rut-C30. N Biotechnol 2013; 30: habitats. Ann Rev Microbiol 1985; 39: 321-46. 183-91. [4] Reisenfeld CS, Schloss PD, Handelsman J. Metagenomics; [30] Nevalainen H, Hekelaar J, Uusitalo J, et al. Transcriptional changes Analysis of microbial communities. Ann Rev Genet 2004; 38: 525- in Trichoderma reesei expressing a heterologous bacterial xynB 52. gene from the thermophile Dictyoglomus thermophilum. Poster, 7th [5] Handelsman J. Metagenomics: Spending our inheritance on the th European Fungal Genetics (ECFG) Conference, 17-20 April, future. Microb Biotechnol 2009; 2: 138-9. 2004. Copenhagen, Denmark. Abstracts VIIIp-30, p. 201. [6] Cowan D, Daniel R, Morgan H. Thermophilic proteases: Properties [31] Miyauchi S, Te’o VSJ, Bergquist PL, et al. Expression of a and potential applications. Trends Biotechnol 1985; 3: 68-72. bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 [7] Coolbear T, Daniel RM, Morgan HW. The enzymes from extreme glycosyl hydrolase gene promoters. N Biotechnol 2013; 30: 523- thermophiles: Bacterial sources, thermostabilities and industrial 30. relevance. Adv Biochem Eng/Biotechnol 1992; 45: 57-98. [32] Miyauchi S, Te’o VSJ, Nevalainen KMH, Bergquist PL. [8] Vieille C, Zeikus GJ. Hyperthermophilic enzymes: Sources, uses Simultaneous expression of the bacterial Dictyoglomus and molecular mechanisms for thermostability. Microbiol Mol Biol thermophilum xynB gene under three different Trichoderma reesei Rev 2001; 65: 1-43. promoters. N Biotechnol 2014; 31: 98-103 [9] Haki GD, Rakshit SK. Developments in industrially important [33] Li S, Yang X, Yang S, et al. Technology prospecting on enzymes: thermostable enzymes: A review. Biores Technol 2003; 89: 17-34. Application, marketing and engineering. Comput Struct Biotechnol [10] De Miguel BT, Barros-Velázquez J, Villa TG. Industrial J 2012; 2: e201209017. applications of hyperthermophilic enzymes: a review. Protein Pept [34] Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H. Lett 2006; 12: 645-51. Proteinase K from Tritirachium album Limber. Eur J Biochem [11] Cowan DA, Fernandez-Lafuente R. Enhancing the functional 1974: 47: 91-7. properties of thermophilic enzymes by chemical modification and [35] McHale RH, Stapleton PM, Bergquist PL. Rapid preparation of immobilization. Enzyme Microb Technol 2011; 49: 326-46. blood and tissue samples for polymerase chain reaction. [12] Peterson ME, Eisenthal R, Danson MJ, et al. A new intrinsic Biotechniques 1991; 10: 20-1. thermal parameter for enzymes reveals true temperature optima. J [36] Borges KM, Bergquist PL. A rapid method for preparation of Biol Chem 2004; 279: 20717-22. bacterial chromosomal DNA in agarose plugs using Thermus [13] Eisenthal R, Peterson ME, Daniel RM, et al. The thermal behavior Rt41A proteinase. Biotechniques 1992; 12: 222-3. of enzyme activity: Implications for biotechnology. Trends [37] McHale R, Bergquist PL, Peek K. PreTaq™: A substitute for Biotechnol 2006; 24: 289-92. Proteinase K. Focus 1993; 15: 16-8. [14] Daniel RM, Danson MJ, Eisenthal R, et al. The effect of [38] Fung MC, Fung KY. PCR amplification of mRNA directly from a temperature on enzyme activity: New insights and their crude cell lysate prepared by thermophilic protease digestion. Nucl implications. Extremophiles 2008; 12: 51-9. Acids Res 1991; 19: 4300. [15] Daniel RM, Danson MJ. A new understanding of how temperature [39] Saul DJ, Williams LC, Toogood HS, Daniel RM, Bergquist PL. affects the catalytic activity of enzymes. Trends Biochem Sci 2010; Sequence of the gene encoding a highly thermostable neutral 35: 584-91. proteinase from Bacillus sp. strain EA1: Expression in Escherichia [16] Daniel RM, Peterson ME, Danson MJ, et al. The molecular basis of coli and characterisation. Biochim Biophys Acta 1996; 1308: 74- the effect of temperature on enzyme activity. Biochem J 2010; 425: 80. 