Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction.

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. KEY POINTS: • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

Structural and molecular dynamics investigations of ligand stabilization via secondary binding site interactions in Paenibacillus xylanivorans GH11 xylanase.

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-ß-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

Synergic activity of Cel8Pa ß-1,4 endoglucanase and Bg1Pa ß-glucosidase from Paenibacillus xylanivorans A59 in beta-glucan conversion.

In the efficient bioconversion of polysaccharides from lignocellulosic biomass, endoglucanases and ß-glucosidases are key enzymes for the deconstruction of ß-glucans. In this work, we focused on a GH8 endoglucanase (Cel8Pa) and a GH1 ß-glucosidase (Bg1Pa) from Paenibacillus xylanivorans A59. Cel8Pa was active on a broad range of substrates, such as ß-glucan from barley (24.5 IU/mg), lichenan (17.9 IU/mg), phosphoric acid swollen cellulose (PASC) (9.7 IU/mg), carboxi-methylcellulose (CMC) (7.3 IU/mg), chitosan (1.4 IU/mg) and xylan (0.4 IU/mg). Bg1Pa was active on cellobiose (C2) and cello-oligosaccharides up to C6, releasing glucose as the main product. When both enzymes were used jointly, there was a synergic effect in the conversion rate of polysaccharides to glucose. Cel8Pa and Bg1Pa presented important properties for simultaneous saccharification and fermentation (SSF) processes in second generation bioethanol production, such as tolerance to high concentration of glucose and ethanol.

Neotropical termite microbiomes as sources of novel plant cell wall degrading enzymes.

In this study, we used shotgun metagenomic sequencing to characterise the microbial metabolic potential for lignocellulose transformation in the gut of two colonies of Argentine higher termite species with different feeding habits, Cortaritermes fulviceps and Nasutitermes aquilinus. Our goal was to assess the microbial community compositions and metabolic capacity, and to identify genes involved in lignocellulose degradation. Individuals from both termite species contained the same five dominant bacterial phyla (Spirochaetes, Firmicutes, Proteobacteria, Fibrobacteres and Bacteroidetes) although with different relative abundances. However, detected functional capacity varied, with C. fulviceps (a grass-wood-feeder) gut microbiome samples containing more genes related to amino acid metabolism, whereas N. aquilinus (a wood-feeder) gut microbiome samples were enriched in genes involved in carbohydrate metabolism and cellulose degradation. The C. fulviceps gut microbiome was enriched specifically in genes coding for debranching- and oligosaccharide-degrading enzymes. These findings suggest an association between the primary food source and the predicted categories of the enzymes present in the gut microbiomes of each species. To further investigate the termite microbiomes as sources of biotechnologically relevant glycosyl hydrolases, a putative GH10 endo-ß-1,4-xylanase, Xyl10E, was cloned and expressed in Escherichia coli. Functional analysis of the recombinant metagenome-derived enzyme showed high specificity towards beechwood xylan (288.1 IU/mg), with the optimum activity at 50 °C and a pH-activity range from 5 to 10. These characteristics suggest that Xy110E may be a promising candidate for further development in lignocellulose deconstruction applications.

Paenibacillus xylanivorans sp. nov., a xylan-degrading bacterium isolated from decaying forest soil.

A xylanolytic bacterial strain, named A59T, was isolated from a forest soil consortium in southern Argentina. Strain A59T is a Gram-stain-positive, facultative anaerobic, endospore-forming and rod-shaped bacterium. Its optimal growth conditions are 30 °C (range, 28-37 °C), pH 7 (range, pH 5-10) and it tolerates up to 7 % of NaCl (range, 2-7 %). Chemotaxonomic analysis revealed that strain A59Tpossesses meso-diaminopimelic acid in the cell wall. It contains menaquinone MK-7 as the predominant isoprenoid quinone and the major fatty acid is anteiso-C15 : 0 (35.1 %), with a moderate amount of C16 : 0 (6.9 %). According to 16S RNA gene sequence analysis, the isolate is phylogenetically placed in the same cluster as Paenibacillus taichungensis BCRC 17757T (99.7 % nucleotide sequence identity) and Paenibacillus pabuli NBRC 13638T (99.1 %) and is closely related to Paenibacillus tundrae A10bT (98.8 %). However, phylogenetic studies based on the housekeeping gyrB gene placed A59T in a separate branch from all other related type strains. Furthermore, the results of whole genome average nucleotide identity analysis (gANI) with related type strains was lower than 91.10 % and the digital DNA-DNA hybridization value was lower than 44.30 %, which are below the threshold values for separating two species. The DNA G+C content was estimated as 46.09 mol%, based on genome sequencing. On the basis of these results, A59T represents a new species of the genus Paenibacillus, and we propose the name Paenibacillusxylanivorans sp. nov. The type strain is A59T (=DSM 107920T=NCIMB 15123T).

A thermostable GH8 endoglucanase of Enterobacter sp. R1 is suitable for ß-glucan deconstruction.

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.

EcXyl43 ß-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

Draft Genome Sequence of Cellulolytic and Xylanolytic Cellulomonas sp. Strain B6 Isolated from Subtropical Forest Soil.

Cellulomonas sp. strain B6 was isolated from a subtropical forest soil sample and presented (hemi)cellulose-degrading activity. We report here its draft genome sequence, with an estimated genome size of 4 Mb, a G+C content of 75.1%, and 3,443 predicted protein-coding sequences, 92 of which are glycosyl hydrolases involved in polysaccharide degradation.

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior.

Draft Genome Sequence of Cellulolytic and Xylanolytic Paenibacillus sp. A59, Isolated from Decaying Forest Soil from Patagonia, Argentina.

Paenibacillus sp. A59 was isolated from decaying forest soil in Argentina and characterized as a xylanolytic strain. We report the draft genome sequence of this isolate, with an estimated genome size of 7 Mb which harbor 6,424 coding sequences. Genes coding for hydrolytic enzymes involved in lignocellulose deconstruction were predicted.

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction.

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.