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Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio‐based product synthesis over more classical petroleum‐based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of “design‐build‐test‐learn” (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new‐to‐industry microorganisms have been investigated as novel cell factories, including the versatile α‐proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly‐β‐hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio‐based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications., Rhodobacter sphaeroides is a purple non‐sulfur bacteria which has served for decades as model laboratory strain for fundamental studies. Nevertheless, in the last decades this microorganism has matured into a versatile microbial cell factory for multiple biotechnological processes. In this work, authors describe this transition by means of the ‘design‐build‐test‐learn’ concept for strain engineering.

Background: Ethyl acetate and hydrogen are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products render the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2. Results: We engineered Escherichia coli, towards improved conversion of formate into hydrogen and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA). Initially 10 strains were evaluated in anaerobic serum bottles with respect to growth, after which four strains were further analyzed. Anaerobic co-production of hydrogen and ethyl acetate via heterologous ethanol acyltransferase (Eat1) was achieved in 1.5-L pH controlled bioreactors. Conclusions: We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70 % of the pathway maximum for ethyl acetate and hydrogen, and propose in-situ product removal via gas stripping as efficient technique to isolate the products of interest.

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and universal genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for universal and inducible control over CRISPR-Cas counterselection. This control delays CRISPR-Cas counterselection, granting more time for the editing event (e.g., by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three bacteria with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a universal gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.

Background Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis. Results This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. Conclusions Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

Additional file 1: Table S1. Overview of pH-controlled batch fermentations in 1.5L Applikon bioreactors with continuous gas stripping. Measured and calculated concentrations of main fermentation products, carbon balance and C-mol yields at end of fermentations are represented as average (AV) with standard deviations (SD) for each duplicate. E. coli BW25113 ΔackAΔldhA (DE3) producing Eat1 variants from pET26b plasmids were grown under anoxic conditions in minimal medium containing 55 mM glucose. Expression of Eat1 was induced by IPTG.

Additional file 2: Figure S2. Lack of correlation between ethyl acetate formation and predicted strength of the RBS controlling the production of K. marxianus trEat1. Ethyl acetate titres were obtained from E. coli BW25113 ΔackAΔldhA (DE3) producing K. marxianus trEat1 variants at 0.01 mM IPTG concentration (Fig. 2b). The translation initiation rates of the RBS were predicted with the RBS calculator [22].

Additional file 1. Additional figures and tables.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli. By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20–30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell’s phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

Background Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from

A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general., Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose–l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose. IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis. These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 Δku80. We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains. IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium

An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 α-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives α-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-β-D-xylopyranoside) and pNPA (p-nytrophenyl-α-L-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50 °C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for α-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were Km of 23 ± 3 mM and kcat of 104 ± 7 s−1. Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its clade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily.

Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors. (C) 2016 Elsevier B.V. All rights reserved.

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00792-017-0939-x) contains supplementary material, which is available to authorized users.

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 A resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 A, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden–Meyerhof and the Entner–Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor. Phylogenetic analysis of the 16S rDNA sequence showed 100% sequence similarity to Pseudomonas stutzeri DSM 50227 and 98.6% sequence similarity to the type strain of P. stutzeri (DSM 5190(T)). The species P. stutzeri possesses a high degree of genotypic and phenotypic heterogeneity. Therefore, eight genomic groups, termed genomovars, have been proposed based upon deltaTm values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA-DNA hybridization. In this study, DNA-DNA hybridization between strain AW-1(T) and P. stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5% similarity. DNA-DNA hybridization between P. stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4% similarity. DNA-DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P. stutzeri. However, strain AW-1(T) and P. stutzeri DSM 50227 are related at the species level. The physiological and biochemical properties of strain AW-1(T) and the two P. stutzeri strains were compared. A common characteristic of P. stutzeri strains is the ability to denitrify. However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate. Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway. Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity. P. stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate. Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P. stutzeri strains. Chlorite dismutase activity was absent in extracts of both P. stutzeri strains but was present in extracts of strain AW-1(T). Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp. nov. The type strain is strain AW-1(T) (= DSM 13592(T) = ATCC BAA-443(T)).

