The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in D-xylose, L-arabinose and steam-exploded sugarcane bagasse.
de Souza, W. R., Maitan-Alfenas, G. P., de Gouvêa, P. F., Brown, N. A., Savoldi, M., Battaglia, E., Goldman, M. H. S., de Vries, R. P. & Goldman, G. H. (2013). Fungal Genetics and Biology, 60, 29-45.
The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production.
A high-throughput platform for screening milligram quantities of plant biomass for lignocellulose digestibility.
Santoro, N., Cantu, S. L., Tornqvist, C. E., Falbel, T. G., Bolivar, J. L., Patterson, S. E., Pauly, M. & Walton, J. D. (2010). BioEnergy research, 3(1), 93-102.
The development of a viable lignocellulosic ethanol industry requires multiple improvements in the process of converting biomass to ethanol. A key step is the improvement of the plants that are to be used as biomass feedstocks. To facilitate the identification and evaluation of feedstock plants, it would be useful to have a method to screen large numbers of individual plants for enhanced digestibility in response to combinations of specific pretreatments and enzymes. This paper describes a high-throughput digestibility platform (HTDP) for screening collections of germplasm for improved digestibility, which was developed under the auspices of the Department of Energy-Great Lakes Bioenergy Research Center (DOE-GLBRC). A key component of this platform is a custom-designed workstation that can grind and dispense 1–5 mg quantities of more than 250 different plant tissue samples in 16 h. The other steps in the processing (pretreatment, enzyme digestion, and sugar analysis) have also been largely automated and require 36 h. The process is adaptable to diverse acidic and basic, low-temperature pretreatments. Total throughput of the HTDP is 972 independent biomass samples per week. Validation of the platform was performed on brown midrib mutants of maize, which are known to have enhanced digestibility. Additional validation was performed by screening approximately 1,200 Arabidopsis mutant lines with T-DNA insertions in genes known or suspected to be involved in cell wall biosynthesis. Several lines showed highly significant (p < 0.01) increases in glucose and xylose release (20–40% above the mean). The platform should be useful for screening populations of plants to identify superior germplasm for lignocellulosic ethanol applications and also for screening populations of mutant model plants to identify specific genes affecting digestibility.
Fast enzymatic saccharification of switchgrass after pretreatment with ionic liquids.
Zhao, H., Baker, G. A. & Cowins, J. V. (2010). Biotechnology progress, 26(1), 127-133.
The pretreatment of cellulose using ionic liquids (ILs) has been shown to be an effective method for improving the enzymatic hydrolysis of cellulose; this technique affords a fast and complete saccharification of cellulose into reducing sugars (Dadi et al., Biotechnol Bioeng. 2006; 95:904–910; Liu and Chen, Chinese Sci Bull. 2006; 51:2432–2436; Zhao et al., J Biotechnol. 2009; 139:47–54). Motivated by these advances, this study examines the effect of IL-pretreatment on the enzymatic hydrolysis of purified xylan (as a model system of hemicellulose) and switchgrass (as a real lignocellulose). The IL-pretreatment resulted in no improvement in the hydrolysis of xylan. The likely reason is that pure xylan has a low degree of polymerization (DP), and is readily biodegraded even without any pretreatment. However, in real cellulosic materials (such as switchgrass), xylan is entrapped within the cellulosic matrix, and cannot be conveniently accessed by enzymes. Our data demonstrate that the IL-pretreatment of switchgrass significantly improved the enzymatic saccharification of both cellulose (96% D-glucose yield in 24 h) and xylan (63% D-xylose yield in 24 h). The compositional analysis of switchgrass suggests a lower lignin content after IL-pretreatment. In addition, the infrared spectrum of regenerated switchgrass indicates a lower substrate crystallinity, whereas the enzyme adsorption isotherm further implies that the regenerated substrate is more accessible to enzymes. This study has further confirmed that IL-pretreatment is an effective tool in enhancing the enzymatic hydrolysis of cellulosic biomass, and allowing a more complete saccharification.
Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway.
Lehmann, D. & Lütke-Eversloh, T. (2011). Metabolic Engineering, 13(5), 464-473.
Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C4) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20 g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33 g/l, resulting in an ethanol to glucose yield of 0.38 g/g.
Process characterization and influence of alternative carbon sources and carbon-to-nitrogen ratio on organic acid production by Aspergillus oryzae DSM1863.
Ochsenreither, K., Fischer, C., Neumann, A. & Syldatk, C. (2014). Applied Microbiology and Biotechnology, 98(12), 5449-5460.
L-Malic acid and fumaric acid are C4 dicarboxylic organic acids and considered as promising chemical building blocks. They can be applied as food preservatives and acidulants in rust removal and as polymerization starter units. Molds of the genus Aspergillus are able to produce malic acid in large quantities from glucose and other carbon sources. In order to enhance the production potential of Aspergillus oryzae DSM 1863, production and consumption rates in an established bioreactor batch-process based on glucose were determined. At 35°C, up to 42 g/L malic acid was produced in a 168-h batch process with fumaric acid as a by-product. In prolonged shaking flask experiments (353 h), the suitability of the alternative carbon sources xylose and glycerol at a carbon-to-nitrogen (C/N) ratio of 200:1 and the influence of different C/N ratios in glucose cultivations were tested. When using glucose, 58.2 g/L malic acid and 4.2 g/L fumaric acid were produced. When applying xylose or glycerol, both organic acids are produced but the formation of malic acid decreased to 45.4 and 39.4 g/L, respectively. Whereas the fumaric acid concentration was not significantly altered when cultivating with xylose (4.5 g/L), it is clearly enhanced by using glycerol (9.3 g/L). When using glucose as a carbon source, an increase or decrease of the C/N ratio did not influence malic acid production but had an enormous influence on fumaric acid production. The highest fumaric acid concentrations were determined at the highest C/N ratio (300:1, 8.44 g/L) and lowest at the lowest C/N ratio (100:1, 0.7 g/L).
Characterization of newly isolated oleaginous yeasts - Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis.
Schulze, I., Hansen, S., Großhans, S., Rudszuck, T., Ochsenreither, K., Syldatk, C. & Neumann, A. (2014). AMB Express, 4, 24.
The yeast strains Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis were isolated from soil samples and identified as oleaginous yeast strains beneficial for the establishment of microbial production processes for sustainable lipid production suitable for several industrial applications. When cultured in bioreactors with glucose as the sole carbon source C. podzolicus yielded 31.8% lipid per dry biomass at 20°C, while T. porosum yielded 34.1% at 25°C and P. segobiensis 24.6% at 25°C. These amounts correspond to lipid concentrations of 17.97 g/L, 17.02 g/L and 12.7 g/L and volumetric productivities of 0.09 g/Lh, 0.1 g/Lh and 0.07 g/Lh, respectively. During the culture of C. podzolicus 30 g/l gluconic acid was detected as by-product in the culture broth and 12 g/L gluconic acid in T. porosum culture. The production of gluconic acid was eliminated for both strains when glucose was substituted by xylose as the carbon source. Using xylose lipid yields were 11.1 g/L and 13.9 g/L, corresponding to 26.8% and 33.4% lipid per dry biomass and a volumetric productivity of 0.07 g/Lh and 0.09 g/Lh, for C. podzolicus and T. porosum respectively. The fatty acid profile analysis showed that oleic acid was the main component (39.6 to 59.4%) in all three strains and could be applicable for biodiesel production. Palmitic acid (18.4 to 21.1%) and linolenic acid (7.5 to 18.7%) are valuable for cosmetic applications. P. segobiensis had a considerable amount of palmitoleic acid (16% content) and may be suitable for medical applications.
Characterisation of dietary fibre components in cereals and legumes used in Serbian diet.
Dodevska, M. S., Djordjevic, B. I., Sobajic, S. S., Miletic, I. D., Djordjevic, P. B. & Dimitrijevic-Sreckovic, V. S. (2013). Food chemistry, 141(3), 1624-1629.
