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The excellent selectivity of the mevalonate kinase-catalyzed phosphorylation of mevalonate simplifies lengthy multi-step routes to (R)-mevalonate-5-phosphate to a one-step biocatalytic reaction, because the phosphate group can be transferred directly and without any additional reaction steps involving introduction and removal of protecting groups. By adjusting the required reaction time for complete conversion, the kinetic resolution of racemic mevalolactone can be easily used for the preparation of (S)-mevalonate and (R)-mevalonate-5-phosphate. A new recombinant mevalonate kinase has been prepared by the expression of the mevalonate kinase gene from the hyperthermophilic archaeon Thermococcus kodakarensis in Escherichia coli and by the subsequent purification. Direct quantitative 31P-NMR kinetic analysis has been utilized to characterize the enantioselectivity of the mevalonate kinase. This method is useful for determining the biocatalyst's utility for the synthesis of enantiomerically pure (R)-mevalonate-5-phosphate as well as for biocatalytic process development.

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

Two conjugative plasmids (CPs) were isolated and characterized from the same ‘Sulfolobus islandicus’ strain, SOG2/4. The plasmids were separated from each other and transferred intoSulfolobus solfataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number and is stably maintained (pSOG2). Plasmid pSOG2 is the firstSulfolobusCP found to have these characteristics. The genomes of both pSOG plasmids have been sequenced and were compared to each other and the availableSulfolobusCPs. Interestingly, apart from a very well-conserved core, 70 % of the pSOG1 and pSOG2 genomes is largely different and composed of a mixture of genes that often resemble counterparts in previously describedSulfolobusCPs. However, about 20 % of the predicted genes do not have known homologues, not even in other CPs. Unlike pSOG1, pSOG2 does not contain a gene for the highly conserved PlrA protein nor for obvious homologues of partitioning proteins. Unlike pNOB8 and pKEF9, both pSOG plasmids lack the so-called clustered regularly interspaced short palindrome repeats (CRISPRs). The sites of recombination between the two genomes can be explained by the presence of recombination motifs previously identified in otherSulfolobusCPs. Like otherSulfolobusCPs, the pSOG plasmids possess a gene encoding an integrase of the tyrosine recombinase family. This integrase probably mediates plasmid site-specific integration into the host chromosome at the highly conserved tRNAGluloci.

Structure-forming fibrous proteins like keratins, gelatins and collagens are degraded only by a few proteases as their tight packing limits access to the potential cleavage sites. To understand the keratin degradation in detail, we describe the first crystal structure of a keratin-degrading enzyme (keratinase), fervidolysin, from Fervidobacterium pennivorans as an immature form with propeptide (PD)-bound. The 1.7 Angstrom resolution crystal structure shows that the protease is composed of four domains: a catalytic domain (CD), two P-sandwich domains (SDs), and the PD domain. A structural alignment shows a distant relationship between the PD-CD substructure of fervidolysin and pro-subtilisin E. Tight binding of PD to the remaining part of the protease is mediated by hydrogen bonds along the domain surfaces and around the active cleft, and by the clamps to SD1 and SD2. The crystal structure of this multi-domain protein fervidolysin provides insights into proenzyme activation and the role of non-catalytic domains, suggesting a functional relationship to the fibronectin (FN)-like domains of the human promatrix metalloprotease-2 (proMMP-2) that degrades the fibrous polymeric substrate gelatin. (C) 2003 Elsevier Ltd. All rights reserved.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

An a-glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3). with a yield of 87.5 U/g of wet biomass. The recombinant cytosolic enzyme was purified to homogeneity with a rapid purification procedure employing only steps of selective and progressive thermal precipitations with a final yield of 75.4% and a purification of 14.5-fold. The properties of this thermophilic a-glucosidase were compared with those of the a-glucosidase of a commercial preparation from Aspergillus niger used in the starch processing.

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.

Small RNA (sRNA) molecules in bacteria play key roles in the regulation of gene expression, in controlling protein functionality, and in guiding defense against invading DNA. sRNAs involved in transcription regulation are divided in a cis - and a trans -encoded class. cis -Encoded sRNAs are encoded by the same genomic locus as their target mRNA, residing either on the transcript they regulate (riboswitches), or derived from the opposite strand (antisense sRNAs). trans -Encoded sRNAs are encoded elsewhere on the genome and require the RNA chaperone Hfq for exerting their function. Other sRNAs regulate nucleic acid-binding proteins by mimicking the nucleic acid sequence and structure, leading to sequestration of the protein. Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) represent yet another class of sRNA that serves as a guide in defense against invading nucleic acids, such as viruses and conjugative plasmids. In addition, prokaryotic Argonaute has recently been proposed to function in antiviral or anti-plasmid defense that is guided by small nucleic acids. In this article, the function and molecular mechanism of the different classes of sRNAs are reviewed.

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.

Archaea are prokaryotes that evolved in parallel with bacteria. Since the discovery of the distinct status of the Archaea, extensive physiological and biochemical research has been conducted to elucidate the molecular basis of their remarkable lifestyle and their unique biology. Here, we discuss how in-depth comparative genomics has been used to improve the annotation of archaeal genomes. Combined with experimental verification, bioinformatic analysis contributes to the ongoing discovery of novel metabolic conversions and control mechanisms, and as such to a better understanding of the intriguing biology of the Archaea.

The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased β-structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surfaces.

The substrate specificity of the beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes. Key words: Archaea, Sulfolobus Genome Project, comparative genomics, bioinformatics.

To characterize the expression and possible regulation of reductive dehalogenation in halorespiring bacteria, a 11.5-kb genomic fragment containing the o -chlorophenol reductive dehalogenase-encoding cprBA genes of the gram-positive bacterium Desulfitobacterium dehalogenans was subjected to detailed molecular characterization. Sequence analysis revealed the presence of eight designated genes with the order cprTKZEBACD and with the same polarity except for cprT . The deduced cprC and cprK gene products belong to the NirI/NosR and CRP-FNR families of transcription regulatory proteins, respectively. CprD and CprE are predicted to be molecular chaperones of the GroEL type, whereas cprT may encode a homologue of the trigger factor folding catalysts. Northern blot analysis, reverse transcriptase PCR, and primer extension analysis were used to elucidate the transcriptional organization and regulation of the cpr gene cluster. Results indicated halorespiration-specific transcriptional induction of the monocistronic cprT gene and the biscistronic cprBA and cprZE genes. Occasional read-through at cprC gives rise to a tetracistronic cprBACD transcript. Transcription of cprBA was induced 15-fold upon addition of the o -chlorophenolic substrate 3-chloro-4-hydroxyphenylacetic acid within 30 min with concomitant induction of dehalogenation activity. Putative regulatory protein binding motifs that to some extent resemble the FNR box were identified in the cprT-cprK and cprK-cprZ intergenic regions and the promoter at cprB , suggesting a role for FNR-like CprK in the control of expression of the cprTKZEBACD genes.

The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta-D-galactopyra noside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans , halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn 916 . To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn 916 . Insertion of one or two copies of Tn 916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA , which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.

Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a five-residue ion-pair network in the hinge region of Pyrococcus furiosus glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C). In order to study the role of this ion-pair network, we introduced it into the T. maritima enzyme using a site-directed mutagenesis approach. The resulting T. maritima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified. Elucidation of the three-dimensional structure of the double mutant N97D/G376 K at 3.0 A, showed that the designed ion-pair interactions were indeed formed. Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant. Melting temperatures of the mutant enzymes N97D, G376 K and N97D/G376 K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme. Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes. Thermal inactivation at 85 degrees C occured more than twofold faster for all mutant enzymes than for the wild-type glutamate dehydrogenase. At temperatures of 65 degrees C and higher, the wild-type and the three mutant enzymes showed identical specific activities. However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was found to be significantly higher than that of the wild-type and N97D enzymes. These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature at which it functions optimally.

Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its alpha- and beta-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine beta-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase.

2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications.

The present work employs far-red resonance Raman spectroscopy (RR) to investigate whether a Cu-Cu bonding interaction exists in Cu{sub A}. The electronic transition of Cu{sub A} near 830 nm provides the opportunity for specific RR observation of the vibrations of this chromophore. We have employed RR with Cu isotopic substitution on genetically modified, solubilized forms of CcO subunit II from Bacillus subtilis and Paracoccus denitrificans. We have also probed the Cu{sub A} site of native beef heart CcO for comparison. The similarity of the CcO and Cu{sub A} fragment (subunit II) spectra suggests that Cu{sub A} in the isolated subunit II has essentially the same structure as that of Cu{sub A} in intact CcO. Thus, the suggested Cu-Cu bonding in Cu{sub A}, if correct, applies both to the isolated subunit II and native CcO. The functional reasons why nature may choose such a structure, or in fact why a two-copper center of any sort is constructed to perform a function which is commonly believed to be simple one-electron transfer, are unclear. 34 refs., 1 fig., 1 tab.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

Regulation of gene expression in the domain Archaea , and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea . We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus . The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria . We show that Lrs14, expressed in Escherichia coli , is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus .

Microbiology is an extraordinary field which has been, and will continue to be, central to the advancement of human cultures. In fact, human cultures and even our planet would not be as they are today if microorganisms and microbiology would not exist. Basic, but at the same time, complex microbiological processes play key roles in biodegradation and nutrient (re)cycling, climate change, disease and biotechnology. Due to its importance, a brief history about microbiology is provided in Chapter 1 and the related (old or new) biotechnological applications are described. The importance of the microorganism-workhorses is highlighted and a “start-at-the-end” approach is recommended for choosing the appropriate microbe for the production of the final product. A brief introduction to clostridia species and their potential for the sustainable production of ester compounds is provided. Lastly, the importance of the revolutionary CRISPR-Cas genome engineering tools is highlighted.Chapter 2 reviews the state-of-the-art technologies used for the microbial production of short and medium chain esters. Several enzymatic processes are described with main focus on the AAT enzymes. In addition, several metabolic engineering processes are reviewed, along with the physical properties of the final ester product. Lastly, an outlook is provided on other products (e.g. alcohols, carboxylic acids and diols) that can be derived from (microbially produced) esters.In Chapter 3, the unique Eat1 enzyme was further characterized using in vitro and in vivo approaches. Two new activities (alcoholysis and thiolysis) were added to the existing activities (AAT, esterase and thioesterase) of Eat1. Alcoholysis appears to be the main activity of the enzyme as it highly prefers to exchange the alcohol moiety of the ester molecule with another free alcohol. This discovery exposes the limitations for using Eat1 as an AAT but also provides insights into the portfolio of ester compounds produced by many yeasts.An alternative approach for microbial ester production is described in Chapter 4. This approach uses the BMCs of C. beijerinckii NCIMB 8052 to encapsulate an AAT enzyme and produce propyl propionate as terminal product. As a proof of principle, the GFP protein is encapsulated in the BMCs by using N-terminal EPs. Unfortunately, due to time restrictions, the encapsulation of AATs and the successful utilization of C. beijerinckii BMCs to produce propyl propionate was not (yet) realized.Chapter 5 describes the development of a simple genome engineering tool for multiplex gene knock-out in C. beijerinckii NCIMB 8052. This tool is based on the CRISPR-FnCas12a system which is only expressed in the presence of the xylose inducer. 25-100% knock-out efficiency was observed when targeting single genes for deletion whereas, 18% knock-out efficiency was apparent when targeting two genes for deletion. The spacer sequence and spacer order in a CRISPR array is also assessed, showing that it highly influences the knock-out efficiency.Low HR efficiency along with strong CRISPR-Cas counterselection limit the application of CRISPR-Cas genome engineering tools in non-model prokaryotes. In Chapter 6, these limitations are mitigated through the development of SIBR. SIBR was developed and used as a widely applicable gene control system that can control the expression of virtually any gene of interest in any mesophilic prokaryote of interest. SIBR was used to control the expression of the FnCas12a protein (collectively termed SIBR-Cas) and allow more time for HR to occur before induction of counterselection. SIBR-Cas was applied in three different bacteria with high editing efficiencies demonstrating the wide applicability of SIBR.&nbsp

