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 ...

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.The central role of catecholic intermediates in aerobic microbial degradation of aromatic compounds is well established. Catechol (1,2-dihydroxybenzene) itself is an intermediate in the degradation of compounds such as benzoate, naphthalene, salicylate, and phenol, and substituted catechols are intermediates in the catabolism of methylated and chlorinated derivatives of these compounds (13,34). A diverse array of enzymes can be elaborated to convert aromatic compounds to central catecholic intermediates. However, the reactions used for oxygenative ring fission of the catechol and the subsequent conversion to Krebs cycle intermediates are limited to one of two metabolic alternatives: those of the ortho-and meta-cleavage pathways. The ortho-cleavage pathways involve ring cleavage between the two hydroxyl groups followed by a well-defined series of reactions leading to P-ketoadipate (reviewed in reference 13). The alternative meta-cleavage pathway involves ring cleavage adjacent to the two catechol hydroxyls, followed by degradation of the ring cleavage product to pyruvate and a short-chain aldehyde (Fig. 1). The use of one pathway or the other is dependent upon the microbial species and/or the nature of the growth substrate.The meta-cleavage pathway was first studied in Pseudomonas strains that can grow at the expense of phenol and cresols (14,29). Since then, the role of the meta-cleavage pathway in aromatic biodegradation by bacteria of many genera, including species of Azotobacter and Alcaligenes and numerous species of Pseudomonas, has been demonstrated (2, 13, 23, 36). In addition, reactions of the lower part of the pathway are involved in the degradation of phenylp...

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmidencoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb (14) and into Pseudomonas strain PB2701 by electroporation with a Bio-Rad Gene Pulser. Ampicillin at 100 ,ug/ml and carbenicillin at 1 to 2 mg/ml were used for selection of plasmid-encoded ,-lactamase in E. coli and Pseudomonas strains, respectively.Plasmids expressing phenol hydroxylase genes. Plasmids were constructed by using the broad-host-range tac expression vectors pMMB66HE and pMMB66EH (8), and derivatives thereof, pMMB66HEA and pMMB66EHA (22), that lack expression of the plasmid-encoded lacdq repressor gene. Subfragments of the DNA shown in Fig. 1

The crystal structure of the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase (DmpG)͞acylating acetaldehyde dehydrogenase (DmpF), which is involved in the bacterial degradation of toxic aromatic compounds, has been determined by multiwavelength anomalous dispersion (MAD) techniques and refined to 1.7-Å resolution. Structures of the two polypeptides represent a previously unrecognized subclass of metal-dependent aldolases, and of a CoA-dependent dehydrogenase. The structure reveals a mixed state of NAD ؉ binding to the DmpF protomer. Domain movements associated with cofactor binding in the DmpF protomer may be correlated with channeling and activity at the DmpG protomer. In the presence of NAD ؉ a 29-Å-long sequestered tunnel links the two active sites. Two barriers are visible along the tunnel and suggest control points for the movement of the reactive and volatile acetaldehyde intermediate between the two active sites. E nzymatic channeling is a process by which intermediates are moved directly between active sites in a sequential reaction pathway without equilibrating with the bulk phase (1, 2). Channeling processes are particularly advantageous over the free diffusion of reaction products through the bulk solvent because they can protect chemically labile intermediates from breakdown, prevent loss of nonpolar intermediates by diffusion across cell membranes, or protect the cell from toxic intermediates. Crystallographic studies on a number of different enzyme systems involved in substrate channeling (3-11) have revealed important structural factors that mediate intersubunit or interdomain communication and facilitate the efficient transfer of intermediates between distant active sites.A bifunctional aldolase-dehydrogenase catalyzes the final two steps of the meta-cleavage pathway for catechol, an intermediate in many bacterial species in the degradation of phenols, toluates, naphthalene, biphenyls and other aromatic compounds (reviewed in ref. 12). Thus, 4-hydroxy-2-ketovalerate aldolase (DmpG; EC 4.1.3.-) and acetaldehyde dehydrogenase (acylating) (DmpF; EC 1.2.1.10) from a methylphenol-degrading pseudomonad convert 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA by way of the intermediate acetaldehyde (Scheme 1).The two enzymes are tightly associated with each other (13,14). Whereas the aldolase appears to be inactive when expressed without the dehydrogenase, the dehydrogenase retains some activity when expressed in the absence of aldolase (14), suggesting that at least the dehydrogenase active site is distinct from that of the aldolase. Several lines of evidence are consistent with channeling of acetaldehyde between the active sites. For example, the conversion of 4-hydroxy-2-ketovalerate to acetyl-CoA occurs Ϸ20 times faster than that of acetaldehyde to acetyl-CoA, and the K m for acetaldehyde exceeds 50 mM, a physiologically irrelevant concentration (J.P., unpublished data). In addition, it has been shown that the aldolase activity is stimulated by the addition of the dehydrogenase cofactor to t...