Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi.

Basidiomycota (basidiomycetes) make up 32% of the described fungi and include most wood-decaying species, as well as pathogens and mutualistic symbionts. Wood-decaying basidiomycetes have typically been classified as either white rot or brown rot, based on the ability (in white rot only) to degrade lignin along with cellulose and hemicellulose. Prior genomic comparisons suggested that the two decay modes can be distinguished based on the presence or absence of ligninolytic class II peroxidases (PODs), as well as the abundance of enzymes acting directly on crystalline cellulose (reduced in brown rot). To assess the generality of the white-rot/brown-rot classification paradigm, we compared the genomes of 33 basidiomycetes, including four newly sequenced wood decayers, and performed phylogenetically informed principal-components analysis (PCA) of a broad range of gene families encoding plant biomass-degrading enzymes. The newly sequenced Botryobasidium botryosum and Jaapia argillacea genomes lack PODs but possess diverse enzymes acting on crystalline cellulose, and they group close to the model white-rot species Phanerochaete chrysosporium in the PCA. Furthermore, laboratory assays showed that both B. botryosum and J. argillacea can degrade all polymeric components of woody plant cell walls, a characteristic of white rot. We also found expansions in reducing polyketide synthase genes specific to the brown-rot fungi. Our results suggest a continuum rather than a dichotomy between the white-rot and brown-rot modes of wood decay. A more nuanced categorization of rot types is needed, based on an improved understanding of the genomics and biochemistry of wood decay.

A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor.

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli, resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75%) to α-amylases from Petrotoga mobilis and Halothermothrix orenii. Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25% (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.

A novel thermostable and glucose-tolerant ß-glucosidase from Fervidobacterium islandicum.

An open reading frame (ORF) encoding the enzyme ß-glucosidase from the extremely thermophilic bacterium Fervidobacterium islandicum has been identified, cloned and sequenced. The bgl1A gene was cloned in a pET-Blue1 vector and transformed in Escherichia coli, resulting in high-level expression of ß-glucosidase FiBgl1A that was purified to homogeneity in a two-step purification. FiBgl1A is composed of 459 amino acid residues and showed high homology to glycoside hydrolase family 1 proteins. It exhibited highest activity towards p-nitrophenyl-ß-D: -glucopyranoside with an optimum activity at pH 6.0 and 7.0 and at 90 °C. The enzyme is resistant to glucose inhibition. Furthermore, it did not require divalent cations for activity, nor was it affected by the addition of p-chloromercuribenzoate (10 mM), EDTA (10 mM), urea (10 mM) or dithiothreitol (10 mM). Addition of surfactants (with the exception of SDS) and a number of solvents enhanced the activity of FiBgl1A. It also displayed remarkable activity across a broad temperature range (80-100 °C). The thermoactivity and thermostability of FiBgl1A and its resistance to denaturing and reducing agents make this enzyme a potential candidate for industrial applications.

Gluconeotrehalose is the principal organic solute in the psychrotolerant bacterium Carnobacterium strain 17-4.

A high proportion of microorganisms that colonise cold environments originate from marine sites; hence, they must combine adaptation to low temperature with osmoregulation. However, little or nothing is known about the nature of compatible solutes used by cold-adapted organisms to balance the osmotic pressure of the external medium. We studied the intracellular accumulation of small organic solutes in the Arctic isolate Carnobacterium strain 17-4 as a function of the growth temperature and the NaCl concentration in the medium. Data on 16S rDNA sequence and DNA-DNA hybridisation tests corroborate the assignment of this isolate as a new species of the bacterial genus Carnobacterium. The growth profiles displayed maximal specific growth rate at 30°C in medium without NaCl, and maximal values of final biomass at growth temperatures between 10 and 20°C. Therefore, Carnobacterium strain 17-4 exhibits halotolerant and psychrotolerant behaviours. The solute pool contained glycine-betaine, the main solute used for osmoregulation, and an unknown compound whose structure was identified as α-glucopyranosyl-(1-3)-ß-glucopyranosyl-(1-1)-α-glucopyranose (abbreviated as gluconeotrehalose), using nuclear magnetic resonance and mass spectrometry. This unusual solute consistently accumulated to high levels (0.35 ± 0.05 mg/mg cell protein) regardless of the growth temperature or salinity. The efficiency of gluconeotrehalose in the stabilisation of four model enzymes against heat damage was also assessed, and the effects were highly protein dependent. The lack of variation in the gluconeotrehalose content observed under heat stress, osmotic stress, and starvation provides no clue for the physiological role of this rare solute.