CRISPR-Cas is an extraordinary prokaryotic adaptive immune system, divided into two classes that each contain three types and a wide variety of subtypes. Class 2 effector proteins are the most exploited because of their compact structures with multi-functional properties. Amongst the class 2 systems, the more recently discovered type V systems appears to be the most diverse type, with new mechanistic features still to be uncovered. The research described in this thesis focusses on the characterization and subsequent development of applications of type V CRISPR-Cas systems, more specifically types V-A and V-U1.Chapter 1 introduces CRISPR-Cas as a sophisticated prokaryotic adaptive immune system. CRISPR-Cas systems. Each CRISPR-Cas system has unique features, but all actively participate in adaptive immunity through a three-step mechanism: adaptation, expression and interference. Although all variants follow the same steps, the different CRISPR-Cas (sub)types are highly diverse with unique structural and functional features at all levels of the mechanism.After describing the underlying molecular mechanism of CRISPR-Cas, an overview is given in chapter 2 on the different genome editing applications. The main focus is on DNA-targeting class 2 effector proteins, such as Cas9, Cas12a and Cas12b. Apart from natural variants, engineered CRISPR-Cas nuclease variants that increase editing precision or regulate nuclease activity were presented as well. One of the biggest bottle necks of genome editing in eukaryotes is the delivery of a specific nuclease. Therefore, different approaches of nuclease/guide delivery were discussed. In addition, various host repair pathways were examined based on the type of DNA damage and the type of repair template available.In chapter 3, the first step of the CRISPR-Cas adaptive immunity, adaptation of the CRISPR memory, is studied in two type V systems, namely type V-A and V-B from Francisella novicida tularensis subsp. novicida U112 and Alicyclobacillus acidoterrestris ATCC 49025, respectively. The type V-A locus encodes Cas12a, Cas4, Cas1 and Cas2, whereas that of V-B encodes Cas12b, a Cas4/1 fusion protein and Cas2. It was found that in type V-A, only Cas1 and Cas2 are required for adaptation, and in type V-B, Cas4/1 and Cas2 are required for adaptation, but Cas4 activity is dispensable. Spacers acquired without a functional Cas4, appeared to target protospacers containing mostly non-conical PAMs. Thus, Cas4 activity is required for PAM selection and acquisition of suitable spacers in both type V-A and V-B. The role of Cas12a in the adaptation process has not been elucidated yet in this chapter, but will be addressed in future studies.Following adaptation, chapter 4 describes crRNA maturation in type V-A. Type V-A crRNA maturation is distinct to that found in type II, where Cas9 requires both a crRNA and a tracrRNA, and gets processed by endogenous RNaseIII after ribonucleoprotein complex formation. This study demonstrated that Cas12a does not require a tracrRNA nor RNase III for crRNA maturation. Instead Cas12a itself is able to process pre-crRNA into mature crRNA using a previously unknown RNase domain found in Cas12a. Having Cas12a able to process its own crRNA is greatly advantageous for genome editing applications and allows for simple simultaneous multi-gene (multiplex) editing using a single CRISPR-array. Using a single CRISPR-array containing four spacers, Cas12a was able to simultaneously edit up to four genes in mammalian cells (ex vivo) and up to three genes in mouse brain cells (in vivo).Apart from processing its own pre-crRNA, another distinct feature of Cas12a is the generation of staggered ends after cleavage of dsDNA. These staggered ends were exploited in chapter 5 to create in a novel genome editing approach in E. coli, termed “cut and paste. Cleavage by Cas12a generates double-stranded DNA breaks with 4-5 nt compatible staggered ends. These staggered ends can be repaired by ligation using T4 ligase. Using cut & paste, a genomic deletion in E. coli was successfully achieved, albeit with a relatively low editing efficiency.To further explore other type V systems, chapter 6 focusses on the characterization of a novel compact type V systems, type V-U1 from Mycolicibacterium mucogenicum CCH10. The type V-U1 CRISPR-Cas locus express a small effector protein MmuCas12u1. MmuCas12u1 is roughly half the size of Cas12a. Despite its small size, MmuCas12u1 seems to retain some functional features also found in Cas12a. Features such as processing its own pre-crRNA, targeting dsDNA and recognizing a 5’-TTN-3’ PAM. The RuvC domain of MmuCas12u1 does not cleave dsDNA, but instead is hypothesized to be involved dsDNA-activated transcriptional silencing. By leveraging this property, MmuCas12u1 has been used for single- and multiplex- transcriptional silencing in E. coli.Chapter 7 described how the fundamental knowledge gained on MmuCas12u1 is used to develop small Mmu base editors (MmuBE). MmuBEs are fusion proteins consisting of MmuCas12u1, cytidine deaminase and uracil glycosylase inhibitor, which is a tool for RNA-guided targeted nucleotide (C à T) substitution. Several MmuBE variants were constructed and characterized in E. coli. Most variants are relatively efficient, with a base editing window consisting of two regions, a PAM-proximal (2-5) and a PAM-distal (13-19) region, with the PAM-proximal region having more edits. In addition, a small-scale pilot experiment also demonstrated on-target base-editing by MmuBE in eukaryotic cells, namely in Saccharomyces cerevisiae. MmuBEs are currently the smallest base editors (genes ~2.8 kb) known, further expanding the current toolbox for prokaryotic base editing, and with great promise for eukaryotic base editing.

Clostridium beijerinckii has been an organism of great interest for industrial biotechnology for about a century because it can degrade a wide range of sugars to produce acetone, butanol, ethanol, and isopropanol, chemicals used in consumer goods (paints, cosmetics, food ingredients) and as alternatives to transport fuel. Still, C. beijerinckii has other less studied physiological traits, such as the formation of intracellular compartments, spores and microcompartments. Therefore, we decided to study sporulation and microcompartment formation in C. beijerinckii using functional genomics.Sporulation is a cell differentiation process that occurs in all Firmicutes. It is triggered when the cells are exposed to an unfavorable environmental condition (presence of oxygen, high pH variation), and yields spores, metabolically inactive cells resistant to harsh conditions (UV, chemicals, heat, oxygen). In Chapter 1, the research that has been done in the last 30 years on sporulation in solvent-producing clostridia, including the latest updates on the molecular regulation of sporulation in solventogenic clostridia, is presented. Common triggers and regulation mechanisms of sporulation in several solventogenic species are identified, underlining the differences and similarities between species. Potential links in the regulation of sporulation and solvent production are pinpointed. This chapter highlights the need for more studies on signal transduction pathways, transcriptional and post-translational regulation of sporulation, and solvent production. A better understanding of the connections between both physiological phenomena would provide us with relevant targets for strain engineering of solventogenic clostridia.Metabolic engineering of Clostridium used to be tedious and hampered the studies on strain physiology. An efficient and fast engineering tool was highly desired. Chapter 2 describes the adaptation of a CRISPR-Cas9 method for use in Clostridium beijerinckii. The tool was initially developed for Clostridium acetobutylicum, but several modifications of the tool were necessary for use in C. beijerinckii. This method allows the deletion and insertion of genes in C. beijerinckii NCIMB 8052 and C. beijerinckii NRRL B593, an isopropanol-producing strain.The method developed in Chapter 2 was used to generate a SpoIIE deficient mutant in C.beijerinckii NCIMB 8052. The mutant phenotype is described in Chapter 3. Morphology, fermentation profile, and transcriptome of the mutant were investigated and compared to the wild-type. Thanks to this mutant strain, we could study the impact of the interruption of the sporulation cycle before forespore formation on other cellular events.The mutant investigated in Chapter 3 was not the only SpoIIE deficient isolate we obtained. Five SpoIIE deficient strains were isolated, and each of them had a slightly different genotype, but all of them were asporogenic. Four of the five strains showed a solventogenic phenotype, and one was deficient in solvent production. The characterization of a non-solventogenic SpoIIE deficient strain is reported in Chapter 4. Morphology, fermentation and transcriptome were compared to the solvent producing ΔspoIIE strain described in Chapter 3.Apart from spores, C. beijerinckii can form other intracellular compartments called bacterial microcompartments (BMCs). Like spores, BMCs are formed due to changes in the environment and, more precisely, when C. beijerinckii metabolizes L-rhamnose. In Chapter 5, the metabolism of C.beijerinckii NRRL B593 on rhamnose was investigated with respect to its fermentation profile, product formation, transcriptome.Chapter 6 summarizes the results described in this thesis and discusses their contribution to Clostridium research in general. Moreover, an outlook on further studies on the physiology of industrially relevant clostridia is given. Based on the work described in this thesis, three interesting future research lines are presented.

DNA contains the information to produce all the proteins a cell needs. However, a cell needs regulatory elements to produce the right protein at the right time in sufficient quantity. To preserve energy a cell does not produce more protein than required. However, for biotechnology applications, a cell needs to produce as much of a specific protein as possible to create an efficient production process. In this thesis, the translation regulating elements were studied to then tune them for reliable high protein yields. The 5’UTR was standardized using a bicistronic design. The 3’UTR was found to significantly impact protein production through a yet unknown mechanism, but surprisingly in a very consistent way. Finally, codon usage was studied extensively and we concluded that the first 9 codons mainly dictate the efficiency of translation and not the overall codon usage. These findings can contribute to more efficient protein production processes in therapeutics, industry and research and might even enable new production processes.

