Thermostable enzymes and thermophilic cell factories may afford economic advantages inFurthermore, we present evidence suggesting that aside from representing a potential 9 reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using 10 classical and molecular genetics. 11Rapid, efficient and robust enzymatic degradation of biomass-derived polysaccharides is 12 currently a major challenge for biofuel production. A prerequisite is the availability of enzymes 13 that hydrolyze cellulose, hemicellulose and other polysaccharides into fermentable sugars at 14 conditions suitable for industrial use. The best studied and most widely used cellulases and to overcome these obstacles is to raise the reaction temperature, thereby increasing hydrolytic 20 rates and reducing contamination risks. AT-rich repetitive regions (Fig. 1). one PL3 and two GH28). Pectin lyases are most active at neutral to alkaline pH whereas GH28 To examine the strategy used by these thermophiles for decomposition of plant cell wall 9 polysaccharides, we used RNA-Seq to compare transcript profiles during growth on barley straw 10 or alfalfa straw to growth on glucose. Alfalfa was chosen to represent dicotyledonous plants, 11 whereas barley was used to represent monocotyledon plants. The conditions. For example, the orthologs in Clades A, B, E, G and P of GH61 are upregulated 8 under growth in complex substrates for both thermophiles (Fig. 2b). An even more striking 9 correlation between transcript levels and orthologs is evident for the GH6 and GH7 cellulases Table 7). 14 Secretomes and exo-proteomes 15In addition to extracellular CAZymes involved in digestion of polysaccharide nutrients, the Thermophilic fungi are major components of the microflora in self-heating composts. They 9 break down cellulose at a faster rate than prodigious, mesophilic cellulase producers such as T. Fig. 8 We also investigated the possibility that thermophilic fungi possess major differences in 27 processes mediating thermophily including heat shock, oxidative stress, membrane biosynthesis, 28 chromatin structure and modification, and fungal cell wall metabolism. We compared the 29 proteins predicted to be involved in these processes in C. globosum, M. thermophila and T. 30terrestris, but were unable to find differences that can convincingly be interpreted as the Fig. 9) Thermophilic fungi are ubiquitous organisms commonly found in decomposing organic matter. 25The biotechnological utility of these fungi has been recognized for many years. enzymes from the thermophiles exhibit higher hydrolytic capacity than their counterparts from 6 mesophiles at temperatures ranging from 30 °C to 60 °C (Fig. 3). One explanation is that the 7 enzymes from the thermophiles possess higher specific activity toward lignocellulosic biomass.8

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Myceliophthora thermophila is a thermophilic fungus whose genome encodes a wide range of carbohydrate-active enzymes (CAZymes) involved in plant biomass degradation. Such enzymes have potential applications in turning different kinds of lignocellulosic feedstock into sugar precursors for biofuels and chemicals. The present study examined and compared the transcriptomes and exoproteomes of M. thermophila during cultivation on different types of complex biomass to gain insight into how its secreted enzymatic machinery varies with different sources of lignocellulose. In the transcriptome analysis three monocot (barley, oat, triticale) and three dicot (alfalfa, canola, flax) plants were used whereas in the proteome analysis additional substrates, i.e. wood and corn stover pulps, were included. A core set of 59 genes encoding CAZymes was up-regulated in response to both monocot and dicot straws, including nine polysaccharide monooxygenases and GH10, but not GH11, xylanases. Genes encoding additional xylanolytic enzymes were up-regulated during growth on monocot straws, while genes encoding additional pectinolytic enzymes were up-regulated in response to dicot biomass. Exoproteome analysis was generally consistent with the conclusions drawn from transcriptome analysis, but additional CAZymes that accumulated to high levels were identified. Despite the wide variety of biomass sources tested some CAZy family members were not expressed under any condition. The results of this study provide a comprehensive view from both transcriptome and exoproteome levels, of how M. thermophila responds to a wide range of biomass sources using its genomic resources.

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The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O2 uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using 14C-glucose as precursor showed the formation of 14C-fructose and 14C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.