|dc.description.abstract||Ophiostoma floccosum and Ophiostoma piliferum are polymorphic ascomycete fungi found throughout the world. Both species are important economically as they are known to colonise timber and cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species, in particular O. piliferum, are used as biological control agents to prevent sapstaining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Other members of the genus include the plant pathogens O. novo-ulmi and O. clavigerum, known to have a severe effect on forest health and economy around the world. O. floccosum and O. piliferum have been demonstrated in the laboratory to be fermented in large volumes and they are particularly suitable as hosts capable of secreting extracellular recombinant proteins.
This research aimed to investigate the transcriptome and molecular functioning of Ophiostoma floccosum and compare this to transcriptomic data available for Ophiostoma piliferum and other Ophiostoma species, O. novo-ulmi, O. clavigerum and O. piceae. This research contributes to the development of O. floccosum and O. piliferum as hosts for protein expression and advances the knowledge of gene expression and molecular functioning in this genus.
To gain insight into the molecular functioning of O. floccosum, an expressed sequence tag (EST) collection from yeast-like growth (blastospores) was created during early phase growth. A total of 1207 EST sequences with an average length of 713 bp were identified. Clustering and assembly of the high-quality EST data set resulted in the identification of 598 unique putative transcripts (UPTs). Functional classification of these UPTs, using both homology searching and ab-initio methods, indicated that the majority of protein transcripts produced were involved in metabolism and cell proliferation. Up-regulation of mitochondrial transcripts involved in respiration and the presence of transcripts homologous to enzymes involved in the tri-carboxylic acid cycle indicated that aerobic respiration was likely the preferred method of ATP production in O. floccosum blastospores. However, the putative identification of genes encoding alcohol dehydrogenases within O. floccosum ESTs and the presence of homologues in other Ophiostoma species would suggest that these Ophiostoma species are also likely to be capable of metabolic functioning under anaerobic conditions.
To identify homologous genes between Ophiostoma species, the O. floccosum EST data set was compared to 20,783 ESTs from other Ophiostoma species including O. piliferum, O. novo-ulmi, O. clavigerum and O. piceae. All UPTs identified within each of the datasets were aligned resulting in the identification of 347 clusters containing EST sequences from more than one Ophiostoma species. Six were identified that had homologues in all of the datasets excluding O. piceae. Three of the six homologous UPTs were predicted to function in core metabolism with two of the UPTs identified as encoding enzymes used in the glycolysis pathway and one encoding a 60S ribosomal protein. The other three homologous UPTs were thought to have a functional role in protein fate and were putatively identified as being a superoxide dismutase, heat-shock protein and a structural alpha-B chain tubulin gene. Of the 347 clusters, 86 of these contained transcripts identified in the O. floccosum EST datasets, and of these 86, only 10 fragments did not align with any significant homology to other fungal sequences contained in the NCBI non redundant database, indicating that the majority these transcripts are conserved in other fungal species.
Predicted genes within the Ophiostoma EST datasets were also investigated to determine codon usage and to identify the presence of genes predicted to encode proteases. Both are important factors in recombinant protein expression. Protease production can severely inhibit the production of recombinant protein in fungal hosts. Based on sequence homology to known proteases, putative proteases were identified in all of the Ophiostoma species investigated with the exception of O. piceae. Homologues for all six peptidase groups were identified including a possible glutamic acid protease and proportionally high numbers of serine and metallo-protease homologues. This research constitutes the first reported findings of putative peptidases in the aspartic, cysteine, glutamic and threonine peptidase families in Ophiostoma species.
Key to the over-expression of recombinant proteins is the optimisation of codons in a cloned gene to better utilise available tRNA species within the recombinant host. No codon bias was apparent between up-regulated and lower frequency transcripts in O. floccosum, O. piliferum, O. clavigerum and O. novo-ulmi. Codon usage was found to be consistent between these Ophiostoma species. However, a large difference between the codon usage in mitochondrially encoded genes compared to nuclear encoded genes in O. floccosum was indicated.
To optimise the efficiency of a recombinant expression system, we sought to identify promoters in both O. floccosum and O. piliferum that may be applied to a vector system. Using EST data, the most up-regulated UPTs identified from O. floccosum and O. piliferum ESTs were a putative subunit 4 of the NADH-ubiquinone oxidoreductase protein (NADH-UR4) and a possible heat-shock protein (HSP), respectively. A unique hydrolase gene was also identified by molecular probing of O. floccosum genomic DNA. This putative 96 kd protein, called PLIP-Lg, was predicted to be a mitochondrial A1 phospholipase based on both nucleotide and predicted amino acid sequence structure and homology. These gene sequences were investigated using genome walking methods to further elucidate nucleotide sequences in the 5' and 3' directions. In silico investigation of the 5' promoter region of the genes identified a number of predicted transcription factor binding sites, including possible TATA boxes identified previously in the promoter region of an O. floccosum protein. Additionally, RT-PCR methods were used to compare the expression of these transcripts throughout growth in both the mycelial and blastospore forms. All three predicted genes were found to be transcribed throughout growth in both morphological forms and, thus, the use of their promoters in a vector system would not be limited to one morphology. However, the level of expression in blastospores compared to mycelial growth varied by up to 20 fold. Therefore, the morphological form of the fungi did influence the level of expression of these genes and is a factor for consideration for future promoter use.
This PhD thesis research provides the first comprehensive investigation into gene expression and the transcriptome of O. floccosum while also providing the first comparative look into similarities between the transcriptomes of several Ophiostoma species. Subsequently, this research adds to the knowledge of metabolic functioning in Ophiostoma species and illustrates the usefulness of EST analysis in determining core molecular functioning within this group. Further to addressing these goals, the research will augment future research into various biotechnological applications for the genus, specifically the development of O. floccosum and O. piliferum as hosts for recombinant protein expression.||en_NZ