Mechanistic strategies of microbial communities regulating lignocellulose deconstruction in a UK salt marsh.

BACKGROUND: Salt marshes are major natural repositories of sequestered organic carbon with high burial rates of organic matter, produced by highly productive native flora. Accumulated carbon predominantly exists as lignocellulose which is metabolised by communities of functionally diverse microbes. However, the organisms that orchestrate this process and the enzymatic mechanisms employed that regulate the accumulation, composition and permanence of this carbon stock are not yet known. We applied meta-exo-proteome proteomics and 16S rRNA gene profiling to study lignocellulose decomposition in situ within the surface level sediments of a natural established UK salt marsh. RESULTS: Our studies revealed a community dominated by Gammaproteobacteria, Bacteroidetes and Deltaproteobacteria that drive lignocellulose degradation in the salt marsh. We identify 42 families of lignocellulolytic bacteria of which the most active secretors of carbohydrate-active enzymes were observed to be Prolixibacteracea, Flavobacteriaceae, Cellvibrionaceae, Saccharospirillaceae, Alteromonadaceae, Vibrionaceae and Cytophagaceae. These families secreted lignocellulose-active glycoside hydrolase (GH) family enzymes GH3, GH5, GH6, GH9, GH10, GH11, GH13 and GH43 that were associated with degrading Spartina biomass. While fungi were present, we did not detect a lignocellulolytic contribution from fungi which are major contributors to terrestrial lignocellulose deconstruction. Oxidative enzymes such as laccases, peroxidases and lytic polysaccharide monooxygenases that are important for lignocellulose degradation in the terrestrial environment were present but not abundant, while a notable abundance of putative esterases (such as carbohydrate esterase family 1) associated with decoupling lignin from polysaccharides in lignocellulose was observed. CONCLUSIONS: Here, we identify a diverse cohort of previously undefined bacteria that drive lignocellulose degradation in the surface sediments of the salt marsh environment and describe the enzymatic mechanisms they employ to facilitate this process. Our results increase the understanding of the microbial and molecular mechanisms that underpin carbon sequestration from lignocellulose within salt marsh surface sediments in situ and provide insights into the potential enzymatic mechanisms regulating the enrichment of polyphenolics in salt marsh sediments. Video Abstract.

Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering.

Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis (XynA), five single mutants (S22E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed in Escherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (Topt) of 55°C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55°C (t50) decreased in the order S22E/N32D>N181R>S22E>Wild-type>S27E=N32D≈N54E. The values of the van't Hoff denaturation enthalpy change (ΔHND), melting temperature (Tm) and heat capacity at constant pressure (ΔCp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328K (ΔG328) S22E/N32D>N181R>S22E>Wild-type>S27E≈N54E>N32D correlates well with the thermotolerance results, and is dominated by changes in ΔHND which is consistent with increased in hydrogen bonding in the thermostable mutants.

Engineering the pattern of protein glycosylation modulates the thermostability of a GH11 xylanase.

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex(8-16)GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.