Browsing by Subject "background activity"

Filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a crucial production organism for enzymes used in industrial applications, such as in feed, food, textile, and biofuel production, due to its ability to secrete high amounts of homologous and heterologous enzymes. Therefore, development of genetic tools to improve the properties of industrial T. reesei strains for even better production yields is essential. In this study, a polyethylene glycol mediated CRISPR-Cas9 transformation method for industrial T. reesei production strains was aimed to be optimised by testing an alternative Cas9 enzyme and varying the stoichiometry and total amount of Cas9 enzyme and single guide RNA in the ribonucleoprotein complex. Correct integration of the gene constructions in the obtained transformants was determined by colony PCR and Southern blot analysis. In addition, two selected background activity encoding genes, endoglucanase 6 and α-glucuronidase 1, were individually deleted from T. reesei xylanase production strain utilising the improved CRISPR-Cas9 transformation protocol. The effect of background activity deletions on the strain growth and protein production were analysed from culture supernatants by pH measurement, Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme activity assays. An improved CRISPR-Cas9 transformation protocol for T. reesei was successfully established basing on high number of transformants and improved DNA integration fidelity. No negative effects were observed in the growth or protein production properties of the background activity deletion strains compared to the xylanase production strain. Thus, further cleansing of T. reesei secretome can be continued to refine the industrial production strains.