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In JoVE (1)
Other Publications (26)
- Plant Physiology
- Fungal Genetics and Biology : FG & B
- Eukaryotic Cell
- Plant Physiology
- DNA Sequence : the Journal of DNA Sequencing and Mapping
- Science (New York, N.Y.)
- Science (New York, N.Y.)
- Proceedings of the National Academy of Sciences of the United States of America
- Critical Care Medicine
- Fungal Genetics and Biology : FG & B
- The Journal of Biological Chemistry
- Molecular Plant-microbe Interactions : MPMI
- Fungal Genetics and Biology : FG & B
- Molecular Plant-microbe Interactions : MPMI
- Bioresource Technology
- Biotechnology for Biofuels
- Biotechnology and Bioengineering
- Eukaryotic Cell
- Biotechnology for Biofuels
- The Journal of Biological Chemistry
- Biotechnology and Bioengineering
- Fungal Genetics and Biology : FG & B
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Articles by Jonathan Walton in JoVE
GENPLAT: Biyokütle Enzim Discovery ve Kokteyl Optimizasyonu Otomatik Platform
Jonathan Walton1,2, Goutami Banerjee2, Suzana Car2
1DOE Plant Research Laboratory, Michigan State University, 2DOE Great Lakes Bioenergy Research Center, Michigan State University
GENPLAT (GLBRC Enzim Platformu) biyokütle bozulması için enzim kokteyller keşif ve optimizasyonu için otomatik bir platform. Bu birden fazla hammadde ve birden fazla bileşen içeren enzimlerin karışımları adapte edilebilir.
Other articles by Jonathan Walton on PubMed
An Extended Physical Map of the TOX2 Locus of Cochliobolus Carbonum Required for Biosynthesis of HC-toxin
Fungal Genetics and Biology : FG & B. Feb, 2002 | Pubmed ID: 11860263
In genetic crosses, HC-toxin production in the filamentous fungus Cochliobolus carbonum appears to be controlled by a single locus, TOX2. At the molecular level, TOX2 is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. All copies of four of the TOX2 genes were previously mapped within a 540-kb stretch of DNA in strain SB111. Subsequently, an additional three TOX2 genes, TOXE, TOXF, and TOXG, have been discovered. In this paper we have mapped all copies of the new genes, a total of seven, and show that except for one of the two copies of TOXE, which was previously shown to be on a chromosome of 0.7 Mb in strain SB111, they are all linked to the previously known TOX2 genes within approximately 600 kb of each other on a chromosome of 3.5 Mb. We show here that this chromosome also contains at least one non-TOX2 gene, EXG2, which encodes an exo-beta1,3-glucanase. EXG2 is still present in strains that have undergone spontaneous deletion of up to approximately 1.4 Mb of the 3.5-Mb chromosome. The results contribute to our understanding of the complex organization of the genes involved in HC-toxin biosynthesis and are consistent with the hypothesis that a reciprocal chromosomal translocation accounts for the pattern of distribution of the TOX2 genes in different C. carbonum isolates.
Eukaryotic Cell. Aug, 2002 | Pubmed ID: 12456002
HC-toxin, a cyclic peptide made by the filamentous fungus Cochliobolus carbonum, is an inhibitor of histone deacetylase (HDAC) from many organisms. It was shown earlier that the HDAC activity in crude extracts of C. carbonum is relatively insensitive to HC-toxin as well as to the chemically unrelated HDAC inhibitors trichostatin and D85, whereas the HDAC activity of Aspergillus nidulans is sensitive (G. Brosch et al., Biochemistry 40:12855-12863, 2001). Here we report that HC-toxin-resistant HDAC activity was present in other, but not all, plant-pathogenic Cochliobolus species but not in any of the saprophytic species tested. The HDAC activities of the fungi Alternaria brassicicola and Diheterospora chlamydosporia, which also make HDAC inhibitors, were resistant. The HDAC activities of all C. carbonum isolates tested, except one non-toxin-producing isolate, were resistant. In a cross between a sensitive isolate and a resistant isolate, resistance genetically cosegregated with HC-toxin production. When fractionated by anion-exchange chromatography, extracts of resistant and sensitive isolates and species had two peaks of HDAC activity, one that was fully HC-toxin resistant and a second that was larger and sensitive. The first peak was consistently smaller in extracts of sensitive fungi than in resistant fungi, but the difference appeared to be insufficiently large to explain the differential sensitivities of the crude extracts. Differences in mRNA expression levels of the four known HDAC genes of C. carbonum did not account for the observed differences in HDAC activity profiles. When mixed together, resistant extracts protected extracts of sensitive C. carbonum but did not protect other sensitive Cochlibolus species or Neurospora crassa. Production of this extrinsic protection factor was dependent on TOXE, the transcription factor that regulates the HC-toxin biosynthetic genes. The results suggest that C. carbonum has multiple mechanisms of self-protection against HC-toxin.