353-60. [40] Moss D, Harbison SA, Saul DJ. An easily-automated, closed-tube [17] Viikari L, Kantelinen A, Sundquist J, Linko M. Xylanases in forensic DNA extraction using a thermostable proteinase. Int J bleaching: from an idea to an industry. FEMS Microbiol Rev 1994; Legal Med 2003; 117: 340-9. 13: 335-50. Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 57

[41] Rainey FA, Donnison AM, Janssen PH, et al. Description of Viikari L, Eds. Washington, D.C.: Am Chem Soc Symp Ser 1996; Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: An 655: 85-100. obligately anaerobic, extremely thermophilic, cellulolytic [62] VanFossen AL, Verhaart MRA, Kengen SMW, et al. Carbohydrate bacterium. FEMS Microbiol Lett 1994; 120: 263-6. utilization patterns for the extremely thermophilic bacterium [42] Huang CY, Patel BK, Mah RE, et al. Caldicellulosiruptor Caldicellulosiruptor saccharolyticus reveal broad growth substrate owensensis sp. nov., an anaerobic, extremely thermophilic, preferences. Appl Environ Microbiol 2009; 75: 7718-24. xylanolytic bacterium. Int J Syst Bacteriol 1998; 48: 91-7. [63] VanFossen AL, Ozdemir I, Zelin SI, et al. Glycoside hydrolase [43] Nielsen P, Mathrani IM, Ahring BK. Thermoanaerobium inventory drives plant polysaccharide deconstruction by the acetigenum spec. nov., a new anaerobic, extremely thermophilic, extremely thermophilic bacterium Caldicellulosiruptor xylanolytic non-spore-forming bacterium isolated from an saccharolyticus. Biotechnol Bioeng 2011; 108: 1559-69. Icelandic hot spring. Arch Microbiol 1993; 159: 460-4. [64] Dam P, Kataeva I, Yang SJ, et al. Insights into plant biomass [44] Svetlichnyi VA, Svetlichnaya TP, Chernykh NA, et al. conversion from thegenome of the anaerobic thermophilic Anaerocellum thermophilum gen. nov sp. nov: An extremely bacterium Caldicellulosiruptor bescii DSM 6725. Nucl Acids Res thermophilic cellulolytic eubacterium isolated from hot springs in 2011; 39: 3240-54. the Valley of Geysers. Microbiology1990; 59: 598-604. (English [65] Ozdemir I, Blumer-Schuette SE, Kelly RM. S-layer homology translation of Mikrobiologiya) domain protein Csac_0678 and Csac_2722 are implicated in plant [45] Bergquist PL, Saul DJ, Gibbs MD, et al. Molecular diversity of polysaccharide deconstruction by the extremely thermophilic thermophilic cellulolytic and hemicellulolytic bacteria. FEMS bacterium Caldicellulosiruptor saccharolyticus. Appl Environ Microbiol Ecol 1999; 28: 99-110. Microbiol 2012; 78: 768-77. [46] Turner P, Mamo G, Karlsson E. Potential and utilization of [66] Tripathi SA, Olson DG, Arygos DA, et al. Development of pyrF- thermophiles and thermostable enzymes in biorefining. Microbiol based genetic system for targeted gene deletion in Clostridium Cell Fact 2007; 6: 9. thermocellum and creation of a pta Mutant. Appl Environ [47] Blumer-Schuette SE, Katayna J, Westpheling J, et al. Extremely Microbiol 2010; 76: 6591-9. thermophilic microorganisms for biomass conversion: Status and [67] Westpheling J, Chung D, Cha M, et al. Methylation in vitro and in prospects. Curr Opinion Biotechnol 2008; 19: 210-7. vivo enables DNA transformation of Caldicellulosiruptor species: [48] Blumer-Schuette SE, Ozdemir I, Mistry D, et al. Complete genome use for metabolic engineering and direct conversion of biomass to sequences for the anaerobic, extremely thermophilic plant biomass- fuels and chemicals. Poster Abstract 2-41. 