Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure.
Dumon, C., Varvak, A., Wall, M. A., Flint, J. E., Lewis, R. J., Lakey, J. H., Morland, C., Luginbühl, P., Healey, S., Todaro, T., DeSantis, G., Sun, M., Parra-Gessert, L., Tan, X., Weiner, D. P. & Gilbert, H. J. (2008). Journal of Biological Chemistry, 283(33), 22557-22564.
Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation MutagenesisTM (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (Tm) 1–8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS, containing seven mutations. EvXyn11TS had a Tm ~ 25°C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.
Functional characterization of a xylose transporter in Aspergillus nidulans.
Colabardini, A. C., Ries, L. N. A., Brown, N. A., dos Reis, T. F., Savoldi, M., Goldman, M. H. S., Menino, J. F., Rodrigues, F. & Goldman, G. H. (2014). Biotechnology for Biofuels, 7(1), 46.
Background: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. Results: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. Conclusions: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.
An evolutionary route to xylanase process fitness.
Palackal, N., Brennan, Y., Callen, W .N, Dupree, P., Frey, G., Goubet, F., Hazlewood, G., P., Healey, S., Kang, Y. E., Kretz, K. A., Lee, E., Xuqiu, T., Tomlinson, G. L., Verruto, J., Wong, V. W. K., Mathur, E. J., Short, J. M., Robertson, D. E. & Steer, B. A. (2004). Protein Science, 13(2), 494–503.
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90°C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61°C to as high as 96°C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).
Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.
Pe´rez-Gonzales´Lez, J. A., De Graaff, L. H., Visser, J. & Ramon, D. (1996). Applied and Environmental Microbiology, 62(6), 2179-2182.
Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains.
Generation of doubled haploid transgenic wheat lines by microspore transformation.
Brew-Appiah, R. A. T., Ankrah, N., Liu, W., Konzak, C. F., Wettstein, D. V. & Rustgi, S. (2013). PLoS One, 8(11), e80155.
Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the To transformants and their stable inheritance in homozygous T1:2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i) pretreatment of immature spikes with CuSO4 solution (500 mg/L) at 4°C for 10 days; (ii) electroporation of plasmid DNA in enlarged microspores by a single pulse of ~375 V; (iii) induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv) co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v) elimination of AGL-1 cells after co-cultivation with timentin (200–400 mg/L).
Initial decay of woody fragments in soil is influenced by size, vertical position, nitrogen availability and soil origin.
Van der Wal, A., De Boer, W., Smant, W. & Van Veen, J. A. (2007). Plant Soil, 301, 189–201.
Fast-growing bacteria and fungi are expected to cause the initial stage of decomposition of woody fragments in and on soils, i.e. the respiration of sugars, organic acids, pectin and easily accessible cellulose and hemi-cellulose. However, little is known about the factors regulating initial wood decomposition. We examined the effect of wood fragment size, vertical position, nitrogen addition and soil origin on initial wood decay and on the relative importance of fungi and bacteria therein. Two fractions of birch wood were used in microcosm experiments, namely wood blocks (dimensions: 3×0.5×0.5 cm) and sawdust (dimensions: 0.5–2 mm). The woody fragments were enclosed in nylon bags and placed on top of- or buried in an abandoned arable soil and in a heathland soil. After 15, 25 and 40 weeks of incubation, fungal biomass was quantified (as ergosterol and chitin content) and bacterial numbers were determined. The results indicated that initial wood decay was mostly caused by fungi; bacteria were only contributing in sawdust in/on abandoned arable soil. Larger fragment size, burial of fragments and nitrogen addition positively influenced fungal biomass and activity. Fungal biomass and decay activities were much lower in woody fragments incubated in/on heathland soil than in those incubated in/on abandoned arable soil, indicating that soil origin is also an important factor determining initial wood decay.
Deciphering transcriptional regulatory mechanisms associated with hemicellulose degradation in Neurospora crassa.