Plant Physiology. May, 2003 | Pubmed ID: 12746531
Quantitative trait loci (QTLs) affecting sugar composition of the cell walls of maize (Zea mays) pericarp were mapped as an approach to the identification of genes involved in cereal wall biosynthesis. Mapping was performed using the IBM (B73 x Mo17) recombinant inbred line population. There were statistically significant differences between B73 and Mo17 in content of xylose (Xyl), arabinose (Ara), galactose (Gal), and glucose. Thirteen QTLs were found, affecting the content of Xyl (two QTLs), Ara (two QTLs), Gal (five QTLs), Glc (two QTLs), Ara + Gal (one QTL), and Xyl + Glc (one QTL). The chromosomal regions corresponding to two of these, affecting Ara + Gal and Ara on maize chromosome 3, could be aligned with a syntenic region on rice (Oryza sativa) chromosome 1, which has been completely sequenced and annotated. The contiguous P1-derived artificial chromosome rice clones covering the QTLs were predicted to encode 117 and 125 proteins, respectively. Two of these genes encode putative glycosyltransferases, displaying similarity to carbohydrate-active enzyme database family GT4 (galactosyltransferases) or to family GT64 (C-terminal domain of animal heparan synthases). The results illustrate the potential of using natural variation, emerging genomic resources, and homeology within the Poaceae to identify candidate genes involved in the essential process of cell wall biosynthesis.
Isolation of the Carbon Catabolite Repressor (CREA) Gene from the Plant-pathogenic Fungus Cochliobolus Carbonum
DNA Sequence : the Journal of DNA Sequencing and Mapping. Apr, 2003 | Pubmed ID: 12825351
The CREA gene has been implicated in glucose repression in several fungi. The product of this gene, CreA, binds to the promoter region of several enzymes and down-regulates gene expression. An ortholog of CREA was isolated and characterized from the maize pathogenic fungus, Cochliobolus carbonum. The deduced amino acid sequence of the C. carbonum CREA gene is very similar to the CreA proteins of Aspergillus niger, Gibberella fujikuroi, Sclerotinia sclerotiorum and Trichoderma reesei, as well as the Mig1 protein of Saccharomyces cerevisiae. And like the other fungal proteins, C. carbonum CreA has two zinc finger regions and a nuclear localization signal. Putative CreA binding sequences were also identified in the 5' region of three C. carbonum cell wall degrading enzyme genes suggesting that the protein may play a role in the regulatory process that controls these enzymes expression.
Science (New York, N.Y.). Mar, 2006 | Pubmed ID: 16574854
Phytochemistry. Jul, 2006 | Pubmed ID: 16839576
HC-toxin is a cyclic tetrapeptide of structure cyclo(D-Pro-L-Ala-D-Ala-L-Aeo), where Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. It is a determinant of specificity and virulence in the interaction between the producing fungus, Cochliobolus carbonum, and its host, maize. HC-toxin qualifies as one of the few microbial secondary metabolites whose ecological function in nature is understood. Reaction to C. carbonum and to HC-toxin is controlled in maize by the Hm1 and Hm2 loci. These loci encode HC-toxin reductase, which detoxifies HC-toxin by reducing the 8-carbonyl group of Aeo. HC-toxin is an inhibitor of histone deacetylases (HDACs) in many organisms, including plants, insects, and mammals, but why inhibition of HDACs during infection by C. carbonum leads to disease is not understood. The genes for HC-toxin biosynthesis (collectively known as the TOX2 locus) are loosely clustered over >500 kb in C. carbonum. All of the known TOX2 genes are present in multiple, functional copies and are absent from natural toxin non-producing isolates. The central enzyme in HC-toxin biosynthesis is a 570-kDa non-ribosomal synthetase encoded by a 15.7-kb open reading frame. Other genes known to be required for HC-toxin encode alpha and beta subunits of fatty acid synthase, which are presumed to contribute to the synthesis of Aeo; a pathway-specific transcription factor; an efflux carrier; a predicted branched-chain amino acid aminotransferase; and an alanine racemase.
