Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter

Abstract

Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.

Novel pentose-regulated promoter of Aspergillus oryzae with application in controlling heterologous gene expression

“…In addition, the identified consensus binding motifs, CGGNTAAW and CGGDTAAW, in the PxyrA sequence could refer to its function that might be regulated by XlnR and AraR transcription factors via xylose and arabinose induction, respectively, as previously described [ 17 , 35 , 36 ]. In addition, the presence of SYGGRG motifs in the PxyrA sequence of A. oryzae indicated that the expression control might participate in the CREA negative regulator-mediated carbon catabolism repression, as observed in several carbon-inducible promoters, such as endoxylanase [ 16 , 17 ], sorbitol-induced [7] and taka-amylase A promoters [ 8 , 9 ].…”

“…With the accumulative knowledge on promoter structure and function, a number of inducible and constitutive promoters have been identified and developed for A. oryzae . For inducible promoters, their regulatory modes are mostly dependent on carbon sources, such as the sorbitol-sensitive ( Psor ) [7] , taka-amylase A ( PamyB ) [ 8 , 9 ] and glucoamylase ( PglaA ) promoters [10] . Although Psor promoter was strictly controllable, its ability in inducing gene expression is rather low as illustrated by green fluorescent protein (GFP) reporter.…”

“…Two maltose/malto-oligosaccharides-inducible promoters, PamyB and PglaA, have frequently been used for fungal systems [11] , [12] , [13] . However, they were not tightly related to the inducible conditions, in which some enzyme expressions under the promoter controls were still detected in the non-induction or the glucose repression conditions [ 7 , 8 , 10 ]. Moreover, it is not practical and feasible, particularly in the industrial application that requires an additional step in the production process, such as cell washing to eliminate the remained glucose repressor before expression induction [11] , [12] , [13] .…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…Transcriptome datasets are used to identify constitutive promoters in bacteria [49] and plants [50]. Oda et al [51] used transcriptome datasets collected under three different conditions to find sorbitol-inducible promoters in A. oryzae. Sibthorp et al [52] used transcriptome datasets obtained under five different culture conditions for the global identification of promoters in A. nidulans.…”

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

“…Fluorescent proteins can be expressed in fungal cells by inserting genes encoding fluorescent proteins into the genome under the control of constitutive or inducible promoters (Oda et al 2016;Pöggeler et al 2003). The green fluorescent protein GFP from the jellyfish Aequorea victoria and its engineered versions with altered spectral properties, such as yellow and cyan-shifted mutants, enhanced brightness versions or pH-sensitive variants have been utilized in fungal cell biology for many years (Bagar et al 2009;Chalfie et al 1994;Chudakov et al 2005;Lorang et al 2001;Rech et al 2007).…”