The typical Serbian diet is characterised by high intake of cereal products and also legumes are often used. The content of total fibre as well as certain fibre fractions was determined in cereals, cereal products, and cooked legumes. The content of total fibre in cooked cereals and cereal products ranged from 2.5 to 20.8 g/100 g, and in cooked legumes from 14.0 to 24.5 g/100 g (on dry matter basis). Distribution of analysed fibre fractions and their quantities differed significantly depending on food groups. Fructans and arabinoxylans were the most significant fibre fractions in rye flakes, and β-glucan in oat flakes, cellulose and resistant starch were present in significant amounts in peas and kidney beans. When the size of regular food portions was taken into consideration, the best sources of total dietary fibre were peas and kidney beans (more than 11 g/serving). The same foods were the best sources of cellulose (4.98 and 3.56 g/serving) and resistant starch (3.90 and 2.83 g/serving). High intake of arabinoxylans and fructans could be accomplished with cooked wheat (3.20 g and 1.60 g/serving, respectively). Oat (1.39 g/serving) and barley flakes (1.30 g/serving) can be recommended as the best sources of β-glucan.
Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan.
Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G. P., Pickersgill, R. & Gilbert, H. J. (1997). The Journal of Biological Chemistry, 272(5), 2942-2951.
In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-β1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-β-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.
Simultaneous uptake of lignocellulose‐based monosaccharides by Escherichia coli.
Jarmander, J., Hallström, B. M. & Larsson, G. (2014). Biotechnology and Bioengineering, 111(6), 1108-1115.
Lignocellulosic waste is a naturally abundant biomass and is therefore an attractive material to use in second generation biorefineries. Microbial growth on the monosaccharides present in hydrolyzed lignocellulose is however associated with several obstacles whereof one is the lack of simultaneous uptake of the sugars. We have studied the aerobic growth of Escherichia coli on D-glucose, D-xylose, and L-arabinose and for simultaneous uptake to occur, both the carbon catabolite repression mechanism (CCR) and the AraC repression of xylose uptake and metabolism had to be removed. The strain AF1000 is a MC4100 derivative that is only able to assimilate arabinose after a considerable lag phase, which is unsuitable for commercial production. This strain was successfully adapted to growth on L-arabinose and this led to simultaneous uptake of arabinose and xylose in a diauxic growth mode following glucose consumption. In this strain, a deletion in the phosphoenolpyruvate:phosphotransferase system (PTS) for glucose uptake, the ptsG mutation, was introduced. The resulting strain, PPA652ara simultaneously consumed all three monosaccharides at a maximum specific growth rate of 0.59 h-1, 55% higher than for the ptsG mutant alone. Also, no residual sugar was present in the cultivation medium. The potential of PPA652ara is further acknowledged by the performance of AF1000 during fed-batch processing on a mixture of D-glucose, D-xylose, and L-arabinose. The conclusion is that without the removal of both layers of carbon uptake control, this process results in accumulation of pentoses and leads to a reduction of the specific growth rate by 30%.
Penicillium purpurogenum produces two GH family 43 enzymes with β-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.
Ravanal, M. C., Alegría-Arcos, M., Gonzalez-Nilo, F. D. & Eyzaguirre, J. (2013). Archives of Biochemistry and Biophysics, 540(1-2), 117-124.
β-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with β-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-β-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, β-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3–pNPXyl, ABF3–pNPAra and XYL1–pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1–pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.
Development and testing of a novel lab-scale direct steam-injection apparatus to hydrolyse model and saline crop slurries.
Guglielmo, S., Dalessandro, A., Maurizio, P., Silvia, C., Maurizio, R., Riccardo, V. & Moresi, M. (2012). Journal of Biotechnology, 157(4), 590-597.