35th Symp Biotechnol degrading bacteria Caldicellulosiruptor hydrothermalis, Caldicel- Fuels Chem 2013; 94. Portland OR. lulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, [68] Kochetkova TV, Rusanov II, Pimenov NV, et al. Anaerobic Caldicellulosiruptor owensensis, and Caldicellulosiruptor lactoa- transformation of carbon monoxide by microbial communities of ceticus. J Bacteriol 2011; 193: 1483-4. Kamchatka hot springs. Extremophiles 2011; 15: 319-25. [49] Blumer-Schuette SE, Giannone RJ, Zuranski JV, et al. [69 ] Nakamura SK, Wakabayashi K, Nakai R, et al. Purification and Caldicellulosiruptor core and pangenomes reveal determinants for some properties of an alkaline xylanase from alkaliphilic Bacillus noncellulosomal thermophilic deconstruction of plant biomass. J sp. strain 41M-1. Appl Environ Microbiol 1993; 59: 2311-6. Bacteriol 2012; 194: 4015-28. [70] Yang VW, Zhuang Z, Elegir G, et al. Alkaline-active xylanase [50] Gibbs MD, Reeves RA, Farrington GK, et al. Multidomain and produced by an alkaliphilic Bacillus sp. isolated from kraft pulp. J multifunctional glycosyl hydrolases from the extreme thermophile Ind Microbiol 1995; 15: 434-41. Caldicellulosiruptor isolate Tok7B.1. Curr Microbiol 2000; 40: [71] Gessesse A, Gashe BA. Production of an alkaline xylanase by an 333-40. alkaliphilic Bacillus sp. isolated from an alkaline soda lake. J Appl [51] Bayer E, Shoham Y, Lamed R. Cellulose-decomposing bacteria Microbiol 1997: 83: 402-6. and their enzyme systems. In: The Prokaryotes 2006; Dworkin M, [72] Chen YL, Tang TY, Cheng KJ. Directed evolution to produce an Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, Eds. alkaliphilic variant from a Neocallimatrix patricianum xylanase. Springer New York 2006; pp. 578-617. Can J Microbiol 2001; 47: 1088-94. [52] Henrissat B, Bairoch A. Updating the sequence-based classification [73] Shibuya H, Kaneko S, Hayashi K. A single amino acid substitution of glycosyl hydrolases. Biochem J 1996; 316: 695-6. enhances the catalytic activity of Family 11 xylanase at alkaline [53] Saul DJ, Williams LC, Grayling RA, et al. celB, a gene coding for pH. Biosci Biotech Biochem 2005; 69: 1492-7. a bifunctional cellulase from the extreme thermophile [74] Mathrani JM, Ahring BK, Anker E. Novel microorganism and "Caldocellum saccharolyticum". Appl Environ Microbiol 1990; 56: novel enzymes. European Patent WO92/18612, 1992. 3117-24. [75] Patel BKC, Morgan HW, Wiegel J, Daniel RM. Isolation of an [54] Gibbs MD, Saul DJ, Luthi E, Bergquist PL. The beta-mannanase extremely thermophilic chemoorganotrophic anaerobe similar to from "Caldocellum saccharolyticum" is part of a multidomain Dictyoglomus thermophilum from New Zealand hot springs. Arch enzyme. Appl Environ Microbiol 1992; 58: 3864-7. Microbiol 1987; 147: 21-4. [55] Te'o VSJ, Saul DJ, Bergquist PL. celA, another gene coding for a [76] Adamson AK, Lindhagen J, Ahring BK. Optimization of multidomain cellulose from the extreme thermophile Caldocellum extracellular