Deletion of creB in Aspergillus oryzae Increases Secreted Hydrolytic Enzyme Activity

Abstract

dAspergillus oryzae has 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 an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains 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 (RTqPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.A spergillus oryzae is a multicellular fungus that has been used for centuries for the production of Asian foods and beverages, including sake (rice wine) and shoyu (fermented soybean). Today it is also used industrially as a source of secreted enzymes, including cellulases, amylases, proteases, ␤-galactosidase, and lipase, and as a host for the production of heterologous proteins (1, 2).creB was identified in the model filamentous fungus Aspergillus nidulans in a screen for mutations that alleviate carbon catabolite repression (CCR) (3). CCR is a mechanism by which genes for the utilization of nonpreferred carbon sources are repressed in the presence of preferred carbon sources; organisms thus avoid wasting energy producing enzymes for the degradation of complex carbon sources when more readily metabolized carbon sources are available. A. nidulans creB mutants grow well and alleviate CCR of various enzymes, including acetyl-coenzyme A (acetyl-CoA) synthetase, isocitrate lyase, acetamidase, and alcohol dehydrogenase (3, 4). In addition, A. nidulans creB mutants show a pleiotropic range of phenotypes apparently unrelated to CCR, including slightly reduced conidiation, reduced growth with quinate, proline, or glucuronate as the sole carbon source, enhanced growth on acetamide or acrylamide, resistance to molybdate, hypersensitivity to acriflavine, and reduced acidification of liquid growth media (3-5). creB encodes a deubiquitinating enzyme (DUB); DUBs are cysteine proteases that specifically cleave ubiquitin from ubiqui...

Carbon Catabolite Repression in Filamentous Fungi

“…Co-immunoprecipitation experiments have shown that CreB and CreC proteins function together as a complex during repression or derepression condition. It was proved that CreB-CreC deubiquitination complex has its role in CCR as deletion of Cre2 and CreB in T. reesei and A. oryzae respectively enhanced the secretion levels of hydrolytic enzymes [ 13 , 144 ]. The CreB-CreC deubiquitination complex eradicates ubiquitin moieties from CreA and other substrates thereby modifying the protein [ 79 ].…”

“…Mutants of CCR in A. nidulans have been mapped to define the genes CreA (Cre = catabolite responsive elements), CreB , CreC and CreD [ 8 , 9 , 10 , 11 ]. In addition, other species of Aspergillus such as A. oryzae and A. flavus have also been adequately studied in relation to CCR [ 12 , 13 ].…”

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

“…Disruption of A. oryzae creB resulted in relief from CCR and increased production of ␣-amylase (23,24). We generated the creB and creD double-disruption mutant strain to investigate the involvement of creD in carbon catabolite derepression in the creB disruption mutant (see Fig.…”

Diverse Regulation of the CreA Carbon Catabolite Repressor in Aspergillus nidulans

“…The CreB-CreC DUB complex has been proposed to be involved in CCR because mutations in CreB and CreC alleviate CCR (Hynes and Kelly 1977). Deletion of creB and cre2 in A. oryzae and T. reesei, respectively, resulted in elevated levels of secreted hydrolytic enzymes (Denton and Kelly 2011;Hunter et al 2013). Furthermore, recent studies have indicated that FbxA, a protein involved in ubiquitination of target proteins, is involved in creA messenger RNA (mRNA) accumulation (Colabardini et al 2012).…”