In this work, a novel laboratory-scale direct steam-injection apparatus (DSIA) was developed to overcome the main drawback of the conventional batch-driven lab rigs, namely the long time needed to heat fiber slurry from room to reaction temperatures greater than 150°C. The novel apparatus mainly consisted of three units: (i) a mechanically-stirred bioreactor where saturated steam at 5–30 bar can be injected; (ii) an automatic on–off valve to flash suddenly the reaction medium after a prefixed reaction time; (iii) a cyclone separator to recover the reacted slurry. This system was tested using 0.75 dm3 of an aqueous solution of H2SO4 (0.5%, v/v) enriched with 50 kg m-3 of either commercial particles of Avicel® and Larch xylan or 0.5 mm sieved particles of Tamarix jordanis. Each slurry was heated to about 200°C by injecting steam at 28 bar for 90 s. The process efficiency was assessed by comparing the dissolution degree of suspended solid (YS), as well as xylose (YX), glucose (YG), and furfural (YF) yields, with those obtained in a conventional steam autoclave at 130°C for 30 or 60 min. Treatment of T. jordanis particles in DSIA resulted in YS and YG values quite similar to those obtained in the steam autoclave at 130°C for 60 min, but in a less efficient hemicellulose solubilization. A limited occurrence of pentose degradation products was observed in both equipments, suggesting that hydrolysis predominated over degradation reactions. The susceptibility of the residual solid fractions from DSIA treatment to a conventional 120 h long cellulolytic treatment using an enzyme loading of 5.4 FPU g-1 was markedly higher than that of samples hydrolysed in the steam autoclave, their corresponding glucose yields being equal to 0.94 and 0.22 g per gram of initial cellulose, respectively. Thus, T. jordanis resulted to be a valuable source of sugars for bioethanol production as proved by preliminary tests in the novel lab rig developed here.
Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse.
van den Brink, J., Maitan-Alfenas, G. P., Zou, G., Wang, C., Zhou, Z., Guimarães, V. M. & de Vries, R. P. (2014). Biotechnology Journal, 9(10), 1329-1338.
Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.
Co-fermentation of acetate and sugars facilitating microbial lipid production on acetate-rich biomass hydrolysates.
Gong, Z., Zhou, W., Shen, H., Yang, Z., Wang, G., Zuo, Z., Hou. Y. & Zhao, Z. K. (2016). Bioresource technology, 207, 102-108.
The process of lignocellulosic biomass routinely produces a stream that contains sugars plus various amounts of acetic acid. As acetate is known to inhibit the culture of microorganisms including oleaginous yeasts, little attention has been paid to explore lipid production on mixtures of acetate and sugars. Here we demonstrated that the yeast Cryptococcus curvatus can effectively co-ferment acetate and sugars for lipid production. When mixtures of acetate and glucose were applied, C. curvatus consumed both substrates simultaneously. Similar phenomena were also observed for acetate and xylose mixtures, as well as acetate-rich corn stover hydrolysates. More interestingly, the replacement of sugar with equal amount of acetate as carbon source afforded higher lipid titre and lipid content. The lipid products had fatty acid compositional profiles similar to those of cocoa butter, suggesting their potential for high value-added fats and biodiesel production. This co-fermentation strategy should facilitate lipid production technology from lignocelluloses.
Effects of an acid/alkaline treatment on the release of antioxidants and cellulose from different agro-food wastes.
Vadivel, V., Moncalvo, A., Dordoni, R. & Spigno, G. (2017). Waste Management, 64, 305-314.
The present investigation was aimed to evaluate the release of both antioxidants and cellulosic fibre from different agro-food wastes. Cost-effective and easily available agro-food residues (brewers’ spent grains, hazelnut shells, orange peels and wheat straw) were selected and submitted to a double-step acid/alkaline fractionation process. The obtained acid and alkaline liquors were analysed for total phenols content and antioxidant capacity. The final fibre residue was analysed for the cellulose, lignin and hemicellulose content. The total phenols content and antioxidant capacity of the acid liquors were higher than the alkaline hydrolysates. Orange peels and wheat straw gave, respectively, the highest (19.70 ± 0.68 mg/gdm) and the lowest (4.70 ± 0.29 mg/gdm) total phenols release. Correlation between antioxidant capacity of the liquors and their origin depended on the analytical assay used to evaluate it. All the acid liquors were also rich in sugar degradation products (mainly furfural). HPLC analysis revealed that the most abundant phenolic compound in the acid liquors was vanillin for brewers’ spent grains, hazelnut shells and wheat straw, and p-hydroxybenzoic acid for orange peels. Wheat straw served as the best raw material for cellulose isolation, providing a final residue with a high cellulose content (84%) which corresponded to 45% of the original cellulose. The applied process removed more than 90% of the hemicellulose fraction in all the samples, while delignification degree ranged from 67% (in hazelnut shells), to 93% (in brewers’ spent grains). It was not possible to select a unique raw material for the release of highest levels of both total phenols and cellulose.