This PhD thesis describes the relation of the structure of RNA with its effect on protein expression and the efficacy of CRISPR-Cas12a. Direct control of protein expression by RNA is done via so-called riboswitches, which change their structure depending on the presence of a ligand or external factors like temperature. A theophylline-responsive riboswitch based on the phage T4 td group I intron is described in detail and a method is proposed to obtain riboswitches based on this intron. This method was used to generate a riboswitch library for 3-methylxanthine, a compound closely related to theophylline, and the unrelated citrulline. The second part focuses on the effect of coding and non-coding mRNA on gene translation. Coding regions that are in close proximity of other coding regions have their translation coupled to each other, and the order of the genes appears to be of surprisingly little influence. The next part investigates the effect of the 3’-UTR and the intergenic region. These non-coding elements may have a lot of influence on the translation rate, by a mechanism that is not yet understood. The last part shines a light on the influence of the RNA structure of the CRISPR-Cas12a crRNA. The pre-crRNA forms a characteristic pseudoknot structure that is required for the recognition by Cas12a, which then turns it into crRNA. The low GC content of the pseudoknot allows this structure to be outcompeted by other potential structures, which renders the pre-crRNA unrecognisable. A design strategy was proposed to improve the efficacy of CRISPR-Cas12a.

Animals are known to possess a large repertoire of immune systems with a high degree of sophistication. On the other hand, the immune systems found in bacteria and archaea appeared to be much more rudimentary. However, the ground-breaking discovery of a novel immune system, CRISPR-Cas, proved otherwise. CRISPR-Cas, is unique in being both adaptive and heritable, and it relies on small RNA molecules that specifically guide the defence system to matching invader DNA sequences. This natural defence system has been successfully repurposed into a valuable molecular technology which is a robust, efficient, easy-to-use method to precisely alter DNA sequences of living organisms. The current CRISPR-based technologies, mostly employ the Cas9 protein, for diverse biotechnological applications. Nevertheless, the natural diversity of CRISPR-Cas systems is remarkably extensive, including systems that target DNA, systems that target RNA, and systems that target both DNA and RNA. The diverse class 2 CRISPR nucleases have unique molecular features that contribute to an expansive toolbox for genome and transcriptome engineering. These nucleases differ greatly in their structure and mechanisms. These differences could be exploited as complementary applications creating numerous CRISPR-based technologies possibly with favourable specificity, efficiency and/or delivery. This thesis explores the diversity of Class 2 CRISPR–Cas systems and provides mechanistic insights into different class 2 nucleases. In addition, it describes potential applications to expand the current repertoire of CRISPR-based technologies. Due to the everlasting arms race between prokaryotes and their viruses, the rapid evolution of CRISPR–Cas systems has resulted in extreme structural and functional diversity. As a result, a plethora of distinct CRISPR–Cas systems are represented in genomes of most archaea and almost half of the bacteria. The key players of this system are the crRNA binding effector complexes, and the associated nuclease domains. CRISPR–Cas systems are currently grouped into two classes each of which is subdivided into three types. Class 1 systems (consisting of types I, III, and IV) use a multi-subunit protein complex to achieve interference, and class 2 systems (consisting of types II, V, and VI) utilize a single multi-domain protein, that have been repurposed for genome editing applications in a wide range of organisms. The mechanism of crRNA maturation in CRISPR–Cas12a systems was unravelled during this thesis. Unlike the type II nuclease Cas9, which utilizes a tracrRNA as well as endogenous RNaseIII for maturation of its dual crRNA/tracrRNA guides, pre-crRNA processing in the Cas12a system proceeds in the absence of tracrRNA or other Cas proteins. It was demonstrated that Cas12a nucleases possess a previously unknown RNase domain that is responsible for cleaving the pre-crRNA to generate the mature crRNAs. The typical cleavage pattern revealed that Cas12a recognizes specific secondary structures and/or motifs on its direct repeats. Furthermore, the ability to autonomously process crRNA has significant implications from a genome editing standpoint, as it provides a simple route to editing multiple genomic loci at a time (multiplex editing). Using a single customized CRISPR array up to four genes in mammalian cells ex vivo and up to three genes in mouse brain cells in vivo were shown to be edited simultaneously. The characterisation of a novel, diminutive type V-U1 Cas protein from Mycolicibacterium mucogenicum (MmuC2c4) was described in this thesis. Type V-U proteins are highly similar to the typical transposon-encoded TnpB-like proteins and each of them (type VU-1 to type VU-5) appear to have originated independently from distinct TnpB families. Akin to most type V proteins, MmuC2c4 was shown to recognize a 5’-TTN-3’ PAM on a double-stranded target DNA. The characterisation of a type II-C Cas9 orthologue of the thermophilic bacterium Geobacillus thermodenitrificans T12, ThermoCas9 is described. This is one of the first reports that provides fundamental insights into a thermophilic CRISPR–Cas9 family member. It was demonstrated that ThermoCas9 is active in vitro between 20 and 70 ℃, that the structure of its sgRNA influences its activity at elevated temperatures, it has a more stringent PAM-preference at lower temperatures, it does not tolerate extensive spacer-protospacer mismatches, and it preferentially cleaves plasmid DNA compared to linear DNA. Furthermore, ThermoCas9 was employed for pyrF gene deletion and transcriptional silencing of ldhL gene at 55 ℃ in Bacillus smithii ET 138 and for pyrF gene deletion at 37 ℃ in Pseudomonas putida. This is the first time Cas9-based bacterial genome editing and silencing tools were used at temperatures above 42 ℃. Four Cas12a orthologues were assessed for their salt tolerance as well as pH- and temperature stability using biochemical assays as described. Subsequently, Francisella tularensis subsp. novicida (FnCas12a) and Eubacterium eligens (EeCas12a) were applied for genome editing in a moderate thermophilic bacterium, Bacillus smithii. It is demonstrated that FnCas12a and EeCas12a are sub-optimally active in vivo at temperatures above 45 ℃. The wide growth temperature range of B. smithii ET 138 was employed for the controllable induction of Cas12a expression at temperatures below the 45 ℃ threshold. It was demonstrated that a mutant can be generated within a short span of 2-3 days. This process can be easily adapted for gene editing applications in a wide variety of both mesophilic and moderate thermophilic organisms. potential to harness the activity of anti-CRISPR (Acr) proteins for controllable bacterial genome engineering was also investigated. The Acr protein from Neisseria meningitidis (AcrIIC1Nme) was employed as an “on/off-switch” to control the activity of thermostable Cas9 orthologues from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9). Initially, it was proven that both ThermoCas9 and GeoCas9 can introduce lethal dsDNA breaks in E. coli at 37 ℃ in a tuneable manner. Next, it was demonstrated that AcrIIC1Nme traps both tested Cas9 orthologues in a DNA-bound, catalytically inactive state. The Cas9/AcrIIC1Nme complexes can promote a transcriptional silencing effect with efficiency comparable to the catalytically “dead” ThermodCas9 and GeodCas9 variants. Finally, a single-vector, tightly controllable and highly efficient Cas9/AcrIIC1Nme-based tool for coupled silencing and targeting in E. coli was developed. This tool may serve as a basis for further developing a controllable genome editing and transcriptional regulation in model as well as non-model microorganisms. Furthermore, a novel biological role and mechanism for the CRISPR–Cas9 system of the pathogen Campylobacter jejuni (CjeCas9) was uncovered. It was demonstrated that upon C. jejuni infection of human cells, CjeCas9 is secreted into the cytoplasm of the infected cells and it can autonomously enter the nucleus. Inside the nucleus, it catalyses metal-dependent and sequence-independent nicking of double stranded DNA, eventually leading to cell death. Genome editing using CjeCas9 was compared with the commonly used Cas9 from Streptococcus pyogenes (SpyCas9), and the latter was shown to be superior in creating indels. It was concluded that the unique catalytic features make CjeCas9 nuclease less suitable for genome editing applications. In conclusion, the research described in this PhD thesis has uncovered novel molecular requirements and mechanisms of several unique Class 2 CRISPR–Cas nucleases. Besides gaining insights into their biochemical mechanism, the potential of Class 2 nucleases has been harnessed for biotechnological applications. Additionally, a unique role and mechanism of CRISPR–Cas in virulence has been elucidated. The characterisation of nucleases such as FnCas12a, EeCas12a, MmuC2c4 and ThermoCas9 opens up exciting possibilities of utilizing them as genome and transcriptome engineering tools.

In order to develop hypotheses presented in the introduction, we have undertaken several experiments, which we describe in the following chapters. The chapters have been published or submitted for publication, however for the purposes of this thesis, some of them have been adapted to make sure the thesis is a coherent stand-alone publication. Some of the chapters have been furthermore updated in order to keep them in accordance with most recent findings in the field. Chapter 2 shows the history of transcriptomics research in thermophiles and hyperthermophiles. We look at a wide range of research questions that transcriptome allows answering and showcase some of the most influential research on thermophile transcriptomics in last decades. Chapter 3 describes the multi-omics toolbox we developed for S. solfataricus research. Standardisation of methodology is a key aspect when it comes to combining experimental work and modelling, we propose a complete collection of methods together with results showing the S. solfataricus Central Carbon Metabolism in shifting temperatures. Chapter 4 focuses on the poorly studied and potentially important membrane proteome of S. solfataricus. We show that better methods allow us elucidating the composition of membrane proteome, and from there, its function. We detect and increased the number of membrane peptides and show a differential protein pattern after cultivation at optimal and at suboptimal temperatures. Chapter 5 addresses the transcription regulation within the S. solfataricus genome. Based on the results from previous chapters we find a putative regulatory sequence responsive to the temperature change and confirm the transcription patterns using RT-qPCR. Chapter 6 looks at experimental evolution as a tool for confirming adaptive traits in S. solfataricus. Looking at sub and super-optimal growth temperatures we try to elucidate whether the selection in fluctuating conditions is selecting for a more generalist growth pattern as opposed to constant selective pressure, which should result in selection for specialists in a given condition. Chapter 7 presents the general conclusions of this thesis focusing on multi approach strategy of looking at biological systems. We try to show how combination of complementary techniques driven by an overreaching hypothesis can aid in finding answers unattainable otherwise. We focus on the links between the previous chapters and propose how findings from one experiment can drive further research.