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α 3 β 3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K m values for perchlorate and chlorate were 27 and

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

The current knowledge on microbial reductive dechlorination of chlorinated ethenes (CEs) and its application are discussed. Physiological studies on CEs dechlorinating microorganisms indicate that ...

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis -1,2-dichloroethene at the optimum temperature of 60 to 65°C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H 2 , formate, or acetate. Fumarate and l -malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H 2 , CO 2 , and succinate. With PCE, less H 2 was formed, suggesting that PCE competed for the reducing equivalents leading to H 2 . PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of ∼60 μM, a high dechlorination rate of 1.1 μmol h −1 mg −1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus , and with the genus Desulfotomaculum (86%).

A predicted esterase ( EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 angstrom resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 angstrom. There are two molecules in the asymmetric unit, with a VM of 2.9 angstrom(3) Da(-1) and 58% solvent content.

The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.

10.1016/j.egypro.2012.09.036 Given the highly reduced state of carbon in glycerol and its availability as a substantial byproduct of biodiesel production, glycerol is of special interest for sustainable biofuel production. Glycerol was used as a substrate for biohydrogen production using the hyperthermophilic bacterium, Thermotoga maritima and Thermotoga neapolitana. Both species metabolized glycerol to mainly acetate and hydrogen. At glycerol concentrations of 2.5 g/L, hydrogen was produced with a yield of 2.75 and 2.65 mol H2/mol glycerol consumed by T. maritima and T. neapolitana respectively. Additionally, the effect of initial pH (ranging between pH 5.0-8.5) and yeast extract concentrations (0.5, 1, 2, 4 g/L) on glycerol fermentation by T. neapolitana was investigated in batch systems. An initial pH value of around 7 was optimal for hydrogen production by T. neapolitana. Lower concentration of yeast extract resulted in a lower H2 production, however increasing the concentration from 2 to 4 g/L did not affect H2 production.

Pyrococcus furiosus harbours several hydrolytic enzyme activities that enable growth on a variety of polymeric substrates like proteins and polysaccharides. Recently, the organism was shown to exhibit an extremely high β-glucosidase activity, apparently involved in hydrolysis of cellobiose. The cytoplasmic enzyme was purified to homogeneity and its properties were compared with those of other glycosidases. This comparison can be summarized as follows: i) the β-glucosidase activity in cell-free extracts is at least 100-fold higher than the P. furiosus α-glucosidase activity; ii) the β-glucosidase comprises 5% of total cell protein as to only 0.3% for the α-glucosidase; iii) with respect to substrate specifity, Km, pI, and pH-optimum the P. furiosus β-glucosidase resembles other β-glucosidases from microorganisms from lower temperature ranges; iv) compared to these β-glucosidases the P. furiosus enzyme exhibits an enhanced general stability; v) a high similarity is observed with a β-galacto/glucosidase from...

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.

The four carbon alcohols butanol and 2,3-butanediol are the longest chain alcohols found as natural major end-products of microbial fermentation. They represent important bulk chemicals widely used in industry as solvents for lacquers, paints and similar, or as intermediates in chemical synthesis reactions. In this chapter the biological methods for the production of butanol, isopropanol and 2,3-butanediol, including advances in strain development and in the fermentation processes, are described in detail to provide the reader with a state-of-the-art view on these fields. In addition, the biological production of other important commercial alcohols (2-butanol, 1,2- and 1,3-propanediol) with the potential to be biologically produced is briefly reviewed.

Reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene and chloroethane (CA) by crude cell extracts of Methanobacterium thermoautotrophicum delta H with H2 as the electron donor was stimulated by Mg-ATP. The heterodisulfide of coenzyme M (CoM) and 7-mercaptoheptanoylthreonine phosphate together with Mg-ATP partially inhibited ethylene production but stimulated CA production compared Mg-ATP alone. The pH optimum for the dechlorination was 6.8 (at 60 degrees C). Michaelis-Menten kinetics for initial product formation rates with different 1,2-DCA concentrations indicated the enzymatic character of the dechlorination. Apparent Kms for 1,2-DCA of 89 and 119 microM and Vmaxs of 34 and 20 pmol/min/mg of protein were estimated for ethylene and CA production, respectively. 3-Bromopropanesulfonate, a specific inhibitor for methyl-CoM reductase, completely inhibited dechlorination of 1,2-DCA. Purified methyl-CoM reductase, together with flavin adenine dinucleotide and a crude component A fraction which reduced the nickel of factor F430 in methyl-CoM reductase, converted 1,2-DCA to ethylene and CA with H2 as the electron donor. In this system, methyl-CoM reductase was also able to transform its own inhibitor 2-bromoethanesulfonate to ethylene.

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).

Cell extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to perform a hydrogen-dependent reduction of factor 390 (F390), the 8-adenylyl derivative of coenzyme F420. Upon resolution of cell extracts, F390-reducing activity copurified with the coenzyme F420-dependent hydrogenase. This indicates that F390 serves as a substrate of that enzyme. Activity towards F390 was approximately 40-fold lower than that towards coenzyme F420 (0.12 and 5.2 mumol.min-1.mg of protein-1, respectively). In addition, cell extracts catalyzed the hydrolysis of F390 to AMP and coenzyme F420. This hydrolysis required the presence of thiols (6 mM) and much ionic strength (1 M KCl) and was reversibly inhibited by oxygen. The reaction proceeded optimally at pH 8.2 and was Mn dependent. Conditions for F390 hydrolysis in cell extracts are in many respects opposite to those previously described for F390 synthesis.

In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H4MPT) is the carrier of the C1 unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H4MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H4MPT, which is reduced in two subsequent coenzyme F420-dependent reactions to 5-methyl-H4MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H4MPT-dependent reactions are discussed.

The conversion of formaldehyde to methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum was stimulated up to 10-fold by catalytic amounts of the heterodisulfide (CoM-S-S-HTP) of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. The stimulation required the additional presence of ATP, also in catalytic concentrations. ATP and CoM-S-S-HTP were mutually stimulatory on the methylcoenzyme M formation and it was concluded that the compounds were both involved in the reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase. Micromolar concentrations of benzyl viologen or cyanocobalamin inhibited the formaldehyde conversion; these compounds, however, strongly stimulated the reduction of CoM-S-S-HTP. The results described here closely resemble observations made on the activation and reduction of CO2 to formylmethanofuran indicating that this step and the reductive activation of the methyltransferase are controlled by some common mechanism.

A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.

Publisher Summary ADP-dependent glucokinase and phosphofructokinase were demonstrated for the first time by Kengen et al . in the hyperthermophile Pyrococcus furiosus . This anaerobic archaeon grows optimally at 100° by fermenting sugar polymers and polypeptides to mainly acetate and alanine. The catabolism of sugars, such as starch, laminarin, maltose, or cellobiose, has been investigated in detail and has led to the discovery of novel type Embden–Meyerhof pathway, involving several enzymatic steps that are different from the classical ones. In addition to ADP-dependent kinases, the pathway involves a one-step, ferredoxin-dependent conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate instead of the conventional two-step, NAD-dependent and ATP-generating conversion. Furthermore, a remarkable AMP-dependent, ATP-generating pyruvate kinase has been described, which is different from the normal ADP-dependent pyruvate kinase. Moreover, the available P. furiosus genome sequence shows that for several other glycolytic enzymes, no homologous sequences can be identified, suggesting that these enzymes may be different from the classical ones as well. This chapter describes the procedures for assay and purification of both kinases and the cloning and expression of their genes.

