Extreme Biocatalysts: From Basic Research to Biotechnological Applications.

Biocatalysis, a cornerstone of modern biotechnology, is poised to revolutionize industrial processes across diverse sectors [...].

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S.acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldariusIMPORTANCEArchaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius, which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level.

Origins of major archaeal clades correspond to gene acquisitions from bacteria.

The mechanisms that underlie the origin of major prokaryotic groups are poorly understood. In principle, the origin of both species and higher taxa among prokaryotes should entail similar mechanisms--ecological interactions with the environment paired with natural genetic variation involving lineage-specific gene innovations and lineage-specific gene acquisitions. To investigate the origin of higher taxa in archaea, we have determined gene distributions and gene phylogenies for the 267,568 protein-coding genes of 134 sequenced archaeal genomes in the context of their homologues from 1,847 reference bacterial genomes. Archaeal-specific gene families define 13 traditionally recognized archaeal higher taxa in our sample. Here we report that the origins of these 13 groups unexpectedly correspond to 2,264 group-specific gene acquisitions from bacteria. Interdomain gene transfer is highly asymmetric, transfers from bacteria to archaea are more than fivefold more frequent than vice versa. Gene transfers identified at major evolutionary transitions among prokaryotes specifically implicate gene acquisitions for metabolic functions from bacteria as key innovations in the origin of higher archaeal taxa.

Salt Stress Response of Sulfolobus acidocaldarius Involves Complex Trehalose Metabolism Utilizing a Novel Trehalose-6-Phosphate Synthase (TPS)/Trehalose-6-Phosphate Phosphatase (TPP) Pathway.

The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals.

2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in the early UV response.

Exposure to UV light can result in severe DNA damage. The alternative general transcription factor (GTF) TFB3 has been proposed to play a key role in the UV stress response in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Reporter gene assays confirmed that tfb3 is upregulated 90-180 min after UV treatment. In vivo tagging and immunodetection of TFB3 confirmed the induced expression at 90 min. Analysis of a tfb3 insertion mutant showed that genes encoding proteins of the Ups pili and the Ced DNA importer are no longer induced in a tfb3 insertion mutant after UV treatment, which was confirmed by aggregation assays. Thus, TFB3 plays a crucial role in the activation of these genes. Genome wide transcriptome analysis allowed a differentiation between a TFB3-dependent and a TFB3-independent early UV response. The TFB3-dependent UV response is characterized by the early induction of TFB3, followed by TFB3-dependent expression of genes involved in e.g. Ups pili formation and the Ced DNA importer. Many genes were downregulated in the tfb3 insertion mutant confirming the hypothesis that TFB3 acts as an activator of transcription. The TFB3-independent UV response includes the repression of nucleotide metabolism, replication and cell cycle progression in order to allow DNA repair.

Insights into the evolutionary conserved regulation of Rio ATPase activity.

Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.

Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose.

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, the growth on different carbon sources was analysed, using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle were observed. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species.

Phosphoglycerate kinase acts as a futile cycle at high temperature.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

The Peculiar Glycolytic Pathway in Hyperthermophylic Archaea: Understanding Its Whims by Experimentation In Silico.

Mathematical models are key to systems biology where they typically describe the topology and dynamics of biological networks, listing biochemical entities and their relationships with one another. Some (hyper)thermophilic Archaea contain an enzyme, called non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), which catalyzes the direct oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate omitting adenosine 5'-triphosphate (ATP) formation by substrate-level-phosphorylation via phosphoglycerate kinase. In this study we formulate three hypotheses that could explain functionally why GAPN exists in these Archaea, and then construct and use mathematical models to test these three hypotheses. We used kinetic parameters of enzymes of Sulfolobus solfataricus (S. solfataricus) which is a thermo-acidophilic archaeon that grows optimally between 60 and 90 °C and between pH 2 and 4. For comparison, we used a model of Saccharomyces cerevisiae (S. cerevisiae), an organism that can live at moderate temperatures. We find that both the first hypothesis, i.e., that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plus phosphoglycerate kinase (PGK) route (the alternative to GAPN) is thermodynamically too much uphill and the third hypothesis, i.e., that GAPDH plus PGK are required to carry the flux in the gluconeogenic direction, are correct. The second hypothesis, i.e., that the GAPDH plus PGK route delivers less than the 1 ATP per pyruvate that is delivered by the GAPN route, is only correct when GAPDH reaction has a high rate and 1,3-bis-phosphoglycerate (BPG) spontaneously degrades to 3PG at a high rate.

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

Investigation of the malE promoter and MalR, a positive regulator of the maltose regulon, for an improved expression system in Sulfolobus acidocaldarius.