Comparative Proteomics of Extracellular Proteins in Vitro and in Planta from the Pathogenic Fungus Fusarium Graminearum
Proteomics. Sep, 2007 | Pubmed ID: 17676664
High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.
The Fusarium Graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization
Science (New York, N.Y.). Sep, 2007 | Pubmed ID: 17823352
We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.
Proceedings of the National Academy of Sciences of the United States of America. Nov, 2007 | Pubmed ID: 18025465
Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.
The Need for Adequate Rate Control Points to Amiodarone As the Drug of Choice for Treating Critically Ill Patients with Acute Onset Atrial Fibrillation
Critical Care Medicine. Nov, 2008 | Pubmed ID: 18941343
Planta. Mar, 2009 | Pubmed ID: 19130077
The cellulose synthase-like (ZmCSL) gene family of maize was annotated and its expression studied in the maize mesocotyl. A total of 28 full-length CSL genes and another 13 partial sequences were annotated; four are predicted to be pseudogenes. Maize has all of the CSL subfamilies that are present in rice, but the CSLC subfamily is expanded from 6 in rice to 12 in maize, and the CSLH subfamily might be reduced from 3 to 1. Unlike rice, maize has a gene in the CSLG subfamily, based on its sequence similarity to two genes annotated as CSLG in poplar. Light regulation of glycan synthase enzyme activities and CSL gene expression were analyzed in the mesocotyl. A Golgi-localized glucan synthase activity is reduced by ~50% 12 h after exposure to light. beta-1,4-Mannan synthase activity is reduced even more strongly (>85%), whereas beta-1,4-xylan synthase, callose synthase, and latent IDPase activity respond only slightly, if at all, to light. At least 17 of the CSL genes (42%) are expressed in the mesocotyl, of which four are up-regulated at least twofold, seven are down-regulated at least twofold, and six are not affected by light. The results contribute to our understanding of the structure of the CSL gene family in an important food and biofuel plant, show that a large percentage of the CSL genes are expressed in the specialized tissues of the mesocotyl, and demonstrate that members of the CSL gene family are differentially subject to photobiological regulation.
Reduced Genomic Potential for Secreted Plant Cell-wall-degrading Enzymes in the Ectomycorrhizal Fungus Amanita Bisporigera, Based on the Secretome of Trichoderma Reesei
Fungal Genetics and Biology : FG & B. May, 2009 | Pubmed ID: 19373972
Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based "shotgun" proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial "cellulase" preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.
The Journal of Biological Chemistry. Jul, 2009 | Pubmed ID: 19389704
The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 220.127.116.11). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.