The hyperthermophilic archaea are a group of phylogenetically related microorganisms which by definition grow optimally at or above 80°C, with a maximal growth temperature of 90°C or higher (5). The majority of these are strictly anaerobic heterotrophs, most of which are obligately dependent upon the reduction of elemental sulfur (S0) to H2S. A limited number of facultative S0-reducing species are able to grow in the absence of S0 by means of an alternative fermentative-type metabolism. An example of this type of organism is Pyrococcus furiosus, which grows optimally at 100°C, with a temperature maximum of 105°C, by the fermentation of peptides and various carbohydrates, including starch, glycogen, β-glucans, cellobiose, and maltose (12). In addition, pyruvate can also be utilized as a carbon and energy source (9, 33). P. furiosus utilizes a modified Embden-Meyerhof pathway for the catabolism of sugars, which involves a pair of unprecedented ADP-dependent kinases (glucokinase and phosphofructokinase) and a unique glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (18, 26, 35, 37). The main products produced during the fermentation of sugars include acetate, CO2, H2, and alanine (19). During growth on either peptides or carbohydrates, reduced ferredoxin is generated (1, 20). Regeneration of oxidized ferredoxin is assumed to be accomplished by three mechanisms: either by S0 reduction to H2S, by proton reduction to H2, or by the formation of alanine. The third alternative, formation of alanine, is found when P. furiosus is grown in the absence of S0. In addition to acetate, a significant amount of alanine is excreted into the medium (19, 33). The amount of alanine produced varies, with an increase in the H2 partial pressure resulting in an increase in the amount of alanine produced. The transamination of pyruvate with glutamate by the action of an alanine aminotransferase (AlaAT) was detected in cell extracts of P. furiosus (19). Furthermore, this activity was shown to be affected by both the partial pressure of hydrogen as well as the available carbon source, suggesting some form of regulation. Glutamate must be replenished through the action of the NADP-dependent glutamate dehydrogenase (GDH). However, there is some controversy concerning the exact role of GDH in the metabolism, since it has been proposed to serve an anabolic role (7, 27), as well as a catabolic role (32). Interestingly, the activity of the GDH reacted similarly to that of AlaAT under the same growth conditions, further suggesting some coordinated regulation of these enzymes (19). The necessary NADPH can be generated by the transfer of reducing equivalents from reduced ferredoxin to NADP+ by the ferredoxin:NADP oxidoreductase activity of the sulfide dehydrogenase. These initial findings suggest that P. furiosus is able to shift its metabolism in response to its environment and in particular the redox potential of the available terminal electron acceptor. In addition to P. furiosus, l-alanine production has been detected in the related archeaon Thermococcus profundus (21), as well as the hyperthermophilic bacteria belonging to the order of the Thermotogales (31). Pyrococcus and Thermococcus are considered to be one of the deepest branches in the domain of the Archaea, with Thermotogales being one of the deepest branches within the domain Bacteria. Based on this finding, it has been proposed that alanine production from sugar fermentation can be regarded as an ancestral metabolic characteristic (31). However, l-alanine production has also been reported during the fermentation of sugars under anaerobic conditions for the intestinal parasite Giardia lamblia (11) and a moderately thermophilic Clostridium species (28), suggesting that this pathway may be more common among the three domains than previously thought. Moreover, homoalanine fermentation was recently established by metabolic engineering in a lactic acid bacterium, indicating that this pathway may function as an electron sink in a wide range of organisms (16). Further analysis of this pathway may provide insight into not only the biology of these organisms, but also the evolution of fermentative metabolism.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway during growth on poly- or disaccharides. Instead of the usual ATP-dependent glucokinase, this pathway involves a novel ADP-dependent (AMP-forming) glucokinase. The level of this enzyme and some other glycolytic enzymes appeared to be closely regulated by the substrate. Growth on cellobiose resulted in a high specific activity of 0.96 units mg-1, whereas on pyruvate a 10-fold lower activity was found. The ADP-dependent glucokinase was purified 1350-fold to homogeneity. The oxygen-stable enzyme had a molecular mass of 93 kDa and was composed of two identical subunits (47 kDa). The glucokinase was highly specific for ADP, which could not be replaced by ATP, phosphoenolpyruvate, GDP, PPi, or polyphosphate. D-Glucose could be replaced only by 2-deoxy-D-glucose, albeit with a low efficiency. The Km values for D-glucose and ADP were 0.73 and 0.033 mM, respectively. An optimum temperature of 105 degrees C and a half-life of 220 min at 100 degrees C are in agreement with the requirements of this hyperthermophilic organism. The properties of the glucokinase are compared to those of less thermoactive gluco/hexokinases.