xylanase production by Dictyoglomus sp. B1 in saccharolyticum. Appl Microbiol Biotechnol 1995; 43: 291-6. continuous culture. Appl Microbiol Biotechnol 1995; 44: 327-32. [56] Park JI, Kent MS, Datta S. Enzymatic hydrolysis of cellulose by [77] Rāttö M, Mathrani IM, Ahring B, et al. Application of thermostable the cellobiohydrolase domain of CelB from the hyperthermophilic xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps. bacterium Caldicellulosiruptor saccharolyticus. Biores Technol Appl Microbiol Biotechnol 1994; 41: 130-3. 2011; 102: 5988-94. [78] Gibbs MD, Reeves RA, Bergquist PL. Cloning, sequencing and [57] Lüthi E, Love DR, McAnulty J, et al. Cloning, sequence analysis expression of a xylanase gene from the extreme thermophile and expression of genes encoding xylan-degrading enzymes from Dictyoglomus Rt46B.1 and the activity of the enzyme on fiber- the thermophile, Caldocellum saccharolyticum. Appl Environ bound substrate. Appl Environ Microbiol 1995; 61: 4403 -8. Microbiol 1990; 56: 1017-24. [58] Morris DD, Gibbs MD, Ford M, et al. Family 10 and 11 xylanase [58] Anderson A, Bergquist PL, Daniel RM, et al. European Patent genes from Caldicellulosiruptor isolate Rt69B.1. Extremophiles EP0921188A2. 09061999. 1999; 3: 103-11. [59] Riedel K, Bronnenmeier K. Intramolecular synergism in an engi- [79] Morris DD, Gibbs MD, Bergquist PL. Cloning of a family G neered exo-endo-1,4-β-glucanase fusion protein. Mol Microbiol xylanase gene (xynB) from the extremely thermophilic bacterium 1998; 28: 767-75. Dictyoglomus thermophilum and activity of the gene product on [60] Ye L, Su X, Schmitz GE, et al. Molecular and biochemical kraft pulp. In: Enzymes for Pulp and Paper Processing; Jeffries TJ, analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional Viikari L, Eds. Am Chem Soc Symp Ser 1996; 655: 101-15. enzyme from the hyperthermophilic bacterium Caldicellulosiruptor [80] Bergquist PL, Gibbs MD, Saul DJ, et al. Isolation and expression bescii. Appl Environ Microbiol 2012; 78: 7048-59. of genes for hemicellulases from extremely thermophilic culturable [61] Bergquist PL, Gibbs MD, Saul DJ, et al. Families and functions of and unculturable bacteria. In: Enzyme Applications in Fiber novel thermophilic hemicellulases in the facilitated bleaching of Processing, Eriksson K-E, Cavaco-Paulo A, Eds. Am Chem Soc pulp. In: Enzymes for pulp and paper processing; Jeffries TJ, Symp Ser 1998; 687: 168-77. 58 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al.

[81] Bergquist PL, Nevalainen KMH, Te’o VSJ, et al. Recombinant [103] Kim Y-S, Yeom S-J, Oh D-K. Characterization of a GH3 family β- enzymes from thermophilic microorganisms produced in fungal glucosidase from Dictyoglomus turgidum and its application to the hosts. Biochem Soc Trans 2004; 32: 293-7. hydrolysis of isoflavone glycosides in spent coffee grounds. J Agric [82] Bergquist PL, Te’o VSJ, Gibbs MD, et al. Production of enzymes Food Chem 2011; 59: 11812-8. from thermophilic microorganisms in fungal hosts. Extremophiles [104] Choi JG, Hong SH, Kim YS, Kim KR, Oh DK. Characterization of 2002; 6: 177-84. a recombinant thermostable D-lyxose isomerase from [83] Walsh DJ, Bergquist PL. Expression and secretion of a Dictyoglomus turgidum that produced D-lyxose from D-xylulose. thermostable xylanase in Kluyveromyces lactis. Appl Environ Biotechnol Lett 2012: 34: 1079-85. Microbiol 1997; 63: 3297-300. [105] Hong