Structural and Biochemical Analysis of Butanol Dehydrogenase From Thermotoga maritima.

Butanol dehydrogenase (BDH) plays a crucial role in butanol biosynthesis by catalyzing the conversion of butanal to butanol using the coenzyme NAD(P)H. In this study, we observed that BDH from Thermotoga maritima (TmBDH) exhibits dual coenzyme specificity and catalytic activity with NADPH as the coenzyme under highly alkaline conditions. Additionally, a thermal stability analysis on TmBDH demonstrated its excellent activity retention even at elevated temperatures of 80°C. These findings demonstrate the superior thermal stability of TmBDH and suggest that it is a promising candidate for large-scale industrial butanol production. Furthermore, we discovered that TmBDH effectively catalyzes the conversion of aldehydes to alcohols and exhibits a wide range of substrate specificities toward aldehydes, while excluding alcohols. The dimeric state of TmBDH was observed using rapid online buffer exchange native mass spectrometry. Additionally, we analyzed the coenzyme-binding sites and inferred the possible locations of the substrate-binding sites. These results provide insights that improve our understanding of BDHs.

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate.

The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0-9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and kcat/Km (598.57 ± 19.97 s-1mM-1) for pN-C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The Km, kcat, and kcat/Km values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s-1, and 2.67 ± 0.02 mM-1 s-1, respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s-1, and 0.02 ± 0.001 mM-1 s-1, respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.

Multifunctional enzymes related to amino acid metabolism in bacteria.

In bacteria, d-amino acids are primarily synthesized from l-amino acids by amino acid racemases, but some bacteria use d-amino acid aminotransferases to synthesize d-amino acids. d-Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial d-amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with d-amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related d- and l-amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima.

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases.

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T. maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T. maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

Orientation of Cel5A and Xyn10B in a fusion construct is important in facilitating synergistic degradation of plant biomass polysaccharides.

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 U µmol-1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90 °C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60 °C for 1 h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose-binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer-dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP-like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin-like protein and further support a domain-swapping step that might have occurred during the evolution of the PBP-like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions.

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7.

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

Characterization of L-arabinose/D-galactose 1-dehydrogenase from Thermotoga maritima and its application in galactonate production.

The first hyperthermophilic L-arabinose/D-galactose 1-dehydrogenase (TmAraDH) from Thermotoga maritima was heterologously purified from Escherichia coli. It belongs to the Gfo/Idh/MocA protein family, prefers NAD+/NADP+ as a cofactor. The purified TmAraDH exhibited maximum activity toward L-arabinose at 75 °C and pH 8.0, and retained 63.7% of its activity after 24 h at 60 °C, and over 60% of its activity after holding a pH ranging from 7.0 to 9.0 for 1 h. Among all tested substrates, TmAraDH exclusively catalyzed the NAD(P)+-dependent oxidation of L-arabinose, D-galactose and D-fucose. The catalytic efficiency (kcat/Km) towards L-arabinose and D-galactose was 123.85, 179.26 min-1 mM-1 for NAD+, and 56.06, 18.19 min-1 mM-1 for NADP+, respectively. TmAraDH exhibited complete oxidative conversion in 12 h at 70 °C to D-galactonate with 5 mM D-galactose. Modelling provides structural insights into the cofactor and substrate recognition specificity. Our results suggest that TmAraDH have great potential for the conversion of L-arabinose and D-galactose to L-arabonate and D-galactonate.

The role of C-terminal helix in the conformational transition of an arginine binding protein.

The thermotoga maritima arginine binding protein (TmArgBP) is a periplasmic binding protein that has a short helix at the C-terminal end (CTH), which is swapped between the two chains. We apply a coarse-grained structure-based model (SBM) and all-atom MD simulation on this protein to understand the mechanism and the role of CTH in the conformational transition. When the results of SBM simulations of TmArgBP in the presence and absence of CTH are compared, we find that CTH is strategically located at the back of the binding pocket restraining the open-state conformation thereby disengaging access to the closed-state. We also ran all-atom MD simulations of open-state TmArgBP with and without CTH and discovered that in the absence of CTH the protein could reach the closed-state within 250 ns, while in its presence, the protein remained predominantly in its open-state conformation. In the simulation started from unliganded closed-state conformation without CTH, the protein exhibited multiple transitions between the two states, suggesting CTH as an essential structural element to stabilize the open-state conformation. In another simulation that began with an unliganded closed-state conformation with CTH, the protein was able to access the open-state. In this simulation the CTH was observed to reorient itself to interact with the protein emphasizing its role in assisting the conformational change. Based on our findings, we believe that CTH not only acts as a structural element that constraints the protein in its open-state but it may also guide the protein back to its open-state conformation upon ligand unbinding.