Factors contributing to the recalcitrance of herbaceous dicotyledons (forbs) to enzymatic deconstruction.

BACKGROUND: Many different feedstocks are under consideration for the practical production of biofuels from lignocellulosic materials. The best choice under any particular combination of economic, agronomic, and environmental conditions depends on multiple factors. The use of old fields, restored prairie, or marginal lands to grow biofuel feedstocks offers several potential benefits including minimal agronomic inputs, reduced competition with food production, and high biodiversity. However, a major component of such landscapes is often herbaceous dicotyledonous plants, also known as forbs. The potential and obstacles of using forbs as biofuel feedstocks compared to the more frequently considered grasses and woody plants are poorly understood. RESULTS: The factors that contribute to the yield of fermentable sugars from four representative forbs were studied in comparison with corn stover. The forbs chosen for the study were lamb's quarters (Chenopodium album), goldenrod (Solidago canadensis), milkweed (Asclepias syriaca), and Queen Anne's lace (Daucus carota). These plants are taxonomically diverse, widely distributed in northern temperate regions including the continental United States, and are weedy but not invasive. All of the forbs had lower total glucose (Glc) content from all sources (cell walls, sucrose, starch, glucosides, and free Glc) compared to corn stover (range 16.2 to 23.0% on a dry weight basis compared to 39.2% for corn stover). When digested with commercial enzyme mixtures after alkaline pretreatment, yields of Glc as a percentage of total Glc were lower for the forbs compared to corn stover. Enzyme inhibition by water-extractable compounds was not a significant contributor to the lower yields. Based on experiments with optimized cocktails of pure glycosyl hydrolases, enzyme imbalance probably accounted for much of the lower yields. Addition of xyloglucanase and α-xylosidase, two enzymes targeting Glc-containing polysaccharides that are more abundant in dicotyledonous plants compared to grasses, enhanced Glc yields from lamb's quarters, but Glc yields were still lower than from corn stover. CONCLUSION: The potential utilization of forb-rich plant communities as biofuel feedstocks must take into account their lower Glc content compared to grasses such as corn stover. Furthermore, new enzyme mixtures tailored to the different cell wall composition of forbs will have to be developed.

Enhancement of fermentable sugar yields by α-xylosidase supplementation of commercial cellulases.

BACKGROUND: Although α-linked xylose is a major constituent of the hemicelluloses of land plants, few secreted α-xylosidases have been described from fungi or bacteria. AxlA of Aspergillus niger is a secreted α-xylosidase that was earlier shown to promote the release of free glucose (Glc) and xylose (Xyl) from substrates containing α-linked xylose, including isoprimeverose (IP), the heptasaccharide subunit of pea xyloglucan (XG), and tamarind XG. RESULTS: The utility of AxlA for enhancing release of free Glc and Xyl in combination with commercial enzyme cocktails from dicotyledonous and monocotyledonous plants was examined. Without AxlA supplementation, a mixture of CTec2 and HTec2 (both of which are derived from T. reesei) did not release significant levels of Glc from pea XG or tamarind XG. This is consistent with their lack of detectable α-xylosidase activity using model substrates. On alkaline hydrogen peroxide-pretreated corn stover, supplementation of CTec2/HTec2 (at a loading of 2.5 mg/g glucan) with AxlA (at a loading of 8 mg/g glucan) increased Glc yields from 82% to 88% of the total available Glc and increased Xyl yields from 55% to 60%. AxlA supplementation also improved Glc yields from corn stover treated with the commercial cellulase Accellerase 1000. The AxlA enhancement was not a general protein effect because bovine serum albumin or bovine gamma-globulin at similar concentrations did not enhance Glc yields from corn stover in response to CTec2/HTec2. Supplementation of CTec2/HTec2 with AxlA did not enhance Glc release from pretreated green or etiolated pea tissue. However, AxlA did enhance Glc and Xyl yields compared to CTec2/HTec2 alone from another dicotyledonous herbaceous plant, Chenopodium album (lamb's quarters). CONCLUSION: Supplementation of commercial cellulase cocktails with AxlA enhances yields of Glc and Xyl from some biomass substrates under some conditions, and may prove useful in industrial lignocellulose conversion.