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) 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 major difference between these 12 materials is that the carbohydrates from barley cell wall are mainly cellulose and hemicellulose 13 with a negligible amount of pectin 11 , whereas alfalfa cell wall contains pectin and xylan in 14 roughly similar proportions, each consisting of 15-20% of total carbohydrates 12, . 15 We observed notable differences between the transcriptional profiles of genes encoding 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 10 ( Supplementary Fig. 7) where the transcript profiles for the orthologs of the two organisms are Table 7). 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. Tables 11-14). On the basis of 24 our comparative analyses of the genomes from two thermophilic fungi, we conclude that their 25 nucleotide and protein features are different from those observed in thermophilic prokaryotes. 26 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. 30 terrestris, but were unable to find differences that can convincingly be interpreted as the Fig. 9). Within the Sordiariales, thermophily 6 is restricted to subgroups of the family Chaetomiaceae. Among fungi more broadly, thermophily 7 also exists in the Zygomycota, but it ...

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat ␤-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.organophosphorus nerve agent ͉ recombinant protein expression ͉ transgenic production H uman plasma butyrylcholinesterase (huBChE) (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of Ϸ340 kDa that is stable in plasma with a half-life of Ϸ12 days (1, 2). Although the physiological function of huBChE is unclear, the enzyme prevents intoxication of animals exposed to organophosphorus (OP) compounds (3, 4). The huBChE enzyme also hydrolyzes many ester-containing drugs, such as cocaine and succinylcholine (5). The toxicity of OP agents is due to irreversible inhibition of acetylcholinesterase and the subsequent continuous stimulation of neurons by acetylcholine (6). Administration of exogenous huBChE, which irreversibly binds OP agents to prevent inactivation of acetylcholinesterase and continuous cholinergic stimulation, is a potential strategy for preventing toxicity from OP agents (4). Although huBChE has been obtained from human plasma by a large scale purification technique, this procedure is severely limited by the volume of human plasma needed (7). It is unlikely that a sufficient amount of enzyme could be purified commercially by this technique. Because of the 1:1 stoichiometry required for protection against exposure to OP agents (8), large quantities of huBChE are needed for effective prophylaxis and treatment of exposure. Compared with other potential enzymatic bioscavengers of OP agents, huBChE has a broad spectrum of activity, a relatively long half-life, and limited, if any, physiological side effects (9). Producing recombinant BChE (rBChE) is an alternative to purification of the enzyme from human plasma. Recombinant huBChE has been expressed in Escherichia coli (10), albeit in a nonfunctional form; mammalian 293T (11); and CHO (12) cells. However, these expression systems cannot economically produce sufficient quantities of rBChE with a residence time similar to native huBChE that would allow development of the enzyme as an agent for prophylaxis against OP poisoning.The production of recombinant proteins by the mammary g...

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

BackgroundHuman butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.ResultsSecretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.ConclusionBoth the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

Isolation of fungal genomic DNA of high quality is required for a number of downstream biotechnology-derived applications such as genome sequencing, microarrays, and digital PCR technologies, to only name a few. In most cases, not only a high molecular weight DNA of superior grade is required but also large quantities. On the other hand, a number of laboratory experiments, such as polymerase chain reaction (PCR) for medical diagnostic or for genotyping, have to be conducted in a limited amount of time and can provide complete results with the use of lower quality DNA. We describe here two different fungal DNA extraction approaches, which are applicable to a wide range of fungal species.First, we adapted a DNA extraction method for PCR-based genotyping which allows analysis of single to hundreds of colonies simultaneously. Cells are disrupted in the presence of sodium dodecyl sulfate and Proteinase K which are then removed by precipitation and centrifugation. The cleared lysate is used for PCR reaction.Secondly, we describe a method to obtain genome sequencing quality grade DNA from fungal liquid cultures. Mycelia are harvested by either filtration or centrifugation. Cells are mechanically disrupted by liquid nitrogen grinding, followed by genomic DNA extraction using the QIAGEN's DNeasy Plant Kit. The quality and quantity of genomic DNA is monitored by fluorometry.