Biosynthesis and Role in Virulence of the Histone Deacetylase Inhibitor Depudecin from Alternaria Brassicicola
Molecular Plant-microbe Interactions : MPMI. Oct, 2009 | Pubmed ID: 19737099
Depudecin, an eleven-carbon linear polyketide made by the pathogenic fungus Alternaria brassicicola, is an inhibitor of histone deacetylase (HDAC). A chemically unrelated HDAC inhibitor, HC toxin, was earlier shown to be a major virulence factor in the interaction between Cochliobolus carbonum and its host, maize. In order to test whether depudecin is also a virulence factor for A. brassicicola, we identified the genes for depudecin biosynthesis and created depudecin-minus mutants. The depudecin gene cluster contains six genes (DEP1 to DEP6), which are predicted to encode a polyketide synthase (AbPKS9 or DEP5), a transcription factor (DEP6), two monooxygenases (DEP2 and DEP4), a transporter of the major facilitator superfamily (DEP3), and one protein of unknown function (DEP1). The involvement in depudecin production of DEP2, DEP4, DEP5, and DEP6 was demonstrated by targeted gene disruption. DEP6 is required for expression of DEP1 through DEP5 but not the immediate flanking genes, thus defining a coregulated depudecin biosynthetic cluster. The genes flanking the depudecin gene cluster but not the cluster itself are conserved in the same order in the related fungi Stagonospora nodorum and Pyrenophora tritici-repentis. Depudecin-minus mutants have a small (10%) but statistically significant reduction in virulence on cabbage (Brassica oleracea) but not on Arabidopsis. The role of depudecin in virulence is, therefore, less dramatic than that of HC toxin.
Fungal Genetics and Biology : FG & B. Mar, 2009 | Pubmed ID: 19146970
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
Effectors, Effectors Et Encore Des Effectors: the XIV International Congress on Molecular-Plant Microbe Interactions, Quebec
Molecular Plant-microbe Interactions : MPMI. Dec, 2009 | Pubmed ID: 19888813
Biopolymers. 2010 | Pubmed ID: 20564017
Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as alpha- and beta-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys crossbridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34-35 amino acids in length and have no predicted signal peptides. The genes for alpha-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable "toxin" region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide.
Bioresource Technology. Dec, 2010 | Pubmed ID: 20678930
A high throughput enzyme assay platform, called GENPLAT, was used to guide the development of an optimized mixture of individual purified enzymes from ten "accessory" and six "core" enzymes. Enzyme mixtures were optimized for release of Glu, Xyl, or a combination of the two from corn stover pretreated by ammonia-fiber expansion (AFEX). Assay conditions were a fixed enzyme loading of 15 mg/g glucan, 48 h digestion, and 50 degrees C. Five of the ten tested accessory proteins enhanced Glu or Xyl yield compared to the core set alone, and five did not. An 11-component mixture containing the core set and five accessory enzymes optimized for Glu released 52.1% of the available Glu, compared to 38.5% with the core set alone. A mixture optimized for Xyl released 39.9% of the Xyl, compared to 26.4% with the core set alone. We predict that there is still considerable opportunity for further improvement of synthetic mixtures. Furthermore, the strategy described here is applicable to the development of more efficient enzyme cocktails for any pretreatment/biomass combination and for detecting enzymes that make a heretofore unrecognized contribution to lignocellulose deconstruction.
Rapid Optimization of Enzyme Mixtures for Deconstruction of Diverse Pretreatment/biomass Feedstock Combinations
Biotechnology for Biofuels. 2010 | Pubmed ID: 20939889
Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.
Biotechnology and Bioengineering. Aug, 2010 | Pubmed ID: 20564609
The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of "accessory" proteins for development of superior enzyme mixtures for biomass conversion.
Colocalization of Amanitin and a Candidate Toxin-processing Prolyl Oligopeptidase in Amanita Basidiocarps
Eukaryotic Cell. Dec, 2010 | Pubmed ID: 20889720
Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the "MSDIN" gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.
Alkaline Peroxide Pretreatment of Corn Stover: Effects of Biomass, Peroxide, and Enzyme Loading and Composition on Yields of Glucose and Xylose
Biotechnology for Biofuels. 2011 | Pubmed ID: 21658263
Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production.
The Journal of Biological Chemistry. Dec, 2011 | Pubmed ID: 22033931
α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.
Scale-up and Integration of Alkaline Hydrogen Peroxide Pretreatment, Enzymatic Hydrolysis, and Ethanolic Fermentation
Biotechnology and Bioengineering. Nov, 2011 | Pubmed ID: 22125119
Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass-to-ethanol pipeline. Here, the feasibility of scaling-up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H(2) O(2) /g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose- and xylose-utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. © 2011 Wiley Periodicals, Inc.
Fungal Genetics and Biology : FG & B. Feb, 2012 | Pubmed ID: 22202811
Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.