The global demand on chemicals and fuels is exponentially increasing. At the same time, the excessive exploitation of fossil-based resources for the coverage of this demand has a high environmental impact, motivating the production of green chemicals and biofuels from renewable resources. Nowadays, the microbial production of green chemicals and fuels gains increasing attention, especially due to the ease in the construction of metabolically engineered microorganisms with high production capacities. This ease was achieved via the development of efficient genome engineering tools. This thesis describes the development of novel genetic tools for mesophilic and thermophilic bacteria, for metabolic engineering purposes. More specifically, here is reported: 1) the development of the first CRISPR-SpCas9-based genome engineering tools for the mesophilic bacteria Rhodobacter sphaeroides and Pseudomonas putida, as well as for the moderate thermophilic bacterium Bacillus smithii, 2) the in vitro characterization of one of the first reported thermotolerant Cas9 homologs, denoted as ThermoCas9, as well the development of the first CRISPR-ThermoCas9-based genome engineering tools for strictly thermophilic bacteria, and 3) the use of the developed tools for the metabolic exploration and exploitation of the moderate thermophilic bacterium B. smithii, towards the characterization of its acetate production pathway and the enhancement of its dicarboxylic acids productivity.

Viruses are very efficient in the delivery of their viral DNA or RNA into cells. Using them as DNA/RNA delivery tools in the lab or in medicine (e.g. gene therapy), however, brings safety issues. Therefore, we designed our own artificial virus. The artificial virus is composed of a simple protein, designed by us, that can bind and fully coat strands of DNA and RNA. Within such an artificial virus particle, the DNA is protected against degradation, potentially allowing it to be delivered into cells. In this thesis, we studied the physical behaviour of these self-designed proteins and used that information to improve their design for delivery into cells. In addition, we made a first effort to design the protein in such a way that it can coat specific DNA strands.

Enzymes are proteins that catalyse chemical reactions. They accelerate the reaction by lowering the activation energy, thereby allowing the equilibrium to be reached more quickly. The molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into molecules known as products. Nearly all metabolic processes in a living cell need enzyme catalysis in order to proceed at rates fast enough to sustain life. In Chapter 1, the relevance of enzymes for society is described. This dates back to more than 8000 years ago, when people unknowingly already made use of enzymatic conversions via fermentation by whole-cell microorganisms to make early forms of bread and beer. In the last two centuries, our knowledge on how enzymes are functioning increased tremendously. They have remarkable features that make them interesting for industrial applications, such as a high specificity and selectivity, and they can make processes ‘greener’ by replacing often polluting or toxic chemical reactions. This resulted in the large scale industrial production and application of enzymes in diverse areas ranging from food industry and detergents to pharma and DNA technology. Currently, there is both the need and the room to increase the number of enzyme applications. A more extensive implementation of enzymes in industry is very important because, sooner or later, we have to make the shift from a fossil fuel based economy to a biobased economy. At this moment, only 5% of all chemical products are produced biologically. For the latter, only 150-170 of the 3000 different types of known enzymes are being applied. It is estimated that only 1% of enzymes is known and, therefore, there is much room to extend the number of industrial enzyme applications. But how to find novel enzymes for these applications? Enzymes can be obtained from nature (natural evolution, screening of metagenomics libraries) or by enzyme engineering via laboratory evolution (screening of random or semi-random enzyme variant libraries) or computational design (in silico generation and screening of enzyme variant libraries followed by experimental verification screens). Together these approaches comprise a multitude of methods to find or generate an enormous amount of genetic diversity. To obtain the desired variants from these large libraries it is essential to have an efficient screening method, in which phenotype and genotype are linked. However, since screening methods are often time-consuming, complicated and/or require expensive equipment, this screening step is the main bottleneck in obtaining novel enzymes. Reporter-based in vivo screening and selection is a new development in order to deal with the large numbers in screening for novel enzymes. This approach is extensively discussed in Chapter 2, including a comparison of other in vivo screening and selection strategies and the various reporter-based mechanisms. In reporter-based approaches, it is not the enzymatic conversion or its product that result in a measurable property, but rather a genetically encoded reporter that gives a discriminating phenotype. Since the enzyme activity is measured indirectly, this approach is independent of the reaction and thus general. A sensor part couples the enzyme activity to the reporter and, hence the reporter choice determines the detection output, e.g. bioluminescence or fluorescence. The cell containing these sensor and reporter components, and functions as a reporter, is called a whole-cell bioreporter or simply bioreporter. As sensor part, various biomolecules are possible, either protein- or RNA-based (riboswitches), but the most popular sensor is a transcriptional regulator. This transcriptional regulator very specifically binds the enzymatic product, which results in a conformational change that modifies its DNA binding capacity and switches on expression of a reporter gene. Consequently, the specificity of the sensor has to be modified for each product. Although the development of this and other reporter-based strategies takes time and effort, the many advantages make this a very powerful screening method. These advantages include: wide applicability, screening for enantioselectivity, signal enhancement, no need for artificial substrates and high-throughput screening. The most commonly used reporter-based screening method combines the reporter GFP with screening by fluorescence activated cell sorting (FACS). Though this is a high-throughput method that has proven very successful in obtaining new and/or improved enzymes, it does entail expensive equipment and experienced people operating this equipment. The aim of this thesis is to make this technology easier by developing a generic and high-throughput in vivo reporter-based system that involves selection instead of screening. The advantage of selection over screening is that only positive cells, containing the active enzyme, stay in the library pool, which allows for a quick reduction of the initially large library size. Although this thesis is not describing the first reporter-based selection system, other systems often are not applicable for a wide range of enzymes. In this thesis, the modular set-up of the system should make it more generic. To show that the developed system can be used in finding novel enzymes, a proof of principle is required. This consist of three aspects: (1) detect a product of an enzymatic activity, (2) apply the system at library scale, and (3) change the specificity of the system to make it applicable for a wide range of enzymatic products and thus different enzymes. In Chapter 3, the first two aspects are demonstrated, while Chapters 4 and 5 each focus on a different approach for the third aspect. Our bioreporter is based on the most common reporter-based strategy, namely the transcriptional regulatory-based strategy, and couples enzymatic activity to growth of the bacterium Escherichia coli. Chapter 3 covers the development of this in vivo transcriptional regulator-based selection system. A high false positive rate is a returning problem for growth-based selection and, therefore, our system was designed with dual reporters, both a selection and a screening reporter. The sensor part of the bioreporter is based on the transcriptional regulator AraC, which is involved in L-arabinose metabolism in E. coli, because AraC has been well studied. Furthermore, protein structures of AraC with and without ligand are available and it has been a topic of several engineering studies. In our system, the AraC sensor binds the product of the enzymatic reaction and switches on transcription of both a selection reporter (LeuB or KmR; enabling growth), for rapid reduction of the initially large library size, and a screening reporter (LuxCDABE; causing bioluminescence), for exclusion of false positives and quantification of positive variants. The characteristics of four different systems, differing in the selection reporter (LeuB or KmR) and in the plasmid origin of replication (low or medium copy number), are compared. The medium copy number system with KmR as selection reporter was found to be the best performing system based on leakiness, maximal signal, dynamic range and sensitivity in both selection and screening. Most importantly, a proof of principle of this system was provided by selecting cells expressing an L-arabinose isomerase derived from mesophilic E. coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with L-arabinose isomerase activity was established by selection and exclusion of false positives by screening. This shows the value of the dual selection and screening system for the detection of both mesophilic and thermophilic enzymes at library scale. However, in order to demonstrate that our bioreporter is generic and can be applied for a wide range of enzymes, its specificity needs to be adaptable towards the product of any enzyme. In Chapters 4 and 5, two different approaches to change the specificity of the bioreporter are presented. In Chapter 4, the replacement of the transcriptional regulator AraC by LacI (the regulator of lactose metabolism in E. coli), is described. The characteristics of four different systems, all having LacI as transcriptional regulator, but varying in the selection reporter (LeuB or KmR) and in the plasmid origin of replication (low or medium copy number), were compared. The low copy system with LeuB as selection reporter was selected as best performing system and using this system, it was demonstrated that previously described weak inducers or anti-inducers can be detected. The newly developed LacI-based system was compared with the original AraC-based system. The LacI-based system has a better sensitivity and a higher fold change of maximal signal over leakiness, but its dynamic range for selection is lower than that of the AraC-based system. Although some optimization is required, the replacement of the transcriptional regulator is rather straightforward due to the modularity of the system. It is a good approach to alter the specificity of the dual selection and screening system and thereby to broaden its range of potential target molecules. A second approach to change the system’s specificity is described in Chapter 5. This approach is based on engineering the ligand specificity of AraC, from L-arabinose to D-xylose, by targeting residues in the ligand binding pocket with combinatorial site-saturation mutagenesis. Others have already successfully modified the specificity of AraC using a GFP- and FACS-based screening of transcriptional-regulator variants. The aim here was to offer a simpler and alternative method by using growth-based selection instead. To this end, the dual reporter system itself was applied for selection and screening of transcriptional-regulator variants. The complete process is described, starting from library design and construction up to kanamycin resistance-based selection and bioluminescence-based screening of these libraries in the presence of D-xylose. Some of the developed AraC variants showed an altered, albeit small, response to D-xylose and several other tested monosaccharides. The selected variants yet have to be investigated in more depth to verify whether their ligand specificity is truly modified. Nonetheless, these variants will be interesting starting points for further engineering and indicate that the right positions in the protein were targeted. However, to obtain variants that give a better response, the selection and screening set-up needs to be optimized. After this optimization step, the same set-up could be used to select not only AraC variants with a better response to D-xylose, but also variants specific to other target molecules. Inhibitory and stimulatory effects of L-arabinose on growth of E. coli were observed during the experimental work with the AraC-based dual reporter system (Chapters 3 and 5). In Chapter 6, these observations are supplemented with follow-up experiments to understand the underlying regulatory mechanisms of these effects. The growth effects caused by L-arabinose are described for the system strain, its parent strain E. coli BW25113 and various single gene knockout strains derived from BW25113. In LB medium, L-arabinose negatively affects growth of wildtype strain BW25113 (lower final OD600), but not of ΔaraC or Δcrp strains. In addition, the effect is stronger in strains with ΔxylA, encoding the first enzyme in D-xylose catabolism. Growth of strains in which wildtype araC is replaced by sacB or by araC variants that encode L-arabinose unresponsive AraC mutants, is still inhibited by L-arabinose. Other related monosaccharides show to various extent also inhibition. In M9 minimal medium, L-arabinose stimulates growth of both BW25113 and ΔaraC strains in the early phase of growth, but ultimately reduces the final OD600 of only BW25113. Based on the different genotypes and phenotypes of the various tested strains, hypothetical regulatory mechanisms that may explain the effects of L-arabinose on growth of E. coli are discussed. To critically question the work presented in this thesis, a general discussion on the developed bioreporter is provided below. The bioreporter is compared to other screening and selection methods and suggestions for further improvements are outlined.

