Exploring a novel ß-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Medium development and production of carotenoids and exopolysaccharides by the extremophile Rhodothermus marinus DSM16675 in glucose-based defined media.

BACKGROUND: The marine thermophilic bacterium Rhodothermus marinus can degrade many polysaccharides which makes it interesting as a future cell factory. Progress using this bacterium has, however, been hampered by limited knowledge on media and conditions for biomass production, often resulting in low cell yields and low productivity, highlighting the need to develop conditions that allow studies of the microbe on molecular level. This study presents development of defined conditions that support growth, combined with evaluation of production of carotenoids and exopolysaccharides (EPSs) by R. marinus strain DSM 16675. RESULTS: Two defined media were initially prepared: one including a low addition of yeast extract (modified Wolfe's medium) and one based on specific components (defined medium base, DMB) to which two amino acids (N and Q), were added. Cultivation trials of R. marinus DSM 16675 in shake flasks, resulted in maximum cell densities (OD620 nm) of 2.36 ± 0.057, cell dry weight (CDW) 1.2 ± 0.14 mg/L, total carotenoids 0.59 × 10-3 mg/L, and EPSs 1.72 ± 0.03 mg/L using 2 g/L glucose in DMB. In Wolfe's medium (supplemented by 0.05 g/L yeast extract and 2.5 g/L glucose), maximum OD620 nm was 2.07 ± 0.05, CDW 1.05 ± 0.07 mg/L, total carotenoids 0.39 × 10-3 mg/L, and EPSs 1.74 ± 0.2 mg/L. Growth trials at 5 g/L glucose in these media either failed or resulted in incomplete substrate utilization. To improve reproducibility and increase substrate utilization, a screening of macroelements (e.g. phosphate) in DMB, was combined with use of trace elements and vitamins of the modified Wolfe's medium. The resulting defined minimal R. marinus medium, (DRM), allowed reproducible cultivations to a final OD620nm of 6.6 ± 0.05, CDW 2.85 ± 0.07 mg/L, a maximum specific growth rate (µmax) of 0.26 h-1, total carotenoids 0.77 × 10-3 mg/L and EPSs 3.4 ± 0.17 mg/L in cultivations supplemented with up to 5 g/L glucose. CONCLUSION: A minimal defined medium (DRM) was designed that resulted in reproducible growth and an almost doubled formation of both total carotenoids and EPSs. Such defined conditions, are necessary for systematic studies of metabolic pathways, to determine the specific requirements for growth and fully characterize metabolite production.

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.

Rhodocaloribacter litoris gen. nov., sp. nov., isolated from an intertidal hot spring.

Red-pigmented strains of non-sporeforming, aerobic, chemoorganotrophic bacteria were isolated from intertidal hot springs in Laugarvík, NW-Iceland. Cells stained Gram-negative and formed pleomorphic rods that often had swollen ends and occurred singly or in filaments. Growth was observed at 40-65 °C (optimum at 60 °C), pH 6-9 (optimum at 6.5-8) and 0.5-5% (optimum at 1-2%) (w/v) NaCl. Strain ISCAR-4553T contained MK-7 as the main respiratory quinone and saturated iso and anteiso branched chains of 17 and 15 carbons as the main cellular fatty acids (83.4%). The G+C content of the DNA is 67.3 mol%. The highest 16S rRNA gene sequence similarity was with the genus Roseithermus (92.0%) and followed by Rhodothermus, Rubrivirga and Rubricoccus (88-90%). Genome and phenotype comparisons supported the affiliation of the novel isolates and the genus Roseithermus to the family Rhodothermaceae of the phylum Rhodothermaeota. The described isolates are proposed to be classified as representatives of a novel species belonging to a novel genus, with the name Rhodocaloribacter litoris gen. nov., sp. nov. The type strain is ISCAR-4553T (=DSM 110790T = ATCC TSD-179T).

Extraction and Modification of Macroalgal Polysaccharides for Current and Next-Generation Applications.

Marine macroalgal (seaweed) polysaccharides are highly promising for next-generation applications in several industries. However, despite the reported comprehensive potential of these polysaccharides, commercial products are scarce on the market. Seaweed cultivations are increasing in number and production quantity, owing to an elevated global trend of utilization interest in seaweed. The extraction of polysaccharides from seaweed generally generates low yields, but novel methods are being developed to facilitate and improve the extraction processes. Current areas of applications for seaweed polysaccharides mainly take advantage of the physicochemical properties of certain polysaccharides, such as gelling, thickening and emulsifying. However, many of the numerous bioactivities reported are still only at research level and lack clinical evidence for commercialization. It has been suggested the construction of smaller units may generate better defined molecules that are more suitable for biomedical applications. Enzymatic modification is a promising tool for the generation of more defined, targeted biomolecules. This review covers; structural differences between the most predominant marine algal polysaccharides, extraction processes, modification alternatives, as well as a summary of current and potential next-generation application areas.

Cultivation technology development of Rhodothermus marinus DSM 16675.

This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.

Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans.

BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for the Construction of Sequence Data Warehouses.

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya. The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

Modification of linear (ß1→3)-linked gluco-oligosaccharides with a novel recombinant ß-glucosyltransferase (trans-ß-glucosidase) enzyme from Bradyrhizobium diazoefficiens.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1→3) linkages preferably and to a lesser extent (ß1→6) or (ß1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1→3)(ß1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.

A CGTase with high coupling activity using γ-cyclodextrin isolated from a novel strain clustering under the genus Carboxydocella.

Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) have mainly been characterized for their ability to produce cyclodextrins (CDs) from starch in an intramolecular transglycosylation reaction (cyclization). However, this class of enzymes can also catalyze intermolecular transglycosylation via disproportionation or coupling reactions onto a wide array of acceptors and could therefore be valuable as a tool for glycosylation.In this paper, we report the gene isolation, via the CODEHOP strategy, expression and characterization of a novel CGTase (CspCGT13) from a Carboxydocella sp. This enzyme is the first glycoside hydrolase isolated from the genus, indicating starch degradation via cyclodextrin production in the Carboxydocella strain. The fundamental reactivities of this novel CGTase are characterized and compared with two commercial CGTases, assayed under identical condition, in order to facilitate interpretation of the results. The comparison showed that the enzyme, CspCGT13, displayed high coupling activity using γ-CD as donor, despite preferentially forming α- and ß-CD in the cyclization reaction using wheat starch as substrate. Comparison of subsite conservation within previously characterized CGTases showed significant sequence variation in subsites -3 and -7, which may be important for the coupling activity.

Novel highly thermostable endolysin from Thermus scotoductus MAT2119 bacteriophage Ph2119 with amino acid sequence similarity to eukaryotic peptidoglycan recognition proteins.

In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an Mr of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His(30), Tyr(58), His(132), and Cys(140)) that form a Zn(2+) binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.

Isolation, growth and genome of the Rhodothermus RM378 thermophilic bacteriophage.

Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 10(11) pfu/ml, and sometimes reached titers as high as 10(13) pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 10(4). The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0% and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages.

Litorilinea aerophila gen. nov., sp. nov., an aerobic member of the class Caldilineae, phylum Chloroflexi, isolated from an intertidal hot spring.

A thermophilic, aerobic, Gram-stain-negative, filamentous bacterium, strain PRI-4131(T), was isolated from an intertidal hot spring in Isafjardardjup, NW Iceland. The strain grew chemo-organotrophically on various carbohydrates. The temperature range for growth was 40-65 °C (optimum 55 °C), the pH range was pH 6.5-9.0 (optimum pH 7.0) and the NaCl range was 0-3 % (w/v) (optimum 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PRI-4131(T) represented a distinct lineage within the class Caldilineae of the phylum http://dx.doi.org/10.1601/nm.550Chloroflexi. The highest levels of sequence similarity, about 91 %, were with Caldilinea aerophila STL-6-O1(T) and Caldilinea tarbellica D1-25-10-4(T). Fermentative growth was not observed for strain PRI-4131(T), which, in addition to other characteristics, distinguished it from the two Caldilinea species. Owing to both phylogenetic and phenotypic differences from the described members of the class Caldilineae, we propose to accommodate strain PRI-4131(T) in a novel species in a new genus, Litorilinea aerophila gen. nov., sp. nov. The type strain of Litorilinea aerophila is PRI-4131(T) ( = DSM 25763(T)  = ATCC BAA-2444(T)).

Improved soluble expression of the gene encoding amylolytic enzyme Amo45 by fusion with the mobile-loop-region of co-chaperonin GroES in Escherichia coli.

The gene encoding the amylolytic enzyme Amo45, originating from a metagenomic project, was retrieved by a consensus primer-based approach for glycoside hydrolase (GH) family 57 enzymes. Family 57 contains mainly uncharacterized proteins similar to archaeal thermoactive amylopullulanases. For characterization of these family members soluble, active enzymes have to be produced in sufficient amounts. Heterologous expression of amo45 in E.coli resulted in low yields of protein, most of which was found in inclusion bodies. To improve protein production and to increase the amount of soluble protein, two different modifications of the gene were applied. The first was fusion to an N-terminal His-tag sequence which increased the yield of protein, but still resulted in high amounts of inclusion bodies. Co-expression with chaperones enhanced the amount of soluble protein 4-fold. An alternative modification was the attachment of a peptide consisting of the amino acid sequence of the mobile-loop of the co-chaperonin GroES of E.coli. This sequence improved the soluble protein production 5-fold compared to His6-Amo45 and additional expression of chaperones was unnecessary.

Metabolic engineering of Thermoanaerobacterium AK17 for increased ethanol production in seaweed hydrolysate.

Sustainably produced renewable biomass has the potential to replace fossil-based feedstocks, for generation of biobased fuels and chemicals of industrial interest, in biorefineries. In this context, seaweeds contain a large fraction of carbohydrates that are a promising source for enzymatic and/or microbial biorefinery conversions. The thermoanaerobe Thermoanaerobacterium AK17 is a versatile fermentative bacterium producing ethanol, acetate and lactate from various sugars. In this study, strain AK17 was engineered for more efficient production of ethanol by knocking out the lactate and acetate side-product pathways. This was successfully achieved, but the strain reverted to acetate production by recruiting enzymes from the butyrate pathway. Subsequently this pathway was knocked out and the resultant strain AK17_M6 could produce ethanol close to the maximum theoretical yield (90%), leading to a 1.5-fold increase in production compared to the wild-type strain. Strain AK17 was also shown to successfully ferment brown seaweed hydrolysate from Laminaria digitata to ethanol in a comparatively high yield of 0.45 g/g substrate, with the primary carbon sources for the fermentations being mannitol, laminarin-derived glucose and short laminari-oligosaccharides. As strain AK17 was successfully engineered and has a wide carbohydrate utilization range that includes mannitol from brown seaweed, as well as hexoses and pentoses found in both seaweeds and lignocellulose, the new strain AK17_M6 obtained in this study is an interesting candidate for production of ethanol from both second and third generations biomass.

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus.

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked ß-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a ß-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6.

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Šresolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.