The Aspergillus nidulans Zn(II) 2 Cys 6 transcription factor AN5673/RhaR mediates L-rhamnose utilization and the production of ¿-L-rhamnosidases

Abstract

Background: Various plant-derived substrates contain L-rhamnose that can be assimilated by many fungi and its liberation is catalyzed by α-L-rhamnosidases. Initial data obtained in our laboratory focussing on two Aspergillus nidulans α-L-rhamnosidase genes (rhaA and rhaE) showed α-L-rhamnosidase production to be tightly controlled at the level of transcription by the carbon source available. Whilst induction is effected by L-rhamnose, unlike many other glycosyl hydrolase genes repression by glucose and other carbon sources occurs in a manner independent of CreA. To date regulatory genes affecting L-rhamnose utilization and the production of enzymes that yield L-rhamnose as a product have not been identified in A. nidulans. The purpose of the present study is to characterize the corresponding α-L-rhamnosidase transactivator. Results: In this study we have identified the rhaR gene in A. nidulans and Neurospora crassa (AN5673, NCU9033) encoding a putative Zn(II) 2 Cys 6 DNA-binding protein. Genetic evidence indicates that its product acts in a positive manner to induce transcription of the A. nidulans L-rhamnose regulon. rhaR-deleted mutants showed reduced ability to induce expression of the α-L-rhamnosidase genes rhaA and rhaE and concomitant reduction in α-L-rhamnosidase production. The rhaR deletion phenotype also results in a significant reduction in growth on L-rhamnose that correlates with reduced expression of the L-rhamnonate dehydratase catabolic gene lraC (AN5672). Gel mobility shift assays revealed RhaR to be a DNA binding protein recognizing a partially symmetrical CGG-X 11 -CCG sequence within the rhaA promoter. Expression of rhaR alone is insufficient for induction since its mRNA accumulates even in the absence of L-rhamnose, therefore the presence of both functional RhaR and L-rhamnose are absolutely required. In N. crassa, deletion of rhaR also impairs growth on L-rhamnose.

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

“…Pardo and Orejas [43] showed by EMSA analysis that the PDR-1 homolog RhaR has a palindromic core-binding motif of a CGG triplet on both sides of an 11 nucleotide long spacer region (CGG-X 11 -CCG). This DNA-binding site is typical for Gal4p-like TFs, which bind their recognition site as a homodimer [44, 45].…”

“…PDR-1 was found to be orthologous to the Zn(II) 2 Cys 6 binuclear cluster TF TRC1 originally identified in P. stipitis [28]. The TRC1-like TFs are highly conserved in Ascomycetes and even several lineages of Basidiomycetes [43]. Functional information regarding this family is limited to TRC1 and its ortholog RhaR from the Aspergilli (Eurotiomycetes) [28, 29, 43].…”

“…The TRC1-like TFs are highly conserved in Ascomycetes and even several lineages of Basidiomycetes [43]. Functional information regarding this family is limited to TRC1 and its ortholog RhaR from the Aspergilli (Eurotiomycetes) [28, 29, 43]. In these organisms, TRC1/RhaR seem to be exclusively regulating the degradation of the pectic sub-domain RG-I or l -Rha catabolism, respectively.…”

Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus Aspergillus niger

“…The activation of structural genes involved in L-rhamnose utilization by A . niger and other fungi have been shown to require the presence of an inducing carbon source which can be either L-rhamnose or pectic polysaccharides [16,18,51]. Assuming that the above also holds for L-rhamnose specific transporters, RhtA was selected by applying stringent differential protein expression criteria.…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…The activation of structural genes involved in L-rhamnose utilization by A. niger and other fungi have been shown to require the presence of an inducing carbon source which can be either L-rhamnose or pectic polysaccharides (Gruben et al, 2014;Koike et al, 2013;Pardo and Orejas, 2014). Assuming that the above also holds for L-rhamnose specific transporters, RhtA was selected by applying stringent differential protein expression criteria.…”