Strategic optimization of xylanase-mannanase combi-CLEAs for synergistic and efficient hydrolysis of complex lignocellulosic substrates.
Bhattacharya, A. & Pletschke, B. I. (2015). Journal of Molecular Catalysis B: Enzymatic, 115, 140-150.
Cost-effective application of lignocellulolytic enzymes holds the key towards commercialization of enzymatic hydrolysis of lignocellulosic biomass. Carrier free immobilization of enzyme(s) offers a lucrative prospect. Combined-cross linked enzyme aggregates (combi-CLEAs) are a novel prospective and this present study addresses the preparation, characterization and application of xylanase-mannanase combi-CLEAS on lime-preteated sugarcane bagasse and milled corn stover. X6-CLEAs, X7-CLEAs, L1-CLEAs and L7-CLEAs were prepared after elaborative optimization of the precipitating agent and glutaraldehyde concentration. The highest activity after precipitation was observed with acetone but following cross-linking with glutaraldehyde less than 60% activity was retained, while more than 60% activity was retained after precipitation with ammonium sulphate and cross-linking with glutaraldehyde. Accessory enzyme activities including α-arabinofuranosidase, β-xylosidase, esterases, β-mannosidase, α-galactosidase and β-glucosidase were also determined. More than an 1.5 fold increase in thermostability compared to the free enzyme was observed over a broad temperature range (50-70°C). Tri-synergy studies and quad synergy studies were used to generate combi-CLEAs with different protein ratios. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at protein ratios corresponding to X6 (33.0%):X7 (17.0%):L1 (17.0%):L7 (33.0%) resulted in a 1.68 fold higher sugar release compared to the quad synergy model using free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs at protein ratios corresponding to X6 (40.0%):X7 (10.0%):L1 (10.0%):L7 (40.0%) resulted in an 1.58 fold higher sugar release compared to the sugar release observed with the quad synergy model using free enzymes. Monomeric sugars constituted 70-75% of reducing sugars released during hydrolysis. The role of accessory enzymes in improving enzyme synergy was clearly shown. The efficiency of combi-CLEAs compared to free enzymes makes them ideal candidates for the prudent and cost-effective commercialization of lignocellulolytic enzymes.
Ethanol effect on metabolic activity of the ethalogenic fungus Fusarium oxysporum.
Paschos, T., Xiros, C. & Christakopoulos, P. (2015). BMC biotechnology, 15(1), 15.
Background: Fusarium oxysporum is a filamentous fungus which has attracted a lot of scientific interest not only due to its ability to produce a variety of lignocellulolytic enzymes, but also because it is able to ferment both hexoses and pentoses to ethanol. Although this fungus has been studied a lot as a cell factory, regarding applications for the production of bioethanol and other high added value products, no systematic study has been performed concerning its ethanol tolerance levels. Results: In aerobic conditions it was shown that both the biomass production and the specific growth rate were affected by the presence of ethanol. The maximum allowable ethanol concentration, above which cells could not grow, was predicted to be 72 g/L. Under limited aeration conditions the ethanol-producing capability of the cells was completely inhibited at 50 g/L ethanol. The lignocellulolytic enzymatic activities were affected to a lesser extent by the presence of ethanol, while the ethanol inhibitory effect appears to be more severe at elevated temperatures. Moreover, when the produced ethanol was partially removed from the broth, it led to an increase in fermenting ability of the fungus up to 22.5%. The addition of F. oxysporum’s system was shown to increase the fermentation of pretreated wheat straw by 11%, in co-fermentation with Saccharomyces cerevisiae. Conclusions: The assessment of ethanol tolerance levels of F. oxysporum on aerobic growth, on lignocellulolytic activities and on fermentative performance confirmed its biotechnological potential for the production of bioethanol. The cellulolytic and xylanolytic enzymes of this fungus could be exploited within the biorefinery concept as their ethanol resistance is similar to that of the commercial enzymes broadly used in large scale fermentations and therefore, may substantially contribute to a rational design of a bioconversion process involving F. oxysporum. The SSCF experiments on liquefied wheat straw rich in hemicellulose indicated that the contribution of the metabolic system of F. oxysporum in a co-fermentation with S. cerevisiae may play a secondary role.