Ester production by microorganisms is of great importance to biotechnological processes. These volatile compounds impart pleasant, fruity aromas to beer, wine, and other fermented products. Yeast in particular are well known for their ability to produce volatile esters. The genetic and enzymatic intricacies of ester production have been the focus of many studies, mainly in S. cerevisiae. Despite this, ester synthesis in yeast is not fully understood. This holds particularly true for yeasts that produce high amounts of ethyl acetate from sugars, such as Kluyveromyces marxianus and Wickerhamomyces anomalus. As was introduced in Chapter 1, this ability has been described more than a century ago, but the enzymatic mechanisms behind the synthesis were unclear. Circumstantial evidence suggested that an alcohol acetyltransferase (AAT) was responsible for the ethyl acetate formation. Ethyl acetate is a versatile commodity chemical that is currently produced in unsustainable processes. Understanding the enzymes responsible for bulk ethyl acetate synthesis in yeast could enable rational design of novel production strains. This, in turn could facilitate the development of biobased ethyl acetate production processes. This thesis has made several important breakthroughs in the field of ester production in yeast, particularly ethyl acetate. The most significant of these was the discovery of the elusive enzyme responsible for bulk ethyl acetate synthesis in yeast. The identification of the ethyl acetate-producing enzyme in W. anomalus is described in Chapter 2. The purified enzyme showed AAT activity with ethanol and acetyl-CoA and was therefore named Ethanol acetyltransferase 1 (Eat1). Production of Eat1 in Escherichia coli enabled efficient ethyl acetate production in E. coli. The enzyme could thus potentially be used to develop new biobased ethyl acetate production processes. However, Eat1 was also able to function as a thioesterase and esterase active against acetyl-CoA and ethyl acetate, respectively. It was observed that the presence of ethanol was able to repress the hydrolytic activities, at which point the AAT activity was dominant. How ethanol can control the activities of Eat1 is unclear. It highlights that sufficient ethanol concentrations must be present to produce ethyl acetate with Eat1. It is also shown that Eat1 homologs are present in other bulk ethyl acetate-producing yeasts. These homologs are only distantly related to known AATs. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family. The discovery of this novel enzyme family was the cornerstone of the research presented in this thesis. The identification enabled further studies on the physiology of Eat1 and bulk ethyl acetate production in the native yeasts in Chapter 3. The cellular location of Eat1 in Kluyveromyces lactis was determined. The enzyme localised to the mitochondria of the yeast. This observation opposed the literature consensus which assumed that bulk ethyl acetate synthesis occurred in the yeast cytosol. Cytosolic acetyl-CoA flux in yeast is low and would presumably not support the synthesis of high amounts of ethyl acetate. The localisation of Eat1 in the mitochondria could better explain how bulk ethyl acetate synthesis occurs. The current hypothesis suggests that bulk ethyl acetate is an overflow product of yeast under iron limited conditions. Under these conditions, the entry of acetyl-CoA into the TCA cycle is impaired and acetyl-CoA accumulates. Eat1 is then proposed to relieve the accumulation by forming ethyl acetate. The TCA cycle and the major flux of acetyl-CoA in yeast are located in the mitochondria, where Eat1 is also located. It is thereby established that bulk ethyl acetate is a mitochondrial product of certain yeasts. Chapter 4 describes the engineering of efficient ethyl acetate production in E. coli using Eat1 as the catalyst. Unlike yeast, the metabolism of E. coli can support the synthesis of ethyl acetate under anaerobic conditions. This removes the need for aeration, which is costly on a large scale. Establishing the anaerobic ethyl acetate pathway was faced with several bottlenecks, and removing them was the main theme of this chapter. The pathway towards ethyl acetate could be improved by disrupting by-product formation and optimising the expression levels of eat1. Further improvements could be made by removing the N-terminal mitochondrial localisation sequence of Eat1. These sequences typically destabilise proteins unless they are removed. These approaches did improve ethyl acetate formation by Eat1 to the point where ethanol levels were no longer sufficient to repress the hydrolytic activities of Eat1. To prevent ester hydrolysis the volatility of ethyl acetate was used to strip it from the fermenter. The result was ethyl acetate production at 63.4 % of the pathway maximum. We then looked beyond ethyl acetate as a bulk chemical in Chapter 5 and investigated the role Eat1 has in general ester formation by yeasts, particularly S. cerevisiae. The formation of esters is industrially relevant also in S. cerevisiae as these compounds contribute to the aroma of fermented foods. S. cerevisiae naturally produces a variety of alcohols and thus provided a convenient platform to compare Eat1 homologs from different yeast. Expression of various eat1 genes resulted in an increase of various in acetate and propionate ester levels. By disrupting the S. cerevisiae eat1 gene the inverse effect was observed. Eat1 therefore seems to contribute to acetate ester synthesis in S. cerevisiae as well. In this chapter, a S. cerevisiae strain was generated where all known AAT genes were disrupted. Opposite to the expectations, ester production persisted in this strain, showing that even more ester-producing mechanisms exist. Chapter 6 focuses on the complex field of ester production as bulk chemicals, from the fundamentals of microbial ester production. Much research has been devoted to understanding the ester-producing processes in microorganisms. This is a daunting task as the structures and functions of esters in microorganisms are in many cases unrelated. Most esters are produced via the AAT reaction. With the exception of Eat1, which was identified in this thesis, other AATs have been studied for a long time. Still, relatively little is known about their structure and catalytic mechanisms, likely because there are no crystal structures available yet. Nevertheless, they have been applied extensively in metabolic engineering of ester production. The AAT reaction is simple. As long as a suitable alcohol and acyl-CoA are provided, the AAT will catalyse the ester formation. The real challenge of engineering ester formation is in the efficient supply of alcohols and acyl-CoA substrates. Some remarkable success has been made in recent years which is outlined in the review. In many cases, esters are less toxic and more readily extractable than the alcohols and acids they are composed of. They could therefore serve as a platform compound. In summary, this thesis contributed significantly to the knowledge of ester synthesis in yeast. The progress made on the production of ethyl acetate specifically can be used to develop new biotechnological, more sustainable production processes for this versatile compound. At the same time, much remains to be discovered.

Microorganisms are under constant threat by their viruses, bacteriophages (phages). In response to this pressure, they have developed multiple strategies to protect from phage infection. Mechanisms that protect microorganisms from phage infection include restriction-modification, BREX, DISARM, and CRISPR-Cas systems. However, phages have developed several mechanisms by which they can evade anti-phage defense systems. One common way to evade these systems by phages is by modifying their nucleic acids (DNA). This thesis encompasses the characterization of several CRISPR-Cas and DISARM proteins and studying the effect of phage DNA modifications on the activity of these proteins.

Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery. Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times. Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis. Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders. In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming. Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.

The growing demand for consumables and energy, combined with increasing consciousness over environmental issues like global warming, faces us with the challenge to find alternatives for fossil resources. Alternative production methods for energy, like windmills, solar panels and hydroelectricity plants, are far developed and have become economically competitive to fossil resourcebased production processes. However, the production of many (bulk) chemicals and products is still dominated by the petroleum industry. One such chemical is lactic acid, a fermentation product of many bacteria and a compound that is gaining interest as a building block for poly lactic acid (PLA). PLA is a polymer used to produce bioplastics, and thereby provides an alternative to petroleumbased plastic production. As described in Chapter 1, economically feasible production of lactic acid is envisioned through consolidated bioprocessing (CBP). In a CBP process, pretreated lignocellulosic biomass is hydrolyzed to fermentable sugars and those sugars are subsequently fermented to desired product in one reaction vessel. The organism of choice for this hydrolyzation and fermentation is preferentially a thermophile, capable of enzyme production and lactic acid fermentation. Species from the genus Geobacillus have many of the desired characteristics, and in Chapter 2 we have enriched and isolated facultative anaerobic (hemi)cellulolytic Geobacillus strains from compost samples. By selecting for growth on both cellulose and xylan, 94 strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible with a transformation efficiency of 1.7×105 CFU/µg of plasmid DNA. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, we demonstrated that strain T12 is capable of direct conversion of beechwood xylan to mainly lactic acid in minimal media. Chapter 3 describes the genome sequencing and several features of G. thermodenitrificans T12. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 kb and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes is similar to those of the denitrification pathways found in strain NG80-2. The host-defence systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system that could potentially be exploited as a genome editing tool for thermophiles. Furthermore, the hemicellulose utilisation (HUS) locus of strain T12 appeared to have orthologues for all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. The HUS-locus associated gene, GtxynA1, encodes an extracellular endoxylanase of strain T12, and belongs to the family 10 glycoside hydrolases (GH10). In Chapter 4, we describe the cloning, expression and characterization of GtXynA1. The recombinant endoxylanase was purified to homogeneity and showed activity between 40°C and 80°C, with an optimum activity at 60°C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60°C. Highest activity was demonstrated towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 can degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endoxylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to be a potential candidate for CBP but lacks the desired endo and exoglucanases needed for the conversion of cellulose. In Chapter 5 we report the heterologous expression of endoglucanases and exoglucanases by G. thermodenitrificans T12, in an attempt to complement the enzymatic machinery of this strain and its suitability for consolidated bioprocessing. A metagenome screen was performed on the metagenome of 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-βglucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having most activity towards p-nitrophenyl-β-dxylopyranoside. Structure modelling of GE40 revealed a protein architecture similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic analysis against 2-HE-cellulose, CM-cellulose and barley β-glucan. In addition, we successfully expressed the earlier characterized Geobacillus sp. 70PC53 endoglucanase celA and the C. thermocellum exoglucanase celK in strain G. thermodenitrificans T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of Geobacillus thermodenitrificans T12 as a host for consolidated bioprocessing. In Chapter 6, we provided more insight in the genetic variation of the hemicellulolytic utilization cluster of G. thermodenitrificans. This variation is far greater than described before and gives ample opportunities for the further development of Geobacillus spp. for hemicellulose degradation. The production of cellulases in Geobacillus species is demonstrated to be successful, and we have expanded on that knowledge with the expression of both endo and exoglucanases from C. thermocellum. However, in line with previous studies, direct cellulose fermentation by geobacilli is not yet achieved, most likely due to insufficient cellulase production and/or secretion. With a rapidly expanding genetic toolbox for thermophiles, now including a thermostable Cas9, we expect that the successful development of Geobacillus spp. for consolidated bioprocessing is just a matter of time.

