Developing Ophiostoma floccosum as a novel expression system

Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system. In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus. A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma.