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L-rhamnose induction of Aspergillus nidulans ?-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

ABSTRACT: BACKGROUND: Little is known about the structure and regulation of fungal ?-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an ?-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative ?-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second ?-L-rhamnosidase gene, (AN10277/rhaA). RESULTS: Amino acid sequence data for the oenological ?-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic ?-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other ?-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression. CONCLUSIONS: The A. nidulans rhaE gene encodes an ?-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans ?-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.

SUBMITTER: Tamayo-Ramos JA

PROVIDER: S-EPMC3312857 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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L-rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

Tamayo-Ramos Juan A JA Flipphi Michel M Pardo Ester E Manzanares Paloma P Orejas Margarita M

Microbial cell factories 20120221

<h4>Background</h4>Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encod ...[more]

PMID: 22353731

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Project description:In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Project description:The study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. Most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. Pectin is another important constituent of plant cell walls, but has received less attention. In relation to the uptake of pectic building blocks, fungal transporters for the uptake of galacturonic acid recently have been reported in Aspergillus niger and Neurospora crassa. However, not a single L-rhamnose (6-deoxy-L-mannose) transporter has been identified yet in fungi or in other eukaryotic organisms. L-rhamnose is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but is also found in diverse plant secondary metabolites (e.g. anthocyanins, flavonoids and triterpenoids), in the green seaweed sulfated polysaccharide ulvan, and in glycan structures from viruses and bacteria. Here, a comparative plasmalemma proteomic analysis was used to identify candidate L-rhamnose transporters in A. niger. Further analysis was focused on protein ID 1119135 (RhtA) (JGI A. niger ATCC 1015 genome database). RhtA was classified as a Family 7 Fucose: H+ Symporter (FHS) within the Major Facilitator Superfamily. Family 7 currently includes exclusively bacterial transporters able to use different sugars. Strong indications for its role in L-rhamnose transport were obtained by functional complementation of the Saccharomyces cerevisiae EBY.VW.4000 strain in growth studies with a range of potential substrates. Biochemical analysis using L-[3H(G)]-rhamnose confirmed that RhtA is a L-rhamnose transporter. The RhtA gene is located in tandem with a hypothetical alpha-L-rhamnosidase gene (rhaB). Transcriptional analysis of rhtA and rhaB confirmed that both genes have a coordinated expression, being strongly and specifically induced by L-rhamnose, and controlled by RhaR, a transcriptional regulator involved in the release and catabolism of the methyl-pentose. RhtA is the first eukaryotic L-rhamnose transporter identified and functionally validated to date.

Project description:BACKGROUND: Various plant-derived substrates contain L-rhamnose that can be assimilated by many fungi and its liberation is catalyzed by ?-L-rhamnosidases. Initial data obtained in our laboratory focussing on two Aspergillus nidulans ?-L-rhamnosidase genes (rhaA and rhaE) showed ?-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 ?-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)2Cys6 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 ?-L-rhamnosidase genes rhaA and rhaE and concomitant reduction in ?-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-X11-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. CONCLUSIONS: To define key elements of the L-rhamnose regulatory circuit, we characterized a DNA-binding Zn(II)2Cys6 transcription factor (RhaR) that regulates L-rhamnose induction of ?-L-rhamnosidases and the pathway for its catabolism in A. nidulans, thus extending the list of fungal regulators of genes encoding plant cell wall polysaccharide degrading enzymes. These findings can be expected to provide valuable information for modulating ?-L-rhamnosidase production and L-rhamnose utilization in fungi and could eventually have implications in fungal pathogenesis and pectin biotechnology.

Dataset Information

L-rhamnose induction of Aspergillus nidulans ?-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

Publications

L-rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

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