In the last few decades microorganisms have been isolated from rather unknown and hostile locations, such as those with high salt concentrations, an extreme pH, or low or high temperatures. Microorganisms isolated from these environments are referred to as extremophiles (1). The most extensively studied group of these extremophiles are the hyperthermophiles, microorganisms that have an optimum temperature for growth above 80 °C (2). Except for two bacterial genera, the Thermotageles and Aquifex , all hyperthermophiles isolated to date belong to the domain of the archaea. The archaea compose together with the bacteria and eucarya the three domains of life (3).Pyrococcus furiosus is a hyperthermophilic archaeon, with an optimal growth temperature of 100 °C that grows heterotrophically on a variety of substrates including peptides and saccharides. For its growth on saccharides it uses a modified version of the Embden-Meyerhof pathway, that involves novel enzymes and unique control mechanisms. The research described in this thesis has mainly focussed on the molecular and biochemical characterization of enzymes involved in the upper part of glycolysis in P. furiosus and related organisms (Fig. 10.1).A brief outline of this study is giving in Chapter 1 . In Chapter 2 sugar metabolism in archaea is reviewed. Recent studies on various modifications in the Entner-Doudoroff and Embden-Meyerhof pathways are discussed, and potential scenarios on the evolution of sugar metabolism are proposed.Figure 10.1 Classical Embden-Meyerhof (EM) pathway vs modified EM-pathway.Classical EM-pathway is operative in bacteria and eucarya. Modifications (*) in the EM-pathway are found in archaea. Enzymes that were under investigation in this thesis are indicated in blocks .Chapter 3 describes the first characterization of an ADP-dependent phosphofructokinase (ADP-PFK). Attempts to purify the ADP-PFK from P. furiosus cell extracts were not successful, because of the difficult purification procedure of this enzyme, which tends to stick to other proteins. Alternative approaches based on anticipated homology with the ADP-dependent glucokinase (ADP-GLK) have resulted in the identification of the gene encoding the ADP-PFK on the P. furiosus genome. The gene encoding the ADP-PFK was functionally expressed in Escherichia coli using a well-established expression system. The production of the ADP-PFK in the mesophilic E. coli allowed for a simple purification procedure consisting of a heat-treatment of the cell extract followed by a single chromatographic step. The purified enzyme was able to phosphorylate fructose-6-phosphate into fructose-1,6-bisphosphate with ADP as phosphoryl group donor. Classical PFKs use ATP or PP i as potential phosphoryl group donor, indicating that the P. furiosus enzyme differs from its canonical counterparts. The enzyme was not regulated by any of the known allosteric modulators of ATP-PFKs, implying that the P. furiosus glycolysis does not possess a typical site of regulation.Sequence analysis on the primary structure of the ADP-PFKs showed no significant sequence similarity with the classical monophyletic PFKs (PFKA). However, high similarity (21% identity) was observed with the ADP-dependent glucokinase (ADP-GLK) from P. furiosus , suggesting that both ADP-dependent sugar kinases are phylogenetically related, and belong to the same enzyme family. Orthologs of the ADP-PFK were identified in genome databases of the closely related P. horikoshii and P. abyssi . Also the paralogous ADP-GLK was present in these Pyrococci. Furthermore, orthologs of the ADP-PFK were identified in the hyperthermophilic methanogen Methanococcus jannaschii and the mesophilic methanogen Methanosarcina mazei ( Chapter 4 ). Based on a combination of genomic comparison and activity measurements it is concluded that ADP-PFKs are not restricted to the Thermococcales , but are present in mesophilic methanogens as well. Interestingly, uncharacterized homologs (presumably ADP-dependent) of this unusual kinase are present in several higher eucarya, including human, mouse and fly. The gene encoding the ADP-PFK from M. jannaschii was expressed in E. coli , and the enzyme was subsequently purified. The biochemical characteristics of the first ADP-PFK from a chemolithoautotrophic archaeon were compared to those of the ADP-PFK from the heterotrophic archaea P. furiosus and Thermococcus zilligii ( Chapter 4 ).In Chapter 5 an ATP-dependent galactokinase (catalyzing the first step of the Leloir pathway) from P. furiosus is described. Therefore, both ADP-dependent sugar kinases and an ATP-dependent sugar kinase appear co-exist in this hyperthermophile. The three dimensional structure of the P. furiosus galactokinase has recently been solved in close collaboration with the group of Prof. David Rice (Sheffield, England). Despite the ADP-dependent sugar kinases, the ATP-dependent galactokinase shares two conserved motifs and a high degree of overall similarity ( ± 32 % identity) to the canonical galactokinases. The galactokinase and the ADP-GLK from P. furiosus were produced in E. coli , and their characteristics were compared to each other and to their canonical counterparts. The kinetic and physical parameters of the heterologously produced ADP-GLK were in good agreement with those of the native ADP-GLK, indicating that the enzyme was successfully produced and folded in E. coli . The affinity for ATP of the galactokinase was extremely high at 90 °C ( K m for ATP of 0.008 mM) compared to the classical galactokinase from mesophiles. However, the affinity for galactose was comparable to that of the canonical enzymes. It was suggested that the extremely high affinity of the galactokinase for ATP might reflect an adaptation to a relative low intracellular ATP concentration in P. furiosus . This might also explain the presence of the ADP-dependent sugar kinases in P. furiosus . Both the ATP-dependent galactokinase and the ADP-GLK showed a high catalytic efficiency for their phosphoryl group donor at 90 °C, compared to their mesophilic counterparts.Chapter 6 describes the purification of a unique phosphoglucose isomerase from P. furiosus , its characterization, isolation of the corresponding gene, and prediction of the structure of the enzyme. The phosphoglucose isomerase was purified from a P. furiosus extract. The N-terminal sequence of the purified enzyme was determined, and the gene, named pgiA , could be identified on the P. furiosus genome. Subsequent expression in E. coli revealed that the gene indeed encoded a phosphoglucose isomerase. The pgiA gene was transcribed as a mono-cistronic messenger, and the transcription start site was mapped. Despite similar substrate specificity and kinetic parameters, no significant sequence similarity was obtained with classical phosphoglucose isomerases. In contrast, the enzyme shares similarity with the CUPIN superfamily (double-stranded beta-helices) that consists of a variety of proteins that are generally involved in sugar binding or protein interaction. This is the first example of a phosphoglucose isomerase that belongs to the CUPIN superfamily, and it is the first characterization of an archaeal phosphoglucose isomerase to date. The novel phosphoglucose isomerase and the two ADP-dependent sugar kinases are examples of an excessive replacement of enzymes in glycolysis, and are a compelling example of convergent evolution.Chapter 7 focuses on two archaeal fructose-1,6-bisphosphate aldolases, i.e fructose-1,6-bisphosphate aldolase from the crenarchaeon Thermoproteus tenax and from the euryarchaeon P. furiosus . The genes encoding these enzymes were identified in the genomes based on sequence similarity with a novel fructose-1,6-bisphosphate aldolase from E. coli . Transcript analyses reveal that the in vivo expression of both genes is induced during sugar fermentation. Subsequently, the genes were expressed in E. coli , and the encoded proteins were purified to homogeneity. Both the archaeal enzymes use a Schiff base mechanism for catalysis similar to the Class I aldolases, in contrast to the Class II aldolases that use metal ions for catalyses. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal fructose-1,6-bisphosphate aldolase. Because this family shows no overall sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I fructose-1,6-bisphosphate aldolase (Class IA). Despite to low sequence similarity between the archaeal type Class I fructose-1,6-bisphosphate aldolases and the classical Class I and Class II aldolases, sequence signatures could be identified resembling the active site region (Lys-191) and the phosphate-binding motif of classical Class I fructose-1,6-bisphosphate aldolases and other members of the (βα)8 barrel superfamilies. This suggests that the archaeal type Class I enzymes are distantly related to the classical Class I fructose-1,6-bisphosphate aldolases, and that they share the same ancestral origin.In Chapter 8 the P. furiosus gluconeogenic fructose-1,6-bisphosphatase is described. The gene was identified in the genome based on the sequence similarity with the recently described Methanococcus jannaschii bi-functional inositol-monophosphatase/fructose-1,6-bisphosphatse. The gene was functionally expressed in E. coli , and the enzyme was subsequently purified to homogeneity. Biochemical characteristics were compared with the homologous gene product from M. jannaschii (MJ0109), revealing distinct characteristics in substrate specificity and inhibitors. The M. jannaschii enzyme is a bi-functional enzyme with high activity on inositol-1-phosphosphate and fructose-1,6-bisphosphate. The P. furiosus enzyme has a more specific substrate specificity with a clear preference for fructose-1,6-bisphosphate. Therefore, the enzyme can be regarded as a true fructose-1,6-bisphosphatase. Sequence analysis of the P. furiosus fructose-1,6-bisphosphatase reveals the enzyme to be more similar to inositol monophosphatases than to fructose-1,6-bisphosphatases (type I), both belonging to the sugar phosphatase superfamily, with similar folding and sequence motifs. Because of the higher similarity of the P. furiosus enzyme to the inositol monophosphatases, and because of its specific preference for fructose-1,6-bisphosphate, the enzyme was proposed to belong to a new sub-family: the euryarchaeal fructose-1,6-bisphosphatase (type IV). This new sub-family shows limited sequence similarity to classical fructose-1,6-bisphosphatase from bacteria and eucarya (type I), and no significant sequence similarity to the bacterial fructose-1,6-bisphosphatases (type II and III).Preliminary results in promoter architecture of genes encoding glycolytic enzymes are described in Chapter 9 . Promoter elements were identified, and a putative glycolytic regulator binding site (ATCACNNNNNGTGAT, where N are random nucleotides) is observed specifically in P. furiosus promoter sequences of glycolytic-enzyme encoding genes. Complete analysis of the P. furiosus genome revealed that this motif is present in 21 promoter sequences. The majority of the genes encode enzymes involved in sugar metabolism. Further research is needed to reveal the function of this putative binding site.In conclusion, this project has resulted in the identification of unique genes encoding novel enzymes of modified glycolytic pathways in archaea. Key enzymes of the pyrococcal glycolytic pathway were shown to be modified in enzyme catalysis, evolution and regulation. In close collaboration with the group of Prof. David Rice (Sheffield, England) significant progress has been made in crystallization of the ADP-PFK and galactokinase from Pyrococcus. Finally, it is postulated that regulation of the glycolytic flux in P.furiosus might involve modulation of gene expression rather than allosteric regulation of enzyme activities. High throughput screening by transcriptomic and proteomic approaches like DNA micro-arrays and 2D-gelelectrophoresis, and generation of knock-out mutants in Pyrococcus will provide more insight in the actual significance of regulation of gene expression in archaeal central metabolism in the near future.