Simultaneous bioethanol distillery wastewater treatment and xylanase production by the phyllosphere yeast Pseudozyma antarctica GB-4 (0).
Watanabe, T., Suzuki, K., Sato, I., Morita, T., Koike, H., Shinozaki, Y., Ueda, H., Koitabashi, M. & Kitamoto, H. K. (2015). AMB Express, 5(1), 36.
Bioethanol production using lignocellulosic biomass generates lignocellulosic bioethanol distillery wastewater (LBDW) that contains a large amount of xylose, making it a potential inexpensive source of xylose for biomaterials production. The main goal of this study was the production of useful enzymes from LBDW during treatment of this wastewater. In this study, we found that xylose strongly induced two yeast strains, Pseudozyma antarctica T-34 and GB-4(0), to produce novel xylanases, PaXynT and PaXynG, respectively. The nucleotide sequence of PaXynT [accession No. DF196774 (GAC73192.1)], obtained from the genome database of strain T-34 using its N-terminal amino acid sequence, was 91% identical to that of PaXynG (accession No. AB901085), and the deduced amino acid sequence is 98% identical. The specific activities of the purified PaXynT and PaXynG were about 52 U/mg. The optimal pH and temperature for both enzymes’ activities were 5.2 and 50°C, respectively. They hydrolyzed xylan to xylose and neither had β-xylosidase (EC 126.96.36.199) activity, indicating that they are endo-β-xylanases (EC 188.8.131.52). With these results, we expect that PaXyns can be employed in saccharizing lignocellulosic biomass materials for the production of useful products just like other endoxylanases. After 72 h of LBDW fed-batch cultivation using a jar-fermentor, strain GB-4(0) produced 17.3 U/ml (corresponding to about 0.3 g/l) of PaXynG and removed 63% of dissolved organic carbon and 87% of dissolved total phosphorus from LBDW. These results demonstrate the potential of P. antarctica for xylanase production during LBDW treatment.
Novel pH-stable glycoside hydrolase family 3 β-xylosidase from Talaromyces amestolkiae: an enzyme displaying regioselective transxylosylation.
Nieto-Domínguez, M., De Eugenio, L. I., Barriuso, J., Prieto, A., de Toro, B. F., Canales-Mayordomo, Á. & Martínez, M. J. (2015). Applied and environmental Microbiology, 81(18), 6380-6392.
This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+ tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-D-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.
Pressure Effects on Lignocellulose‐Degrading Enzymes.
Kirsch, C., Surendran, S. & Smirnova, I. (2016). Chemical Engineering & Technology, 39(4), 786-790.
The effect of elevated pressure on the activity and stability of industrially relevant lignocellulose-degrading enzymes and their mixtures was investigated. It was observed that even at moderate pressure the tested enzymes can be applied at higher temperature as at ambient pressure without significant activity loss. No negative influence of pressure on the enzyme stability was detected in the pressure range investigated. It can be concluded that pressure can improve the stability of lignocellulose-digesting enzymes, enabling higher reaction temperatures and, therefore, higher reaction rates. This might be a useful tool in industrial applications of lignocellulose-digesting enzymes.
Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens.
Warnock, N. D., Wilson, L., Canet-Perez, J. V., Fleming, T., Fleming, C. C., Maule, A. G. & Dalzell, J. J. (2016). International Journal for Parasitology, 46(8), 473-477.
Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.
Development of a new method for D-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.
Sánchez-Moreno, I., García-Junceda, E., Hermida, C. & Fernández-Mayoralas, A. (2016). Journal of Biotechnology, 234, 50-57.
The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD+ and a strong preference for D-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250 days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150 days of incubation at 4°C. Based on these characteristics, the capability of XylB in D-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of D-xylose of 10 mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568 mg/dL and 1.89 mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of D-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.