In this study, the regulator MalR (Saci_1161) of the TrmB family from Sulfolobus acidocaldarius was identified and was shown to be involved in transcriptional control of the maltose regulon (Saci_1660 to Saci_1666), including the ABC transporter (malEFGK), α-amylase (amyA), and α-glycosidase (malA). The ΔmalR deletion mutant exhibited a significantly decreased growth rate on maltose and dextrin but not on sucrose. The expression of the genes organized in the maltose regulon was induced only in the presence of MalR and maltose in the growth medium, indicating that MalR, in contrast to its TrmB and TrmB-like homologues, is an activator of the maltose gene cluster. Electrophoretic mobility shift assays revealed that the binding of MalR to malE was independent of sugars. Here we report the identification of the archaeal maltose regulator protein MalR, which acts as an activator and controls the expression of genes involved in maltose transport and metabolic conversion in S. acidocaldarius, and its use for improvement of the S. acidocaldarius expression system under the control of an optimized maltose binding protein (malE) promoter by promoter mutagenesis.

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

BACKGROUND: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging. RESULTS: Here, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. CONCLUSIONS: By reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

A cool tool for hot and sour Archaea: proteomics of Sulfolobus solfataricus.

In recent years, much progress has been made in proteomic studies to unravel metabolic pathways and basic cellular processes. This is especially interesting for members of the Archaea, the third domain of life. Archaea exhibit extraordinary features and many of their cultivable representatives are adaptable to extreme environments. Archaea harbor many unique traits besides bacterial attributes, such as size, shape, and DNA structure and eukaryal characteristics like information processing. Sulfolobus solfataricus P2, a thermoacidophilic archaeal representative, is a well-established model organism adapted to low-pH environments (pH 2-3) and high temperatures (80°C). The genome has a size of 3 Mbp and its sequence has been deciphered. Approximately 3033 predicted open reading frames have been identified and the genome is characterized by a great number of diverse insertion sequence elements. In unraveling the organisms' metabolism and lifestyle, proteomic analyses have played a major role. Much effort has been directed at this organism and is reviewed here. With the help of proteomics, unique metabolic pathways were resolved in S. solfataricus, targets for regulatory protein phosphorylation identified, and cellular responses upon virus infection as well as oxidative stress analyzed.

Atypical protein kinases of the RIO family in archaea.

Atypical protein kinases of the RIO (right open reading frame) kinase family are found in all three domains of life, emphasizing their essential function. In all archaeal genomes sequenced to date, typically two, but at least one, members of the RIO kinase family have been identified. Although the function of RIO kinases in Archaea remains to be resolved, bioinformatics analysis (e.g. comparison of the phylogenetic distribution and gene neighbourhood analysis, as well as interaction analysis) in combination with the available phosphoproteome study of Sulfolobus solfataricus provided some first hints to the possible function as well as revealed some putative target proteins for RIO kinases in Archaea. This study suggests a possible function of archaeal RIO kinases in RNA and/or DNA binding/processing translation initiation or ribosomal biogenesis resembling the assumed physiological role in yeast.

Application of the endogenous CRISPR-Cas type I-D system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are widely distributed among bacteria and archaea. In this study, we demonstrate the successful utilization of the type I-D CRISPR-Cas system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Given its extreme growth conditions characterized by a temperature of 75°C and pH 3, an uracil auxotrophic selection system was previously established, providing a basis for our investigations. We developed a novel plasmid specifically designed for genome editing, which incorporates a mini-CRISPR array that can be induced using xylose, resulting in targeted DNA cleavage. Additionally, we integrated a gene encoding the ß-galactosidase of Saccharolobus solfataricus into the plasmid, enabling blue-white screening and facilitating the mutant screening process. Through the introduction of donor DNA containing genomic modifications into the plasmid, we successfully generated deletion mutants and point mutations in the genome of S. acidocaldarius. Exploiting the PAM (protospacer adjacent motif) dependence of type I systems, we experimentally confirmed the functionality of three different PAMs (CCA, GTA, and TCA) through a self-targeting assessment assay and the gene deletion of upsE. Our findings elucidate the application of the endogenous Type I-D CRISPR-Cas system for genetic engineering in S. acidocaldarius, thus expanding its genetic toolbox.

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus.

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Šresolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Šresolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.

Thermostable and O2-Insensitive Pyruvate Decarboxylases from Thermoacidophilic Archaea Catalyzing the Production of Acetaldehyde.

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.

Workflows for optimization of enzyme cascades and whole cell catalysis based on enzyme kinetic characterization and pathway modelling.

To move towards a circular bioeconomy, sustainable strategies for the utilization of renewable, non-food biomass wastes such as lignocellulose, are needed. To this end, an efficient bioconversion of d-xylose - after d-glucose the most abundant sugar in lignocellulose - is highly desirable. Most standard organisms used in biotechnology are limited in metabolising d-xylose, and also in vitro enzymatic strategies for its conversion have not been very successful. We herein discuss that bioconversion of d-xylose is mostly hampered by missing knowledge on the kinetic properties of the enzymes involved in its metabolism. We propose a combination of classical enzyme characterizations and mathematical modelling approaches as a workflow for rational, model-based design to optimize enzyme cascades and/or whole cell biocatalysts for efficient d-xylose metabolism.