Haloorganics such as chlorophenols and chlorinated ethenes are among the most abundant pollutants in soil, sediments and groundwater, mainly caused by past and present industrial and agricultural activities. Due to bioaccumulation and toxicity, these compounds threaten the integrity of the environment, and human and animal health. A recently discovered, phylogenetically diverse, group of anaerobic so-called halorespiring bacteria is able to couple the reductive dehalogenation of various haloorganic compounds to energy conservation and hence to growth, significantly contributing to in situ dehalogenation processes in anoxic environments. The observed persistence of halogenated pollutants in untreated ecosystems and the accumulation of degradation intermediates during bioremediation, however, indicated the need for engineering of process conditions and/or augmentation with efficient degraders. This thesis describes genetic approaches towards a thorough understanding of the molecular basis of anaerobic reductive dehalogenation in order to enable the further optimization of clean up procedures for contaminated anoxic environments.The Gram-positive Desulfitobacterium dehalogenans , capable of degrading o -chlorophenols, PCE and hydroxylated PCB's, was used as model organism throughout a major part of this study. The key enzyme o -chlorophenol reductive dehalogenase (CPR) was isolated and characterized at the biochemical and molecular levels, and comparison with known chlorophenol- and chloroalkene-reductive dehalogenases indicated that these enzymes constitute a yet unknown, but evolutionary ancient family of corrinoid-containing iron-sulfur proteins, which in addition share a twin-arginine signal sequence. Transcriptional analysis of the CPR-encoding gene cluster revealed that dehalogenation activity is strongly regulated at the transcriptional level. Efficient gene cloning, and random and specific gene inactivation systems were developed to enable (i) the elucidation of additional components involved in the anaerobic dehalogenation process and (ii) the study of their structure and function within the respiratory network. Halorespiration-deficient mutants were isolated following random chromosomal integration, and their characterization lead to the identification of genes encoding proteins possibly involved in structure, maturation and regulation of respiratory complexes. Reductive dehalogenase-encoding gene targeted (RT-)PCR-based molecular approaches were developed that were useful for the detection of reductive dehalogenation potential and activity in pure cultures of potentially halorespiring microorganisms.The results obtained in this study provide valuable knowledge on the molecular basis of anaerobic reductive dehalogenation and might serve as a sound basis for the further exploitation of halorespiring bacteria as dedicated degraders in biological remediation processes.

Hyperthermophilic Archaea are microorganisms that grow optimally above 80°C. To be able to live at these temperature extremes their cell components display extreme resistance towards thermal degradation. This characteristic is an attractive feature for use of their enzymes in industrial processes. Examples of thermozymes with potential applications in food and pharmaceutical industry are b -Glycosidases, enzymes that specifically hydrolize b -linked glycosidic bonds, which are present in for instance cellulose or the milk sugar lactose. .Using the b -glucosidase CelB from Pyrococcus furiosus as a model enzyme, molecular determinants of substrate recognition and catalysis in b -glycosidases have been studied by rational design and directed evolution approaches. A 3D model of CelB was established, and its active site was compared to that of a related enzyme with a distinct specificity, the 6-phospho-b -galactosidase LacG of Lactococcus lactis. The substrate specificity of CelB was adjusted by engineering a phosphate-binding site, which resulted in a significant improvement in the hydrolysis of 6-phospho-b-glycosides. In a second study, the active sites of b -glucosidase CelB was compared to that of a b -mannosidase BglB from Pyrococcus horikoshii , and the substrate affinities and activities of the two enzymes could be swapped by exchange of unique residues in their active sites.The b -glucosidase CelB of P. furiosus was also compared to that of the related b -glycosidase LacS of the hyperthermophile Sulfolobus solfataricus. While the enzymes are very similar regarding catalytic mechanism and substrate specificity, they have not been stabilized to withstand high temperatures in the same way. While CelB is relatively sensitive to detergents, LacS is readily inactivated in the presence of salts. This strongly suggests that CelB is mainly stabilized by hydrophobic interactions, while ion-pair interactions contribute most to the stability of LacS.In one of the first laboratory evolution studies on proteins from a hyperthermophile, CelB has been optimized for low-temperature catalysis. In several CelB mutants this was accomplished with retention of wild-type stability. Increased activity at low temperatures seemed to result from mutations that increase protein flexibility. In a second directed evolution study, the genes coding for CelB and LacS were shuffled to functional b -glycosidase hybrids. The hybrids of this DNA shuffling experiment were screened for thermostability and hydrolysis of lactose at 70 °C. Several thermostable high-performance mutants were isolated and characterized. The hybrids consisted of an N-terminal LacS stretch, followed by a CelB core. This resulted in a hybrid active site structure, which could explain the altered catalytic properties.Finally, site-directed CelB mutants from the previous mentioned studies have been tested for their ability to catalyze oligosaccharide synthesis. Indeed, several variants showed increased yields in galacto-oligosaccharide synthesis with lactose as a substrate, compared to wild-type CelB.The studies described in the thesis are illustrative for the differences in protein engineering by rational design versus by directed evolution. While rational design can give an initial change in activity or substrate specificity, directed evolution is more likely to be successful for fine-tuning of enzyme properties.&nbsp