SY, Lim YR, Kim YS, Oh DK. Molecular characterization of [84] Poon DK, Webster YP, Withers SG, et al. Characterizing the pH- a thermostable L-fucose isomerase from Dictyoglomus turgidum dependent stability and catalytic mechanism of the family 11 that isomerizes L-fucose and D-arabinose. Biochimie 2012; 94: xylanase from the alkalophilic Bacillus agaradhaerens. Carbohyd 1926-34. Res 2003; 338: 415-21. [106] Ju Y-H, Oh D-K. Characterization of a recombinant L-fucose [85] Gibbs MD, Nevalainen KMH, Bergquist PL. Degenerate isomerase from Caldicellulosiruptor saccharolyticus that oligonucleotide gene shuffling (DOGS): A method for enhancing isomerizes L-fucose, D-arabinose, D-altrose and L-galactose. the frequency of recombination with family shuffling. Gene 2001; Biotechnol Lett 2010; 32: 299-304. 271: 13-20. [107] Lin C-J, Tseng W-C, Fang T-Y. Characterization of a thermophilic [86] Bergquist PL, Reeves RA, Gibbs MD. Degenerate Oligonucleotide L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus Gene Shuffling (DOGS) and Random Drift Mutagenesis (RNDM): ATCC43494. J Agric Food Chem 2011; 59: 8702-6. two complementary techniques for enzyme evolution. Biomol [108] Kim, Y-S, Shin K-C, Lim Y-R, et al. Characterization of a Engineering 2005; 22: 63-72. recombinant L-rhamnose isomerase from Dictyoglomus turgidum [87] Gibbs MD, Reeves RA, Sunna A, et al. Sequencing and expression and its application for L-rhamnulose production. Biotechnol Lett of a β-mannanase from the extreme thermophile Dictyoglomus 2013; 35: 259-64. tyhermophilum Rt46B.1 and characteristics of the recombinant [109] Kim J-E, Kim Y-S, Kang L-W, et al. Characterization of a enzyme. Curr Microbiol 1999; 39: 351-7. recombinant cellobiose 2-epimerase from Dictyoglomus turgidum [88] Comfort DA, Chhabra SR, Connors SB, et al. Strategic biocatalysis that epimerizes and isomerizes β-1,4- and α-1,4-gluco- with hyperthermophilic enzymes. Green Chem 2004; 6: 459-65. oligosaccharides. Biotechnol Lett 2012; 34: 2061-8. [89] Armstrong CD. Method of fracturing using a mannohydrolase [110] Lee G-W, Kim K-R, Oh D-K. Production of rare gingenosides breaker. US Patent 8,058,212, 2011. (compound Mc, compound Y and aglycon protopanaxadiol) by β- [90] Nielsen HB, Mladenovska Z, Ahring BH. Bioaugmentation of a glucosidase from Dictyoglomus turgidum that hydrolyzes β-linked, two-stage thermophilic (68 °C/55 °C) anaerobic digestion concept but not α-linked, sugars in ginsenosides. Biotechnol Lett 2012; 34: for improvement of the methane yield from cattle manure. 1679-86. Biotechnol Bioeng 2007; 97: 1638-43. [111] Gibbs MD, Reeves RA, Mandelman D, et al. Molecular diversity [91] Fukusumi S, Kamizono A, Horinouchi S, et al. Cloning and and catalytic activity of Thermus DNA polymerases. Extremophiles nucleotide sequence of a heat-stable amylase gene from an 2009; 13: 817-26. anaerobic thermophile, Dictyoglomus thermophilum. Eur J [112] Ghadessy FJ, Ong JL, Holliger P. Directed evolution of polymerase Biochem 1988; 174: 15-21. function by compartmentalized self-replication. Proc Nat Acad Sci [92] Horinouchi S, Fukusumi S, Oshima Y, et al. Cloning and USA 2001; 98: 4552-7. expression in Escherichia coli of two additional amylase genes of a [113] Ghadessy FJ, Ramsey N, Boudsocq F, et al. Generic expansion of strictly anaerobic thermophile, Dictyoglomus thermophilum, and the substrate spectrum of a DNA polymerase by directed