Summary
Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression ...of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β‐glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1‐modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1‐dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high‐throughput sequencing (‘ChIP‐seq’) showed that lae1 expression was not obviously correlated with H3K4 di‐ or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.
Centromeres of filamentous fungi Smith, Kristina M; Galazka, Jonathan M; Phatale, Pallavi A ...
Chromosome research,
07/2012, Volume:
20, Issue:
5
Journal Article
Peer reviewed
Open access
How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20 years, the idea of centromeres as precise genetic loci has been replaced by the ...realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived “point centromeres” of the budding yeast Saccharomyces cerevisiae and its close relatives are counterexamples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared among various groups of organisms. For that reason, it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes, and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use.
The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in ...Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.
Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic ...hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.
High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing ...centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.
Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this ...organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.
Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about ...potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.
Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this ...organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.
In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish
the critical task of correct cell division. In some organisms, centromeres are
composed of a euchromatic central core ...region embedded in a stretch of
heterochromatin and the inheritance and maintenance of centromeres are controlled
by dynamic epigenetic phenomena. Although the size of centromeres differs between
organisms, its organization, and the placement of euchromatic and heterochromatic
regions is conserved from the fission yeast, Schizosaccharomyces pombe, to
humans, Homo sapiens. However, relatively little is known about centromeres in the
filamentous fungi from the Ascomycota, representing the largest group of fungi and
fungal pathogens. Further, studies from humans, flies, yeast and plants have shown
that the inheritance of centromeres is not strictly guided by centromeric DNA content,
which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to
align ans assemble the sequenced contigs of centromeric regions of higher
eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad)
analysis has helped to map the centromeres of the filamentous fungus Neurospora
crassa early on. The research presented in this dissertation used N. crassa as a
model to focus on characterizing different features of centromeres with an emphasis
on the centromere-specific histone H3 (CenH3) protein. Data included here represent
the first study on centromere-specific proteins in Neurospora, and demonstrate that
the central core of the centromeres are heterochromatic, showing enrichment of silent
histone marks, which is in contrast to the centromere arrangement in fission yeast.
The CenH3 protein, whose deposition on the genome licenses formation or
maintenance of centromeres, shows highly divergent N-terminal regions and a
conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of
CenH3 is also observed in the Ascomycota, which provides an opportunity for
functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with
CenH3 genes from other species within the Ascomycota. The results from this
experimental approach provide good measures for (1) determining the specific
regions of CenH3 required for the assembly of centromeres during meiotic and mitotic
cell divisions and (2) analyzing the resistance to changes in the organization of
centromeres in N. crassa.
The genetic analysis showed that the divergent N-terminal region is essential
for the proper assembly of centromeres, and that the conserved carboxy-terminus of
CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq
analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3-
GFP) from certain N. crassa centromeres does not result in obvious phenotypic
defects, e.g. diminished growth or evidence for aneuploidy. Further, the low
enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined
during meiosis, which results in irreversible and progressive decreases in enrichment.
It remains to be determined if this process is random as far as selection of
centromeres is concerned. Together the results presented here suggest that during
meiosis more stringent structural requirements for centromere assembly apply and
that these are dependent on CenH3, and that depletion of CenH3 from centromeres
does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae.
Graduation date: 2013
PDF
