Venkataramanan Subramanian

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO(2)) the recalcitrant fused-ring high molecular weight (4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

P450ome of the white rot fungus Phanerochaete chrysosporium: structure, evolution and regulation of expression of genomic P450 clusters

The model white rot fungus Phanerochaete chrysosporium has the extraordinary ability to degrade (to CO(2)) lignin and detoxify a variety of chemical pollutants. Whole genome sequencing of this fungus has revealed the presence of the largest P450ome in fungi comprising approx. 150 P450 genes, most of which have unknown function. On the basis of our genome-wide structural and evolutionary analysis, these P450 genes could be classified into 12 families and 23 subfamilies and under 11 fungal P450 clans. The analysis further revealed an extensive gene clustering with a total of 16 P450 clusters constituted of up to 11 members per cluster. In particular, evidence and role of gene duplications and horizontal gene transfer in the evolution of these P450 clusters have been discussed using two of the P450 families [CYP63 and CYP505 (where CYP is cytochrome P450)] as examples. In addition, the observed differential transcriptional induction of the clustered members of the CYP63 gene family, in response to different xenobiotic chemicals and carbon sources, indicated functional divergence within the P450 clusters, of this basidiomycete fungus.

Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

This article describes novel ways that algae may adjust metabolite trafficking when specific branches of fermentation metabolism are blocked. This rerouting of metabolites allows for continued glycolytic energy production under anoxic conditions, which is critical for the cell’s survival. Mechanisms associated with this reengineering of metabolism are almost completely unexplored. Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

A Mutant in the ADH1 Gene of Chlamydomonas reinhardtii Elicits Metabolic Restructuring during Anaerobiosis

The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO2 was significantly reduced, but fermentative H2 production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.

Role of P450 monooxygenases in the degradation of the endocrine-disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium.

ABSTRACT The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (∼75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant.

Immunoproteomic identification of secretory and subcellular protein antigens and functional evaluation of the secretome fraction of Mycobacterium immunogenum, a newly recognized species of the Mycobacterium chelonae-Mycobacterium abscessus group.

Mycobacterium immunogenum has been associated with occupational pulmonary disease hypersensitivity pneumonitis (HP). The aim of this study was to identify immunogenic proteins (antigens) in this pathogen as a first step toward understanding its virulence factors and role in HP etiology. Immunoproteomic profiling of secreted and subcellular protein fractions using a combination of two-dimensional electrophoresis (2-DE), immunoblotting, and matrix-assisted laser desorption/ionization-Time of flight (MALDI-TOF) led to the identification of 33 immunoreactive proteins, comprising of 4 secretory, 6 cell wall-associated, 11 membranous, and 12 cytosolic proteins. Of these, eight immunoreactive proteins represented homologues of the known mycobacterial antigens, namely heat shock protein GroEL, antigen 85A, elongation factor Tu (EF-Tu), L-asparaginase, polyketide synthase, PE-PGRS, PPE, and superoxide dismutase (SOD). Global functional search revealed that the remaining 25 novel mycobacterial antigens in M. immunogenum showed homology with hypothetical proteins (11 antigens) and other bacterial proteins (14 antigens) with a role in virulence, survival, and/or diverse metabolic functions. To understand immunogenicity of the secretome in M. immunogenum, the major protein spot on the secretome 2D-gel (consisting of multiple secretory antigens such as OtsB and CtpA, among others) was eluted and subjected to functional characterization in terms of induction of innate immune response in murine alveolar macrophages. The secretome eluate caused up-regulation of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-18 and down-regulation of the anti-inflammatory cytokine IL-10, implying a potential of the secreted antigens to cause host immune response underlying the M. immunogenum-induced lung disease HP. This is the first report on identification of antigens in M. immunogenum as well as on the potential of its secretome proteins to induce host response. The identified antigens could have likely roles in virulence and/or diagnosis and serve as potential targets for drug, biocide, and/or vaccine development.

Physiological regulation, xenobiotic induction, and heterologous expression of P450 monooxygenase gene pc-3 (CYP63A3), a new member of the CYP63 gene cluster in the white-rot fungus Phanerochaete chrysosporium.

In order to characterize the functional diversity in CYP63 cluster of tandemly linked P450 genes (pc-1, pc-2, and pc-3) in Phanerochaete chrysosporium, here we report the functional characterization of pc-3 (CYP63A3), a newly cloned member of this group. pc-3 expression was favored in nutrient-limited versus nutrient-rich media in 3–6-day-old cultures and was upregulated by starch as a carbon source or by oxygenation of cultures. pc-3 was induced by various xenobiotics in defined nutrient-limited (3–9-fold) and nutrient-rich (2–5-fold) cultures. Particularly, a range of unsubstituted and substituted aliphatic hydrocarbons (alkanes and fatty acids) induced the expression under the two nutrient conditions albeit in a differential manner. Interestingly, pc-3 was also inducible by certain oxygenated mono aromatics (nitrophenol, benzoate, and resorcinol), lower molecular weight (2 to 4 ring size) polycyclic aromatic hydrocarbons (PAHs) and alkali-treated lignin derivatives in nutrient-rich malt extract cultures. The study further establishes that the three CYP63 genes (CYP63A1, A2, and A3) are independently regulated despite being members of the tandem gene cluster with high gene structural similarity (13–14 introns) and protein sequence homology (59–85%). The pc-3 cDNA (1,812 bp) was expressed in E. coli as a His-tagged protein (∼ 74 kDa). This constitutes the first report on heterologous expression of a P450 monooxygenase enzyme from this model white-rot fungus.