evolution. their nucleotide sequences with extremely low guanine-plus- Nature Biotechnol 2004; 22: 755-9. cytosine contents. Eur J Biochem 1988; 176: 243-53. [114] Loakes D, Gallego J, Pinheiro VB, et al. Evolving a polymerase for [93] Mathrani JM, Ahring BK. Isolation and characterization of a hydrophobic base analogues. J Amer Chem Soc 2009; 131: 14827- strictly xylan-degrading Dicyloglomus from a man-made, 37. thermophilic anaerobic environment. Arch Microbiol 1991; 157: [115] Loakes D, Holliger P. Polymerase engineering: towards the 13-7. encoded synthesis of unnatural biopolymers. Chem Commun [94] McCarthy AA, Morris DD, Bergquist PL, et al. Crystal structure of (Camb) 2009; 31: 4619-31. a highly thermostable β-1,4-xylanase from Dictyoglomus thermo- [116] Leconte AM, Chen L, Romesberg EF. Polymerase evolution: philum Rt46B.1 at 1.8Å resolution. Acta Cryst D 2000; 56: 1367- Efforts towards expansion of the genetic code. J Amer Chem Soc 75. 2005; 127: 12470-1. [95] Wouters J, Georis J, Engher D, et al. Crystallographic analysis of [117] Leconte AM, Patel MP, Sass LE, et al. Directed evolution of DNA family 11 endo-β-1,4-xylanase Xyl1 from Streptomyces strain S38. polymerases for next-generation sequencing. Angew Chem Int Ed Acta Cryst D 2001; D57: 1813-9. 2010; 49: 5921-4. [96] Bergquist PL, Gibbs MD, Morris DD, et al. Hyperthermophic [118] Cramer A, Whitehorn EA, Tate E, et al. Improved green xylanases. Meth Enzymol 2001; 330: 301-8. fluorescent protein by molecular evolution using DNA shuffling. [97] Zhang C, Liu MS, Xing XH. Temperature influence on Nat Biotechnol 1996; 14: 315-9. fluorescence intensity and enzyme activity of the fusion protein of [119] d’Abbadie M, Hofreiter M, Vaisman A, et al. Molecular breeding GFP and hyperthermophilic xylanase. Appl Microbiol Biotechnol of polymerases for amplification of ancient DNA. Nat Biotechnol 2009; 84: 511-7. 2007; 25: 939-43. [98] Zhang W, Lou K, Li G. Expression and characterization of the [120] Baar C, d’Abbadie M, Vaisman A, et al. Molecular breeding of Dictyoglomus thermophilum Rt46B.1 xylanase gene (xynB) in polymerase for resistance to environmental inhibitors. Nucl Acids Bacillus subtilis. Appl Biochem Biotechnol 2010; 160: 1484-95. Res 2011; 39: e51. [99] Borkhardt BJ, Harholt J, Ulvskov P, et al. Autohydrolysis of plant [121] Jones MD, Foulkes NS. Reverse transcription of mRNA by xylans by apoplastic expression of thermophilic bacterial endo- Thermus aquaticus DNA polymerase. Nucl Acids Res 1989; 17: xylanases. Plant Biotechnol J 2010; 8: 363-74. 8387-8. [100] Sunna A, Chi F, Bergquist P. Characterization of a linker peptide [122] Myers TW, Gelfand DH. Reverse transcription and DNA with high affinity towards silica-containing materials. N Biotechnol amplification by Thermus thermophilus DNA polymerase. 2013; 30: 485-92. Biochem 1991; 30: 7661-6. [101] Brumm PJ, Hermanson S, Luedtke J, et al. Identification, cloning [123] DiStefano JJ, Busier RG, Mallabar LM, et al. Polymerization and and characterization of Dictyoglomus turgidum CelA, an RNAse H activities of the reverse transcriptases from avian endoglucanase with cellulase and mannanase activity. J Life Sci myeloblastosis, human immunodeficiency, and Moloney murine 2011; 5: 488-96. leukemia viruses are functionally uncoupled. J Biol Chem 1991; [102] Brumm PP, Hermanson S, Hochstein B, et al. Mining 266: 7423-31. Dictyoglomus turgidum for enzymatically active carbohydrases. [124] Harrison GP, Mayo MS, Hunter E, et al. Pausing of reverse Appl Biochem Biotechnol 2011; 163: 205-14 transcriptase on retroviral RNA templates is influenced by Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Current Biotechnology, 2014, Volume 3, No. 1 59