Hyperthermophilic organisms are able to survive and reproduce optimally between 80°C and 113°C. Most of them belong to the domain of the Archaea, although several hyperthermophilic Bacteria have been described. One of the major questions regarding hyperthermophiles concerns the molecular mechanisms that determine the extreme stability of their macromolecules. In particular, enzymes on the one hand should be flexible in order to efficiently perform catalysis, while on the other hand they should be sufficiently rigid in order to prevent thermal unfolding and inactivation. In other words, they have to be thermostable as well as thermoactive. Therefore, the research described in this thesis has been focused on analysing the molecular determinants of enzyme thermostability and thermoactivity.In the first chapter of this thesis, hyperthermophilic microorganisms are introduced, and the origin of the hyperthermophilic phenotype is discussed in the light of established and emerging phylogenetic, biochemical and structural biological considerations. Furthermore, thermodynamic and kinetic protein stability is explained, and an overview is given of the strategies that have been suggested to confer thermostability to enzymes from hyperthermophiles, by analysing amino acid sequences, homology-based structural models, and three-dimensional structures. One of the proposed general strategies for achieving hyperthermostability relates to the number of ion-pairs and their organisation in extensive networks in hyperthermostable enzymes. While biochemical studies have shown that isolated ion-pairs at room temperature do not contribute significantly to protein stability, several theoretical considerations indicate that arrangements of ion-pairs into networks at high temperatures may play an important role in protein stabilisation. In contrast to the need for maximum stability at high temperatures, it is the requirement for flexibility that enables an enzyme to efficiently perform its catalytic function. The mechanisms that determine hyperthermostability on the one hand, hyperthermoactivity on the other hand, and the interplay between these two characteristics, have been investigated in the research described in this thesis, using three different approaches of protein engineering, namely (i) domain swapping, (ii) site-directed mutagenesis, and (iii) random mutagenesis and directed evolution.In order to study the molecular determinants of enzyme thermostability and thermoactivity, we decided to include representatives of both hyperthermophilic Archaea and Bacteria in the analysis. Pyrococcus furiosus was selected as hyperthermophilic Archaeon, since it optimally grows at 100°C . Moreover, P.furiosus is very well studied with regard to its physiology, biochemistry and genomics. As representative of the hyperthermophilic Bacteria we choose Thermotoga maritima that grows optimally at 80°C . Both organisms are able to ferment mixtures of polypeptides and a variety of oligosaccharides. One of the enzymes that was chosen as model, isβ-glucosidase, a glycosyl hydrolase involved in the hydrolysis of disaccharides and oligosaccharides into the corresponding monomeric sugars that are further metabolised. The second model enzyme is glutamate dehydrogenase (GDH), a key enzyme in a pathway that is involved in the disposal of reducing equivalents that are generated during sugar fermentation. In both P.furiosus and T.maritima these two key enzymes are expected to perform similar functions.GDH is extensively introduced in Chapter 2 and is a useful model enzyme since it is well studied, catalyses an important reaction, and has been isolated and characterised from many organisms in all three kingdoms of life, ranging from psychrophiles to hyperthermophiles. Multiple amino acid sequences are available, as well as high resolution three-dimensional structures from GDH of various sources including the hyperthermophiles P.furiosus and T.maritima . Furthermore, the gdh genes from these hyperthermophiles can be easily overexpressed in Escherichia coli , allowing efficient enzyme purification and mutagenesis (Chapter 3). GDH is in general a hexameric enzyme, composed of six identical subunits that are arranged in two trimers that are stacked upside down on top of each other . Each subunit is composed of a substrate and a nicotinamide cofactor binding domain, linked by a flexible hinge region and separated by a deep cleft in which the active site is located. During catalysis, the hinge region mediates the opening and closing of the cleft by a rotation of the cofactor-domain with respect to the substrate-binding domain. In this way an adequate hydrophobic environment for hydride transfer during catalysis is created.Chapter 3 describes the functional expression of the gdh gene from P.furiosus in E.coli . The P.furiosus GDH amounted to 20 % of total E.coli cell protein, with the majority of the expressed protein in the hexameric conformation. Following activation by a heat-treatment, the GDH that could be purified from E.coli was indistinguishable from that purified from P.furiosus . The role of the GDH substrate and cofactor binding domains in conferring thermoactivity and thermostability were studied by exchanging them between the GDHs of the hyperthermophilic archaeon P.furiosus and the mesophilic bacterium C.difficile . Hybrid genes were constructed and successfully expressed in E.coli . One of the resulting hybrid proteins, containing the glutamate-binding domain of the C.difficile and the cofactor-binding domain of the P.furiosus enzyme, did not show detectable activity. In contrast, the complementary hybrid, containing the P.furiosus glutamate and the C.difficile cofactor binding domain was found to be a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the C.difficile enzyme. Compared to the C.difficile GDH, the latter archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.These results indicate (i) that the cofactor-binding domain is structurally independent from the substrate-binding domain with respect to affinity for the coenzyme, and (ii) that thermal and chemical stability may be uncoupled.The elucidation of the three-dimensional structure of GDH from P.furiosus and its comparison with the homologous GDH from the mesophilic bacterium C.symbiosum , has highlighted the formation of extensive ion-pair networks at domain and subunit interfaces as a possible explanation for the superior thermostability of the archaeal enzyme . In order to assess whether this is a more general stabilising strategy which is not only employed in GDH from P.furiosus , but also in enzymes from other hyperthermophilic Archaea and Bacteria, an homology-based modelling study was carried out using GDH amino acid sequences derived from species spanning a wide spectrum of optimal growth temperatures. Chapter 4 describes the observed correlation between the amount and extent of ion-pair networks and the thermal stability of the studied enzymes, which is consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.In order to analyse the role of ion-pair networks in more detail, we decided to rationally engineer the networks that are found in the GDH from P.furiosus , into the GDH from T.maritima , in which most of the networks are largely reduced or absent and which is much less thermostable than the pyrococcal GDH . In the study that is described in Chapter 5, we focused on the flexible hinge region that is connecting the two domains. While in GDH from P.furiosus a five-residue ion-pair network is present, this is reduced to two single ion-pairs in T.maritima GDH. Using a site-directed mutagenesis approach, the missing charged residues were introduced into T.maritima GDH.The resulting mutant GDHs as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified. Elucidation of the three-dimensional structure of the double mutant N97D/G376K at 3.0 Å, showed that the designed ion-pair interactions were indeed formed. Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant. Melting temperatures of the mutant enzymes N97D, G376K and N97D/G376K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme. Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes. Thermal inactivation at 85°C occured more than two-fold faster for all mutant enzymes than for the wild-type GDH. At temperatures of 65°C and higher, the wild-type and the three mutant enzymes showed identical specific activities. However, at 58°C the specific activity of N97D/G376K and G376K was found to be significantly higher than that of the wild-type and N97D enzymes. These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature- or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature at which it functions optimally.Chapter 6 focuses on the role of an 18-residue ion-pair network, which is present at the subunit interface of the GDH from P.furiosus . This network has been studied by introducing four new charged amino acid residues into the subunit interface of GDH from T.maritima. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T.maritima GDH showed that all anticipated ion-pairs were formed and that a 16-residue ion-pair network was present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilising single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilising mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85°C, a 3°C higher temperature optimum for catalysis, and a 0.5°C higher apparent melting temperature than that of wild type GDH. These findings confirm the hypothesis that large ion-pair networks do indeed stabilise enzymes from hyperthermophilic organisms.Regarding subunit interfaces in GDH, a more or less complete picture of the role of ion-pair networks has emerged by now. The modelling study that involved GDHs derived from different sources spanning the complete temperature spectrum, showed a progressive increase in formation of networks at the subunit interfaces with increasing temperature. The removal of central ion-pairs from the large 18-residue ion-pair network in P. kodakaraensis GDH has been reported to reduce the half-life for thermal inactivation.Furthermore, there are now multiple examples of reconstituted large networks in less thermostable GDHs that significantly increase half-life values for thermal inactivation and apparent melting temperatures (Chapter 6). Ion-pair networks therefore play an important role in the thermostabilisation and maintenance of a correct structure of this enzyme. However, except for the studies described in this thesis (Chapter 5 and 6), three-dimensional structures of mutated GDHs have not been determined. This implies that only for the charged residues introduced into the subunit interface and hinge region of T.maritima GDH it has actually been proven that the predicted networks are indeed formed.The role of ion-pairs in the flexible hinge region of GDH is less well resolved. These networks are apparently involved in determining thermoactivity and kinetic parameters, which is consistent with the hinge region being involved in positioning of the domains with regard to each other during catalysis. However, the introduction of a 6-residue ion-pair network in the hinge of T.maritima GDH did not increase the thermostability of this enzyme. This result may reflect the considerable differences between the archaeal and the bacterial enzyme, which share 55% amino acid identity. This consideration is supported by the fact that (i) in Thermococcus litoralis GDH, which has a much higher homology (87% identity) with P.furiosus GDH, a restored ion-pair network was found to be stabilising only in combination with a second site mutation , and (ii) stabilisation of the T.maritima GDH subunit interface is only observed after the combination of multiple, destabilising single mutations (Chapter 6).Apparently, different strategies for stabilisation have evolved within the hyperthermostable members of this protein family. While the subunit interfaces of the most thermostable, archaeal members are highly charged, the subunit interface of the T.maritima GDH is optimised by hydrophobic interactions . In spite of this, we have shown that it is possible to successfully introduce an archaeal feature into a bacterial enzyme that is stabilised by a different mechanism. The electrostatic optimisation of the subunit interfaces in the archaeal GDHs, on the one hand, and the hydrophobic optimisation of the bacterial GDH subunit interface on the other hand, is not a universal division. Remarkably, exactly the opposite situation has been observed in the superoxide dismutase family, which shows electrostatic optimisation of the subunit interfaces in the enzyme from the bacterium Aquifex pyrophilus , and hydrophobic optimisation in the enzyme of the archaeon Sulfolobus acidocaldarius .The thesis continues with an overview of the current knowledge on the well-studied second model enzyme, theβ-glucosidase CelB from P.furiosus (Chapter 7). CelB is involved in the hydrolysis ofβ-(1,4)-linked disaccharides during growth on sugars, and is highly thermostable and thermoactive, with a half-life of thermal inactivation of 85 hours at 100°C and an optimum temperature for catalysis of 102-105°C . Heterologous expression and enzyme purification procedures have been developed that allow for routine site-directed mutagenesis and in vitro applications (Chapter 7).Directed evolution is a potent approach to evolve a desired property into a biocatalyst, as well as a promising method to study structure-function and structure-stability relationships. So far, this approach was restricted to model enzymes from mesophilic and thermophilic sources. Chapter 7 describes the development of a directed evolutionary approach for the thermostable CelB by random mutagenesis, in vitro recombination and a rapid screening procedure. The constructed library contains over 6000 random CelB clones with an average of 1 or 2 amino acid substitutions per enzyme. The ability of CelB to hydrolyse chromogenic glucose- and galactose derivatives was used to isolate mutants with a changed ratio in the hydrolysis of these enantiomeric sugars. One of the isolated mutants was characterised in detail and found to contain a single amino acid substitution of asparagine to serine at position 206 in the active site. In the wild-type enzyme this asparagine forms a hydrogen bond with the hydroxyl group of the substrate at the C2 position. Biochemical analysis of the mutant using different substrates provided insight in the active-site architecture of CelB, and indicated that the interactions between CelB and its substrates are similar to those observed in three-dimensional structures of mesophilic homologues complexed with substrate analogues.Chapter 8 describes a three-dimensional model of CelB and the validation of this model by engineering substrate specificity. The model is based on 3.3 Å X-ray diffraction data and on the tetramericβ-glycosidase LacS from Sulfolobus solfataricus and the monomeric 6-phospho-β-galactosidase from Lactococcus lactis (LacG) as search models. CelB shows high structural homology with LacS and contains subunits that adopt the common (αβ) 8 barrel motif and are arranged in a slightly twisted tetramer. The difference in substrate specificity betweenβ-glycosidases and 6-phospho-β-glycosidases seems to be determined by three residues in the active site that are conserved within, but not between, both types of glycosidases. This hypothesis was tested by replacing these residues in CelB with the corresponding substitutes, as present in LacG. Mutant CelB containing all three substitutions showed a reduced catalytic activity on non-phosphorylated sugars and increased efficiency on 6-phospho-β. These results indicate that the phosphate-binding site is functional at elevated temperatures.Low-temperature activity and substrate specificity of CelB was studied in Chapter 9 using the random CelB library described in Chapter 7. Screening of this library at room temperature on the artificial substrate pNp-glucose resulted in the isolation and characterisation of mutants with up to three-fold increased rates of hydrolysis of this aryl-glucoside. Amino acid substitutions were identified in the active site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterisation of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilisation of the enzyme. Kinetic characterisation of active site mutants showed that in all cases the catalytic efficiency at 20°C on p -nitrophenol-βD-glucose as well as on the disaccharide cellobiose, was up to two-fold increased. In most cases, this was achieved at the expense ofβ-galactosidase activity at 20°C, and of total catalytic efficiency at 90°C. Substrate specificity is affected by many of the observed amino acid substitutions, only some of which are located in the vicinity of the active site. The largest change in the ratio of p -nitrophenol-β-D-glucopyranoside/ p -nitrophenol-β-D-galactopyranoside hydrolysis is a 7.5-fold increase, which was observed in N415S CelB. This asparagine at position 415 interacts with active site residues that stabilise the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing and axial in galactose-containing substrates.In conclusion, this random screening approach using a hyperthermostable enzyme resulted in the identification of residues that are critical in determining thermostability, low-temperature activity, and substrate recognition. It was shown that low-temperature activity may be engineered into a hyperthermostable enzyme without affecting its extreme stability. Several mutants display evolved properties that could enable them to perform more efficiently in industrial or biotechnological applications than the wild-type CelB. It is feasible that N415S CelB with its increased catalytic efficiency on cellobiose but unchanged stability, or T371A CelB with its much better affinity for cellobiose, could be applied instead of wild-type CelB in biosensors for the detection of cellobiose at low temperatures. Not only is directed evolution a potent approach to evolve a desired property into a biocatalyst of potential industrial interest, but, as is shown here, it may enhance our understanding of the mechanisms determining enzyme catalysis, stability, and substrate recognition at physiological or extreme conditions.In Chapter 10 the molecular basis for the extreme thermostability of CelB has been addressed by comparative analysis of the three-dimensional model of CelB and the crystal structure of the homologousβ-glycosidase LacS from S.solfataricus . Furthermore, this chapter describes the biochemical characterisation of wild-type CelB and mutants thereof, containing substitutions or deletions of charged residues at the C-terminus, which may be involved in the formation of ion-pair networks. All mutant CelB enzymes show decreased optimal temperatures for activity and increased rates of thermal inactivation, indicating that ion-pairs at the C-terminus of CelB play a role in determining the extreme thermostability of this enzyme. Moreover, increased thermal inactivation of CelB in the presence of sodium chloride and at extreme pHs, supports the role of electrostatic interactions in this enzyme. The CelB structure indicates that part of CelB stability is derived from structural adaptations that allow oligomerisation and from the presence of solvent-filled, hydrophilic cavities, which have earlier been identified in LacS. However, structural comparison of several large ion-pair networks that are present in LacS, reveals that a five-residue and a six-residue membered network is not present in CelB and a large 16-membered network at the subunit interface, extensively cross-linking the subunits, has been fragmented.The picture emerging from the structural and biochemical characterisation of wild-type, site-directed, and random mutants of CelB, is that, compared to homologous enzymes from mesophilic sources, active site architecture, substrate recognition, acid-base catalysis and transition state stabilisation have been preserved, while thermoactivity and thermostability have been adapted to the high-temperature conditions in which the enzyme has to function optimally.An evaluation of the obtained results and conclusions against the original aims as set in the introduction of this thesis reveals the following. With the GDH model we have established and validated a strategy for enzyme stabilisation that seems to be employed by many different hyperthermophiles. Furthermore, we have shown that these features can be successfully engineered into less thermostable enzymes, in order to raise their thermostability. In addition, we have increased our knowledge of enzyme catalysis and substrate recognition at high temperatures and in relation with low-temperature catalysis, using site-directed mutagenesis and directed evolution of the hyperthermostableβ-glucosidase. The directed evolutionary approach, in particular, has been shown to be a potent technique which will in the near future rapidly increase our knowledge of enzyme characteristics and may be employed to increase the biotechnological potential of the enzymes under study.