Sun, J., Tian, C., Diamond, S. & Glass, N. L. (2012). Eukaryotic Cell, 11(4), 482-493.
Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to N. crassa grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in N. crassa, we identified a TF (XLR-1; xylan degradation regulator 1) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in Aspergillus and Trichoderma species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of xlr-1 in N. crassa abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.
Isolation and characterization of a Bacillus licheniformis strain capable of degrading zearalenone.
Yi, P. J., Pai, C. K., & Liu, J. R. (2011). World Journal of Microbiology and Biotechnology, 27(5), 1035-1043.
The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.
Biochemical characterization of an endoxylanase from Pseudozyma brasiliensis sp. nov strain GHG001 isolated from the intestinal tract of Chrysomelidae larvae associated to sugarcane roots.
Borges, T. A., De Souza, A. T., Squina, F. M., Riaño-Pachón, D. M., Dos Santos, R. A. C., Machado, E., Oliveira, J. R. L. D. C., Damásio, A. R. L. & Goldman, G. H. (2014). Process Biochemistry, 49(1), 77-83.
Endo-xylanases play a key role in the hydrolysis of xylan and recently they have attracted much attention due to their potential applications on the biofuel and paper industries. We isolated a Pseudozyma brasiliensis sp. nov. strain from the intestinal tract of Chrysomelidae larvae that parasitize sugarcane roots. This basidiomycetous yeast produces a xylanase designated PbXynA which was purified and characterized. The molecular weight of PbXynA is 24 kDa, it belongs to the GH11 family and its optimum pH and optimum temperature are 4.0 and 55°C, respectively. PbXynA has as secondary structure predominantly β-sheets and sigmoidal kinetic behavior with elevated speed conversion from substrate-to-products (Vmax = 2792.0 μmol product/min/mg protein). It is highly activated by bivalent cations such as Ca2+, however in the presence of Cu2+ xylanase activity was inhibited. It has a high specific activity and produces xylooligosaccharides that have a variety of industrial applications, indicating PbXynA has a great biotechnological potential.
CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans.
Tamayo, E. N., Villanueva, A., Hasper, A. A., De Graaff, L. H., Ramón, D. & Orejas, M. (2008). Fungal Genetics and Biology, 45(6), 984-993.
The Aspergillus nidulans xlnR gene encodes a Zn2 Cys6 transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X22, X24 and X34, and β-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X22) and xlnB (encodes X24) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished increAd30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.
Induction of mannanase, xylanase, and endoglucanase activities in Sclerotium rolfsii.
Sachslehner, A., Nidetzky, B., Kulbe, K. D. & Haltrich, D. (1998). Applied and Environmental Microbiology, 64(2), 594-600.
Induction of mannanase, xylanase, and cellulase (endoglucanase) synthesis in the plant-pathogenic basidiomycete Sclerotium rolfsii was studied by incubating noninduced, resting mycelia with a number of mono-, oligo-, and polysaccharides. The simultaneous formation of these three endoglycanases could be provoked by several polysaccharides structurally resembling the carbohydrate constituents of lignocellulose (e.g., mannan and cellulose), by various disaccharide catabolites of these lignocellulose constituents (e.g., cellobiose, mannobiose, and xylobiose), or by structurally related disaccharides (e.g., lactose, sophorose, and galactosyl-β-1,4-mannose), as well as by L-sorbose. Synthesis of mannanase, xylanase, and endoglucanase always occurred concomitantly and could not be separated by selecting an appropriate inducer. Various structurally different inducing carbohydrates promoted the excretion of the same multiple isoforms of endoglycanases, as judged from the similar banding patterns obtained in zymogram analyses of enzyme preparations obtained in response to these different inducers and resolved by analytical isoelectric focusing. Whereas enhanced xylanase and endoglucanase formation is strictly dependent on the presence of suitable inducers, increased levels of mannanase are excreted by S. rolfsii even under noninducing, derepressed conditions, as shown in growth experiments with glucose as the substrate. Significant mannanase formation commenced only when glucose was exhausted from the medium. Under these conditions, only very low, presumably constitutive levels of xylanase and endoglucanase were formed. Although the induction of the three endoglycanases is very closely related in S. rolfsii, it was concluded that there is no common, coordinated regulatory mechanism that controls the synthesis of mannanase, xylanase, and endoglucanase.
Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes.
Skovgaard, P. A. & Jørgensen, H. (2013). Journal of Industrial Microbiology & Biotechnology, 40(5), 447-456.
Lignocellulolytic enzymes are among the most costly part in production of bioethanol. Therefore, recycling of enzymes is interesting as a concept for reduction of process costs. However, stability of the enzymes during the process is critical. In this work, focus has been on investigating the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65°C and up to 5 % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55°C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation.
High-level heterologous expression of Bacillus halodurans putative xylanase Xyn11A (BH0899) in kluyveromyces lacti.
Wamalwa, B. M., Zhao, G., Sakka, M., Shiundu, P. M., Kimura, T. & Sakka, K. (2007). Bioscience, Biotechnology, and Biochemistry, 71(3), 688-693.
The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.
Diversity of termitomyces associated with fungus-farming termites assessed by cultural and culture-independent methods.
Makonde, H. M., Boga, H. I., Osiemo, Z., Mwirichia, R., Stielow, J. B., Göker, M. & Klenk, H. P. (2013). PLoS One, 8(2), e56464.
Background: Fungus-cultivating termites make use of an obligate mutualism with fungi from the genus Termitomyces, which are acquired through either vertical transmission via reproductive alates or horizontally transmitted during the formation of new mounds. Termitomyces taxonomy, and thus estimating diversity and host specificity of these fungi, is challenging because fruiting bodies are rarely found. Molecular techniques can be applied but need not necessarily yield the same outcome than morphological identification. Methodology: Culture-dependent and culture-independent methods were used to comprehensively assess host specificity and gut fungal diversity. Termites were identified using mitochondrial cytochrome oxidase II (COII) genes. Twenty-three Termitomyces cultures were isolated from fungal combs. Internal transcribed spacer (ITS) clone libraries were constructed from termite guts. Presence of Termitomyces was confirmed using specific and universal primers. Termitomyces species boundaries were estimated by cross-comparison of macromorphological and sequence features, and ITS clustering parameters accordingly optimized. The overall trends in coverage of Termitomyces diversity and host associations were estimated using Genbank data. Results and Conclusion: Results indicate a monoculture of Termitomyces in the guts as well as the isolation sources (fungal combs). However, cases of more than one Termitomyces strains per mound were observed since mounds can contain different termite colonies. The newly found cultures, as well as the clustering analysis of GenBank data indicate that there are on average between one and two host genera per Termitomyces species. Saturation does not appear to have been reached, neither for the total number of known Termitomyces species nor for the number of Termitomyces species per host taxon, nor for the number of known hosts per Termitomyces species. Considering the rarity of Termitomyces fruiting bodies, it is suggested to base the future taxonomy of the group mainly on well-characterized and publicly accessible cultures.
Novel modular endo-β-1,4-xylanase with transglycosylation activity from Cellulosimicrobium sp. strain HY-13 that is homologous to inverting GH family 6 enzymes.
Kim, D. Y., Ham, S. J., Kim, H. J., Kim, J., Lee, M. H., Cho, H. Y., Shin, D. H., Rhee, Y. H., Son, K. H. & Park, H. Y. (2012). Bioresource Technology, 107, 25-32.
The gene (2304-bp) encoding a novel xylanolytic enzyme (XylK2) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 β-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylK2δFn3-CBM 2 displayed high transferase activity (788.3 IU mg-1) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylK2δFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-β-1,4-xylanase activity of XylK2δFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50 mM xylobiose.