secondary structures both 5’ and 3’ of the catalytic site. Nucl Acids [146] Bell PJL, Nevalainen H, Morgan WH, et al. Rapid cloning of Res 1998; 26: 3433-42. thermoalkalophilic lipases from Bacillus spp. using PCR. [125] Cadwell RC, Joyce GF. Randomization of genes by PCR Biotechnol Lett 1999; 21: 1003-6. mutagenesis. PCR Meth Appl 1992; 2: 28-32. [147] Bell PJL, Sunna A, Gibbs MD, et al. Prospecting for novel lipases [126] Leung DW, Chen E, Goeddel DV. A method for random genes using PCR. Microbiol 2002; 148: 2283-91. mutagenesis of a defined DNA segment using a modified [148] Kim HK, Park SY, Oh TK. Purification and partial characterization polymerase chain reaction. Technique 1989; 1: 11-5. of thermostable carboxyl esterase from Bacillus sterothermophilus [127] Shandilya H, Griffiths K, Flynn EK, et al. Thermophilic bacterial L1. J Microbiol Biotechnol 1997; 7: 37-42. DNA polymerases with reverse-transcriptase activity. [149] Kim HK, Park SY, Lee HK, et al. Thermostable lipase of Bacillus Extremophiles 2004; 8: 243-51. sterothermophilus: production, purification and calcium-dependent [128] Sauer KBM, Marx A. Evolving thermostable reverse transcriptase thermostability. Biosci Biotechnol Biochem 2000; 64: 280-6. activity in a DNA polymerase scaffold. Angew Chem Int Ed 2006; [150] Royter M, Schmidt M, Elend C, et al. Thermostable lipases from 45: 7633-5. the extreme thermophiliic anaerobic bacteria Thermoanaerobacter [129] Schönbrunner N, Fiss EH, Budker O, et al. Chimeric thermostable thermohydrosulfuricus SOL1 and Caldoanaerobacter subterraneus DNA polymerases with reverse transcriptase and attenuated 3’-5’ subsp. tengcongensis. Extremophiles 2009; 13: 769-83. exonuclease activity. Biochem 2006; 45: 12786-95. [151] Chow J, Kovacic F, Dall Antonia Y, et al. The metagenome- [130] Ong JL, Loakes D, Jaroslawski S, et al. Directed evolution of DNA derived enzymes LipS and LipT increase the diversity of known polymerase, RNA polymerase and reverse transcriptase activity in lipases. PLoS One 2012; 7: e47655. a single polypeptide. J Mol Biol 2006; 361: 537-50. [152] Sunna A, Hunter L, Hutton C, et al. Biochemical characterization [131] Cozens C, Pinheiro VB, Vaisman A, et al. A short adaptive path of a recombinant thermoalkalophilic lipase and assessment of its from DNA to RNA polymerases. Proc Nat Acad Sci USA 2012; substrate enantioselectivity. Enz Microbial Technol 2002; 31: 472- 109: 8067-72. 6. [132] Barry ER, Bell SD. DNA replication in the Archaea. Microbiol [153] Hutchins LM, Hunter L, Ehya N, et al. Highly enantioselective Mol Biol Rev 2006; 70: 876-87. recombinant thermoalkalophilic lipases from Geobacillus and [133] Beattie TR, Bell SD. Molecular machines in archaeal DNA Bacillus sp. Tetrahedron Assym 2004; 15: 2975-80. replication. Curr Opinion Chem Biol 2011; 15: 614-9. [154] Bajpal P. Application of enzymes in the pulp and paper industry. [134] Borns M. DNA polymerase fusions and uses thereof. European Biotechnol Prog 1999; 15: 147-55. Patent 1616033, 2006. [155] Salamad M, Wiegel J. Lipases from extremophiles and potential for [135] Anderson PJ, Steffens LD, Urlacher MT, et al. Mutant polymerases industrial applications. Adv Appl Microbiol 2007; 61: 253-83. for sequencing and genotyping. European Patent 1805303, 2007. [156] Kelly JR, Rubin AJ, Davis JH, et al. Measuring the activity of [136] Sato Y, Nishiwaki K, Shimada N, et al.Polypeptides having DNA BioBrick promotors using an in vivo reference standard. J Biol Eng polymerase activity. US Patent 8,334,105, 2013. 2009; 3: 4. [137] Perler FB, Kumar S, Kong H. Thermostable DNA polymerases. [157] Khalil AS, Lu TK, Bashor CJ, et al. A synthetic biology framework Adv Protein Chem 1996; 48: 377-434. for programming eukaryotic transcription functions. Cell 2012; [138] Southworth MW, Kong H, Kucera RB, et al. Cloning of 150: 647-58. thermostable DNA poymerases from hyperthermophilic marine [158] Moon TS, Lou C, Tamsir A, et al. Genetic programs constructed Archaea with emphasis on Thermococcus sp. 9°N-7 and mutations from layered logic gates in single cells. Nature 2012; 491: 249-53. affecting 3’-5’ exonuclease activity. Proc Nat Acad Sci USA 1996; [159] Gibson DG, Benders GA, Andrews-Pfannoch C, et al. Complete 93: 5281-5. chemical synthesis, assembly, and cloning of a Mycoplasma [139] Niehaus F, Frey B, Antranikian G. Cloning and characterization of genitalium genome. Science 2008; 309: 1215-20. a thermostable α-DNA polymerase from the hyperthermophilic [160] Gibson DG, Young L, Chuang R-Y, et al. Enzymatic assembly of archaeon Thermococcus TY. Gene 1997; 204: 153-8. DNA molecules up to several hundred kilobases. Nat Methods [140] Perler FB, Comb DA, Jack WE, et al. Intervening sequences in an 2009; 6: 343-5. Archaea DNA polymerase gene. Proc Nat Acad Sci USA 1992; 98: [161] Gibson DG, Smith HO, Hutchinson CA, et al. Chemical synthesis 5577-91. of the mouse mitochondrial genome. Nat Methods 2010; 7: 901-3. [141] Takagi M, Nishioka M, Kakihara HM, et al. Characterization of a [162] Gibson DG, Glass JI, Lartigue C, et al. Creation of a bacterial cell DNA polymerase from Pyrococcus sp. strain KOD1 and its controlled by a chemically synthesized genome. Science 2010; 319: application to PCR. Appl Environ Microbiol 1997; 63: 4504-10. 1215-20. [142] Griffiths K, Nayak S, Park K, et al. New high fidelity polymerases [163] Wakarchuk WW, Campbell RL, Sung WL, et al. Mutational and from Thermococcus species. Prot Express Purif 2007; 52: 19-30. crystallographic analyses of the active site residues of the Bacillus [143] Hasan F, Ali SA, Hameed A. Industrial applications of microbial circulans xylanase. Protein Sci 1994; 3: 467-75. lipases. Enz Microbial Technol 2006; 39: 235-51. [164] Wakarchuk WW, Sung WL, Campbell RL, et al. [144] Handelsman T, Shoham Y. Production and characterization of an Thermostabilization of the Bacillus circulans xylanase by the extracellular thermophilic lipase from a thermophilic Bacillus sp. J introduction of disulfide bond. Protein Engin 1994; 7: 1379-86. Gen Appl Microbiol 1994; 40: 435-43. [165] McIntosh LP, Hand G, Johnson PE, et al. The pK(a) of the general [145] Schmidt-Dannert C, Sztager H, Stöcklem W, et al. Screening, acid/base carboxyl group of glycosidase cycles during catalysis: A purification and properties of a thermophilic lipase from Bacillus 13C-NMR study of Bacillus circulans xylanase. Biochem 1996; 35: thermocatenulatus. Biochim Biophys Acta 1994; 1214: 43-53. 9958-66.

Received: May 23, 2013 Revised: September 25, 2013 Accepted: October 16, 2013