Markku Saloheimo

Improvement of Foreign-Protein Production in Aspergillus niger var. awamori by Constitutive Induction of the Unfolded-Protein Response

ABSTRACT Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

Characterization of Secretory Genes ypt1/yptA and nsf1/nsfA from Two Filamentous Fungi: Induction of Secretory Pathway Genes of Trichoderma reesei under Secretion Stress Conditions

ABSTRACT Two genes involved in protein secretion, encoding the Rab protein YPT1/YPTA and the general fusion factor NSFI/NSFA, were characterized from two filamentous fungi, Trichoderma reesei and Aspergillus niger var. awamori. The isolated genes showed a high level of conservation with their Saccharomyces cerevisiae and mammalian counterparts, and T. reesei ypt1 was shown to complement yeast Ypt1p depletion. The transcriptional regulation of the T. reesei ypt1, nsf1, and sar1 genes, involved in protein trafficking, was studied with mycelia treated with the folding inhibitor dithiothreitol (DTT) and with brefeldin A, which inhibits membrane traffic between the endoplasmic reticulum and Golgi complex. The well-known inducer of the yeast and T. reesei unfolded protein response (UPR), DTT, induced the nsf1 gene and the protein disulfide isomerase gene, pdi1, in both of the experiments, and sar1 mRNA increased in only one experiment under strong UPR induction. The ypt1 mRNA did not show a clear increase during DTT treatment. Brefeldin A strongly induced pdi1 and all of the intracellular trafficking genes studied. These results suggest the possibility that the whole secretory pathway of T. reesei could be induced at the transcriptional level by stress responses caused by protein accumulation in the secretory pathway.

Next generation biotherapeutic production system: The filamentous fungus Trichoderma reesei

espanolEn este trabajo se abordo el estudio quimico de la fraccion lipidica del hongo Hydnellum ferrugineum mediante tecnicas espectroscopicas y cromatograficas, en el cual fueron identificados: n-alkanos naturales (C10 – C30), esteroides con el esqueleto del ergosterol (ergosta-7-en-3-ona, ergosta-7,22-dien-3-ona, ergosta-7-en-3β-ol y ergosta-7,22-dien-3β-ol), dos triterpenos pentaciclicos (friedelina y taraxerol), acidos grasos saturados (C11, C12, C13, C14, C15, C16, C17, y C18), oleato de etilo y linoleato de etilo. Es de esperar que esta informacion quimicoespecifica aporte nuevos datos para la clasificacion quimico-taxonomica de este hongo EnglishIn this work we explored a chemical study of the lipid fraction of the fungus Hydnellum ferrugineum through spectroscopic and chromatographic techniques, in which the following were identified: natural n-alkanes (C10 – C30); steroids with an ergosterol skeleton (ergosta-7-en-3-one, ergosta-7,22-dien-3-one, ergosta-7-en-3β-ol and ergosta-7,22-dien-3β-ol); two pentacyclic triterpenes (friedeline and taraxerol); saturated fatty acids (C11, C12, C13, C14, C15, C16, C17, and C18); ethyl oleate; and ethyl linoleate. This chemical-specific information is expected to provide new data for the chemical-taxonomic classification of this fungusTwo fungi were isolated from the Yellowstone national park using Millipore filtration systems equipped with 0.22 µm membrane filters. The filters were transported back to the lab, cut into small pieces, placed on Nutrient Agar plates and incubated at 37°C for 72 hours. Single colonies of YNP6-TSU and YNP7-TSU were selected and inoculated on new PDA media to have a pure culture for characterization. Three approaches were conducted to assist the identification process. The morphological assessment was done by using the agar block technique to examine the structure of the fungi and the spores without disrupting them. It was found that YNP6-TSU has septate hyphae and the spores are inside an ascus, and YNP7-TSU has coenocytic hyphae with spores inside sporangia. To assess the effect of the temperature, two approaches were conducted. To determine the optimal temperature, three agar media PDA, SAM and V8 were used to grow the fungi in 25, 30, 37, 45 and 50oC for 10 days. To test the growth in higher temperature, PDA broth media was used to grow fungi at 55, 60, 65 and 70oC in water bath for 10 days as well. It was concluded that at 37oC and with PDA as a media, the growth of both fungi was at a higher rate, while there was no growth at 50oC andxa0higher. In the molecular process, fungi were grown on PDA for seven days and mycelia were harvested for DNA extraction, PCR and sequencing. The results of sequencing and blasting showed 100% match for YNP6-TSU to Verruconis calidifluminalis and for YNP7-TSU to Rhizopus microspores. Future studies will include the determination of the optimal PH for growth of the fungi in three broth media and the extraction and purification of the brown pigment from Verruconis calidifluminalis (YNP6-TSU).