Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Taxonomy and redescription of the Swamp Antechinus, Antechinus minimus (E. Geoffroy) (Marsupialia: Dasyuridae)

We provide a taxonomic redescription of the dasyurid marsupial Swamp Antechinus, Antechinus minimus (Geoffroy, 1803). In the past, A. minimus has been classified as two subspecies: the nominate A. minimus minimus (Geoffroy, 1803), which is found throughout much of Tasmania (including southern Bass Strait islands) and A. minimus maritimus (Finlayson, 1958), which is found on mainland Australia (as well as some near-coastal islands) and is patchily distributed in mostly coastal areas between South Gippsland (Victoria) and Robe (South Australia). Based on an assessment of morphology and DNA, we conclude that A. minimus is both distinctly different from all extant congeners and that the two existing subspecies of Swamp Antechinus are appropriately taxonomically characterised. In our genetic phylogenies, the Swamp Antechinus was monophyletic with respect to all 14 known extant congeners; moreover, A. minimus was well-positioned in a large clade, together with all four species in the Dusky Antechinus complex, to the exclusion of all other antechinus. Within A. minimus, between subspecies there were subtle morphological differences (A. m. maritimus skulls tend to be broader, with larger molar teeth, than A. m. minimus, but these differences were not significant); there was distinct, but only moderately deep genetic differences (3.9–4.5% at mtDNA) between A. minimus subspecies. Comparatively, across Bass Strait, the two subspecies of A. minimus are morphologically and genetically markedly less divergent than recently recognised species pairs within the Dusky Antechinus complex, found in Victoria (A. mimetes) and Tasmania (A. swainsonii) (9.4–11.6% divergent at mtDNA)

Senescence in fungi: the view from Neurospora

Some naturally occurring strains of fungi cease growing through successive subculturing, i.e., they senesce. In Neurospora, senescing strains usually contain intramitochondrial linear or circular plasmids. An entire plasmid or its part(s) integrates into the mtDNA, causing insertional mutagenesis. The functionally defective mitochondria replicate faster than the wild-type mitochondria and spread through interconnected hyphal cells. Senescence could also be due to spontaneous lethal nuclear gene mutations arising in the multinucleated mycelium. However, their phenotypic effects remain masked until the nuclei segregate into a homokaryotic spore, and the spore germinates to form a mycelium that is incapable of extended culturing. Ultimately the growth of a fungal colony ceases due to dysfunctional oxidative phosphorylation. Results with senescing nuclear mutants or growth-impaired cytoplasmic mutants suggest that mtDNA is inherently unstable, requiring protection by as yet unidentified nuclear-gene-encoded factors for normal functioning. Interestingly, these results are in accord with the endosymbiotic theory of origin of eukaryotic cells.

Simultaneous Utilization of Glucose and Sucrose by Thermophilic Fungi

The utilization of mixtures of glucose and sucrose at nonlimiting concentrations was studied in batch cultures of two common thermophilic fungi, Thermomyces lanuginosus and Penicilium duponti. The sucrose-utilizing enzymes (sucrose permease and invertase) in both fungi were inducible. Both sugars were used concurrently,regardless of their relative proportion in the mixture. At the optimal growth temperature (50C), T.lanuginosus utilized sucrose earlier than it did glucose, but at a suboptimal growth temperature (30°C) the two sugars were utilized at nearly comparable rates. The coutilization of the two sugars was most likely possible because (i) invertase was insensitive to catabolite repression by glucose, (ii) the activity and affinity of the glucose transport system were lowered when sucrose was included in the growth medium, and (iii) the activity of the glucose uptake system was also subject to repression by high concentrations of glucose itself. The concurrent utilization of the available carbon sources by thermophilic fungi might be an adaptive strategy for opportunistic growth in nature under conditions of low nutrient availability and thermal fluctuations in the environment.

Studies of fungi associated with Tomicus piniperda L. in Finland

The Scots pine bark beetle, Tomicus piniperda is a secondary colonizer of pine and other conifers. It is a native species from Europe and Asia that was recently introduced in North America. Although it is necessary to understand this insect's interactions with other organisms, few studies have focussed on its fungal associates. This study focused on the effect of latitude in the occurrence of fungi associated with T. piniperda. T. piniperda were collected from Pinus sylvestris in Northern (Rovaniemi) and Southern (Hyytiala) Finland. Both endo- and epi- mycota were isolated. The fungi were identified using a combination of morphological features and molecular data. The results revealed a great diversity of fungi species associated with T. piniperda, with a total of 3073 isolates representing 23 species. The most frequently isolated fungi in the bark beetles from Northern Finland were Beauvaria bassiana, Kuraishia sp. and Penicillium sp. whereas P. brevicompactum and Mortierella sp. were mostly observed in the South. Ophiostoma canum and O. minus were also observed. The number of isolates per insect in the north was 2.83 epi- and 2.38 for endo-mycota fungus. In the south, the number of isolates per insect was 4.1 for epi- and 3.5 for endo-mycota. Statistical analysis indicated that there was significant differences in fungal populations associated with the beetles in Southern and Northern Finland. There was however no significant difference between the epi- and endo-mycota fungal populations. The highest richness and diversity of the fungal species was observed in the South. However, the overall fungal diversity index analysis revealed that the mycobiota was undersampled.

Taxonomy and phylogeny of the manna lichens and allied species (Megasporaceae)

This dissertation is focused on the taxonomy, phylogeny, and ecology of the vagrant, erratic and allied terricolous and saxicolous species of the genera Aspicilia A. Massal. and Circinaria Link (Megasporaceae), particularly those traditionally referred to as manna lichens . The group has previously been defined on the basis of few morphological characters. The phylogeny of the family Megasporaceae is inferred from the combined dataset of nuLSU and mtSSU sequences. Five genera Aspicilia, Circinaria, Lobothallia, Megaspora, and Sagedia are recognized. Lobothallia is sister of the four other genera, while Aspicilia and Sagedia form the next clade. All these genera have small asci with eight spores. Circinaria is a sister genus of Megaspora, and these two have in common asci with (1 4) 6 8 large spores. Circinaria forms a monophyletic group and sphaerothallioid species form a monophyletic group within Circinaria. The presence of certain morphological characters such as pseudocyphellae, thickness of cortex and medulla layers, as well as ecological differences in sphaerothallioid species distinguish it from some other crustose species, especially those containing aspicilin and characterised by thin cortex and medulla layers, conidium length c. 6 12 µm and absence of pseudocyphellae. If sphaerothallioid species are accepted as a distinct genus, the rest of the Circinaria species would remain as a paraphyletic assemblage. Currently, the genus Circinaria includes all the sphaerothallioid species and its generic position is confirmed and accepted. Thus, it is proposed as a correct generic name also for the manna lichens described originally in other genera. Phylogeny at the species level was studied using nrITS sequence data. Traditionally, morphological characters have been used for the recognition of species. They were re-evaluated in the light of molecular data. Since characters such as vagrant, erratic and crustose growth forms proved to be misleading for the recognition of some species, a combination of several characters (including molecular data) is recommended. Vagrant growth form seems to have evolved several times among the distantly related lineages and even within a single population. The reasons behind the high plasticity in the external morphology of the sphaerothallioid Circinaria remain, however, unknown. Six new species are recognized: Aspicilia tibetica, Circinaria arida, C. digitata nom provis., C. gyrosa nom. provis., C. rogeri nom. provis., and C. rostamii nom. provis. Based on an analysis of nrITS dataset, three new erratic, vagrant and crustose species were also recognized, but these require additional study. The results also reveal that C. elmorei and C. hispida are not monophyletic as currently understood. In addition, 13 new combinations in the genus Circinaria are proposed.

Thermophilic fungi: An assessment of their potential for growth in soil

An attempt has been made to forecast the potential of thermophilic fungi to grow in soil in the laboratory and in the field in the presence of a predominantly mesophilic fungal flora at usual temperature. The respiratory rate of thermophilic fungi was markedly responsive to changes in temperature, but that of mesophilic fungi was relatively independent of such changes. This suggested that in a thermally fluctuating environment, thermophilic fungi may be at a physiological disadvantage compared to mesophilic fungi. In mixed cultures in soil plates, thermophilic fungi outgrew mesophilic fungi under a fluctuating temperature regime only when the amplitude of the fluctuating temperatures was small and approached their temperature optima for growth. An antibody probe was used to detect the activity of native or an introduced strain of a thermophilic fungus, Thermomyces lanuginosus, under field conditions. The results suggest that although widespread, thermophilic fungi are ordinarily not an active component of soil microflora. Their presence in soil most likely may be the result of the aerial dissemination of propagules from composting plant material.

Unified Taxonomy for Reference Ontology of Shape Features in Product Model

This paper presents a unified taxonomy of shape features. Such taxonomy is required to construct ontologies to address heterogeneity in product/shape models. Literature provides separate classifications for volumetric, deformation and free-form surface features. The unified taxonomy proposed allows classification, representation and extraction of shape features in a product model. The novelty of the taxonomy is that the classification is based purely on shape entities and therefore it is possible to automatically extract the features from any shape model. This enables the use of this taxonomy to build reference ontology.

Biotechnological potential of plant-associated endophytic fungi:hope versus hype

The potential of endophytes, particularly endophytic fungi, capable of demonstrating desirable functional traits worth exploitation using red biotechnology is well established. However, these discoveries have not yet translated into industrial bioprocesses for commercial production of biopharmaceuticals using fungal endophytes. Here, we define the current challenges in transforming curiosity driven discoveries into industrial scale endophyte biotechnology. The possible practical, feasible, and sustainable strategies that can lead to harnessing fungal endophyte-mediated pharmaceutical products are discussed.