Deletion ofcreBin Aspergillus oryzae Increases Secreted Hydrolytic Enzyme Activity

ABSTRACTAspergillus oryzaehas been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of anAspergillus nidulansstrain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show thatAspergillus oryzaecontains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in bothAspergillus nidulansandAspergillus oryzaeare mirrored at the transcript level, indicating transcriptional regulation. We report thatAspergillus oryzaeDAR3699, originally isolated from soy fermentation, has a similar phenotype to that of acreBdeletion mutant of the RIB40 strain, and it contains a mutation in thecreBgene. Collectively, the results forAspergillus oryzae,Aspergillus nidulans,Trichoderma reesei, andPenicillium decumbensshow that deletion ofcreBmay be broadly useful in diverse fungi for increasing production of a variety of enzymes.