Justin Powlowski

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Crystal structure of a bifunctional aldolase-dehydrogenase: Sequestering a reactive and volatile intermediate

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.

Curation of characterized glycoside hydrolases of fungal origin.

Fungi produce a wide range of extracellular enzymes to break down plant cell walls, which are composed mainly of cellulose, lignin and hemicellulose. Among them are the glycoside hydrolases (GH), the largest and most diverse family of enzymes active on these substrates. To facilitate research and development of enzymes for the conversion of cell-wall polysaccharides into fermentable sugars, we have manually curated a comprehensive set of characterized fungal glycoside hydrolases. Characterized glycoside hydrolases were retrieved from protein and enzyme databases, as well as literature repositories. A total of 453 characterized glycoside hydrolases have been cataloged. They come from 131 different fungal species, most of which belong to the phylum Ascomycota. These enzymes represent 46 different GH activities and cover 44 of the 115 CAZy GH families. In addition to enzyme source and enzyme family, available biochemical properties such as temperature and pH optima, specific activity, kinetic parameters and substrate specificities were recorded. To simplify comparative studies, enzyme and species abbreviations have been standardized, Gene Ontology terms assigned and reference to supporting evidence provided. The annotated genes have been organized in a searchable, online database called mycoCLAP (Characterized Lignocellulose-Active Proteins of fungal origin). It is anticipated that this manually curated collection of biochemically characterized fungal proteins will be used to enhance functional annotation of novel GH genes. Database URL: http://mycoCLAP.fungalgenomics.ca/

On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

DmpK from Pseudomonas sp. strain CF600 represents a group of proteins required by phenol-degrading bacteria that utilize a multicomponent iron-containing phenol hydroxylase. DmpK has been overexpressed in Escherichia coli and purified to homogeneity; it lacks redox cofactors and was found to strongly inhibit phenol hydroxylase in vitro. Chemical cross-linking experiments established that DmpK binds to the two largest subunits of the oxygenase component of the hydroxylase; this may interfere with binding of the hydroxylase activator protein, DmpM, causing inhibition. Since expression of DmpK normally appears to be much lower than that of the components of the oxygenase, inhibition may not occur in vivo. Hence, the interaction between DmpK and the oxygenase manifested in the inhibition and cross-linking results prompted construction of E. coli strains in which the oxygenase component was expressed in the presence and absence of a low molar ratio of DmpK. Active oxygenase was detected only when expressed in the presence of DmpK. Furthermore, inactive oxygenase could be activated in vitro by adding ferrous iron, in a process that was dependent on the presence of DmpK. These results indicate that DmpK plays a role in assembly of the active form of the oxygenase component of phenol hydroxylase.

Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family

Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes.

A Mercuric Ion Uptake Role for the Integral Inner Membrane Protein, MerC, Involved in Bacterial Mercuric Ion Resistance

Bacterial detoxification of mercuric ion depends on the presence of one or more integral membrane proteins (MerT and/or MerC) whose postulated function is in transport of Hg2+ from a periplasmic Hg2+-binding protein (MerP) to cytoplasmic mercuric reductase. In this study, MerC from the Tn21-encoded mer operon was overexpressed and studied in vesicles and in purified form to clarify the role played by this protein in mercuric ion resistance. MerC-containing vesicles were found to take up mercuric ion independently of MerP. Since uptake correlated with the level of MerC expression was unaffected by osmotic pressure, and was only partially decreased in the presence of 0.05% Triton X-100, the observed uptake appears to represent mainly binding to MerC. Binding was inhibited by thiol-specific reagents, consistent with an essential role for cysteine residues. The essential thiol groups were inaccessible to hydrophilic thiol reagents, whereas hydrophobic reagents completely abolished Hg2+ binding. These observations are consistent with the predicted topology of the protein, wherein all 4 cysteine residues are either in the cytoplasm or the bilayer. A role for MerC in Hg2+ transport is thus also likely. Based on these results, a modified model for bacterial Hg2+ transport is proposed.

Conservation of regulatory and structural genes for a multi-component phenol hydroxylase within phenol-catabolizing bacteria that utilize a meta-cleavage pathway

Pseudomonas sp. strain CF600 can degrade phenol and some of its methylated derivatives via a plasmid (pVI150)-encoded pathway. The metabolic route involves hydroxylation by a multi-component phenol hydroxylase and a subsequent meta-cleavage pathway. All 15 structural genes involved are clustered in an operon that is regulated by a divergently transcribed transcriptional activator. The multi-component nature of the phenol hydroxylase is unusual since reactions of this type are usually accomplished by single component flavoproteins. We have isolated and analysed a number of marine bacterial isolates capable of degrading phenol and a range of other aromatic compounds as sole carbon and energy sources. Southern hybridization and enzyme assays were used to compare the catabolic pathways of these strains and of the archetypal phenol-degrader Pseudomonas U, with respect to known catabolic genes encoded by Pseudomonas CF600. All the strains tested that degraded phenol via a meta-cleavage pathway were found to have DNA highly homologous to each of the components of the multicomponent phenol hydroxylase. Moreover, DNA of the same strains also strongly hybridized to probes specific for pVI150-encoded meta-pathway genes and the specific regulator of its catabolic operon. These results demonstrate conservation of structural and regulatory genes involved in aromatic catabolism within strains isolated from diverse geographical locations (UK, Norway and USA) and a range of habitats that include activated sludge, sea water and fresh-water mud.

Semantic text mining support for lignocellulose research

BackgroundBiofuels produced from biomass are considered to be promising sustainable alternatives to fossil fuels. The conversion of lignocellulose into fermentable sugars for biofuels production requires the use of enzyme cocktails that can efficiently and economically hydrolyze lignocellulosic biomass. As many fungi naturally break down lignocellulose, the identification and characterization of the enzymes involved is a key challenge in the research and development of biomass-derived products and fuels. One approach to meeting this challenge is to mine the rapidly-expanding repertoire of microbial genomes for enzymes with the appropriate catalytic properties.ResultsSemantic technologies, including natural language processing, ontologies, semantic Web services and Web-based collaboration tools, promise to support users in handling complex data, thereby facilitating knowledge-intensive tasks. An ongoing challenge is to select the appropriate technologies and combine them in a coherent system that brings measurable improvements to the users. We present our ongoing development of a semantic infrastructure in support of genomics-based lignocellulose research. Part of this effort is the automated curation of knowledge from information on fungal enzymes that is available in the literature and genome resources.ConclusionsWorking closely with fungal biology researchers who manually curate the existing literature, we developed ontological natural language processing pipelines integrated in a Web-based interface to assist them in two main tasks: mining the literature for relevant knowledge, and at the same time providing rich and semantically linked information.

Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila.

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.