A Cytoplasmic NAD(P)H-Dependent Polysulfide Reductase with Thiosulfate Reductase Activity from the Hyperthermophilic Bacterium Thermotoga maritima.

Thermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments. IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

Identification of a novel d-amino acid aminotransferase involved in d-glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

In Vitro One-Pot 3-Hydroxypropanal Production from Cheap C1 and C2 Compounds.

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.

Structural Basis of Redox-Sensing Transcriptional Repressor Rex with Cofactor NAD+ and Operator DNA.

The transcriptional repressor Rex plays important roles in regulating the expression of respiratory genes by sensing the reduction-oxidation (redox) state according to the intracellular NAD+/NADH balance. Previously, we reported on crystal structures of apo, NAD+-bound, and NADH-bound forms of Rex from Thermotoga maritima to analyze the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. In this study, the crystal structure of Rex in ternary complex with NAD+ and operator DNA revealed that the N-terminal domain of Rex, including the helix-turn-helix motif, forms extensive contacts with DNA in addition to DNA sequence specificity. Structural comparison of the Rex in apo, NAD+-bound, NADH-bound, and ternary complex forms provides a comprehensive picture of transcriptional regulation in the Rex. These data demonstrate that the conformational change in Rex when binding with the reduced NADH or oxidized NAD+ determines operator DNA binding. The movement of the N-terminal domains toward the operator DNA was blocked upon binding of NADH ligand molecules. The structural results provide insights into the molecular mechanism of Rex binding with operator DNA and cofactor NAD+/NADH, which is conserved among Rex family repressors. Structural analysis of Rex from T. maritima also supports the previous hypothesis about the NAD+/NADH-specific transcriptional regulation mechanism of Rex homologues.

High-temperature behavior of hyperthermostable Thermotoga maritima xylanase XYN10B after designed and evolved mutations.

A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3-3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower Km. In XYNB and XYNC, the additional mutations over XYNA decreased the thermostability and temperature optimum, while elevating the Km. In XYNC, two large side-chains were introduced into the protein's interior. Micro-differential scanning calorimetry (DSC) showed that the melting temperature (Tm) dropped in XYNB and XYNC from 104.9 °C to 93.7 °C and 78.6 °C, respectively. The detrimental mutations showed that extremely thermostable enzymes can tolerate quite radical mutations in the protein's interior and still retain high thermostability. The analysis of mutations (F806H and F806V) in a hydrophobic area lining the substrate-binding region indicated that active site hydrophobicity is important for high activity at extreme temperatures. Although polar His at 806 provided higher stability, the hydrophobic Phe at 806 provided higher activity than His. This study generates an understanding of how extreme thermostability and high activity are formed in GH10 xylanases. KEY POINTS: • Characterization and molecular dynamics simulations of TmXYN10B and its mutants • Explanation of structural stability of GH10 xylanase.

Changes in the Distribution of Membrane Lipids during Growth of Thermotoga maritima at Different Temperatures: Indications for the Potential Mechanism of Biosynthesis of Ether-Bound Diabolic Acid (Membrane-Spanning) Lipids.

Membrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32, and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn-1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids. IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.

Molecular dynamics simulation study on Thermotoga maritima EngA: GTP/GDP bound state of the second G-domain influences the domain-domain interface interactions.

EngA, a GTPase involved in the late steps of ribosome maturation, consists of two GTP binding domains (G-domains) [GD1, GD2] and a C-terminal domain. The combination of GTP/GDP in G-domains dictates its binding to the ribosomal subunits by altering its conformation. Studies and comparisons on the available structures of EngA enable us to understand the correlation between nucleotide bound states and its conformation. Using all-atom molecular dynamics (MD) simulations, we have explored the conformational behavior of EngA from Thermotoga maritima (TmDer) upon binding the various combinations of GTP and GDP. Analyses of Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) emphasize the importance of the second G-domain nucleotide bound state. RMSD and Rg exhibit slightly lower values when GTP is embedded in GD2 compared to GDP. These lower values are due to Sw-II of GD2, which has been observed from RMSF plot. Further investigation on the effects of GD2 nucleotide bound state using Principal Component Analysis (PCA) and Free Energy Landscape (FEL) analysis manifests an allosteric connection between GD2 nucleotide bound state and the GD1-KH interface. This is further validated by extracting electrostatic interactions and H-bonds at the GD1-KH interface. In silico mutations at the GD1 interface of KH domain affect the Sw-II mobility of GD2 by showing inverted behavior. This suggests using the second G-domain as an antibacterial target and further simulation studies on different species of EngA are to be explored.Communicated by Ramaswamy H. Sarma.

Acetylornithine aminotransferase TM1785 performs multiple functions in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system.

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System.

The effects of water activity (aw), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90-95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst.

Engineering Thermotoga maritima ß-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis.

Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a ß-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-ß-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-ß-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-ß-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.