Direct Ethanol Production from Ionic Liquid-Pretreated Lignocellulosic Biomass by Cellulase-Displaying Yeasts.
Yamada, R., Nakashima, K., Asai-Nakashima, N., Tokuhara, W., Ishida, N., Katahira, S., Kamiya, N., Ogino, C. & Kondo, A. (2017). Applied Biochemistry and Biotechnology, 182(1), 229-237.
Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.
Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments.
Daly, P., Munster, J. M., Blythe, M. J., Ibbett, R., Kokolski, M., Gaddipati, S. et al. (2017). Biotechnology for Biofuels, 10(1), 35.
Background: The capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails. Results: We investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitated identification of temporally limited gene induction patterns. Conclusions: The presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.
GH11 xylanase increases prebiotic oligosaccharides from wheat bran favouring butyrate-producing bacteria in vitro.
Ravn, J. L., Thøgersen, J. C., Eklöf, J., Pettersson, D., Ducatelle, R., van Immerseel, F. & Pedersen, N. R. (2017). Animal Feed Science and Technology, 226, 113-123.
Alternative solutions to optimise intestinal health in monogastric animals have become essential since the ban of antimicrobials in animal feed. In this study, the prebiotic potential of a commercial feed GH11 xylanase was investigated in vitro. Enzymatic degradation of arabinoxylan (AX), the substrate present in wheat bran cell walls, was visualised using immuno-microscopy techniques. The arabinoxylooligosaccharides (AXOS) generated by the enzyme were analysed by non-starch polysaccharide (NSP) analysis, mass spectrometry (MS) and carbohydrate chromatography to investigate how AXOS glycan complexity and enzyme dosage may affect fermentation patterns in a wheat-based diet. Using a 10 mg EP/kg dosage of xylanase, AXOS with an average degree of polymerisation (avDP) of 10 were generated, while using a higher enzyme dosage (50 mg EP/kg) avDP shifted to 4-8. For both enzyme concentrations, AXOS had an arabinose/xylose ratio of ~0.4. Wheat bran incubated without or with xylanase was simultaneously fermented by broiler cecal bacteria in vitro and short chain fatty acid production was monitored. A small (but significant) increase in butyrate production by addition of xylanase was shown to be dose-dependent and increased by 2 mM (P < 0.05) compared to control by adding 50 mg EP/kg enzyme dosage. Butyrate-producing bacterial genera Faecalibacterium and Intestinimonas were significantly increased in fermentation reactions of wheat bran with GH11 xylanase addition while Bacteroidetes levels were significantly lowered. Supernatants from fermentation reactions of wheat bran incubated with and without xylanase and cecal microbiota were tested in an intestinal epithelial layer permeability assay using Caco-2 cells stimulated with LPS. The xylanase addition to the bran incubated with cecal content of broilers reversed LPS-induced epithelial layer resistance losses. The GH11 xylanase was able to solubilise and degrade wheat bran AX to yield low avDP AXOS that can be fermented by cecal microbiota, resulting in microbiota shifts and beneficial effects on transepithelial resistance in vitro.
Improvement of ectoine productivity by using sugar transporter-overexpressing Halomonas elongata.
Tanimura, K., Matsumoto, T., Nakayama, H., Tanaka, T. & Kondo, A. (2016). Enzyme and Microbial Technology, 89, 63-68.
We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar.
Characterization of a novel pH-stable GH3 β-xylosidase from Talaromyces amestolkiae: An enzyme displaying regioselective transxylosylation.
Nieto-Domínguez, M., de Eugenio, L. I., Barriuso, J., Prieto, A., de Toro, B. F., Canales-Mayordomo, Á. & Martínez, M. J. (2015). Applied and Environmental Microbiology, AEM-01744.
This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1 could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intron-less gene belonging to the Glycoside Hydrolase Gene Family 3 (GH 3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow synthesizing oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+ tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-D-xylopyranoside as substrate. The catalytic efficiencies for xylooligosaccharides hydrolysis were remarkably high, making it suitable for different applications in food and bioenergy industries.