Identification of the genes encoding the catalytic steps corresponding to LRA4 (l‐2‐keto‐3‐deoxyrhamnonate aldolase) and l‐lactaldehyde dehydrogenase in Aspergillus nidulans: evidence for involvement of the loci AN9425/lraD and AN0544/aldA in the l‐rhamnose catabolic pathway

Abstract

Summary l‐rhamnose is found in nature mainly as a component of structural plant polysaccharides and can be used as a carbon source by certain microorganisms. Catabolism of this sugar in bacteria, archaea and fungi occurs by two routes involving either phosphorylated or non‐phosphorylated intermediates. Unlike the corresponding pathway in yeasts, the metabolic details of the non‐phosphorylated pathway in filamentous fungi are not fully defined. The first three genes (lraA, lraB and lraC) of the non‐phosphorylated pathway in Aspergillus nidulans have recently been studied revealing dependence on lraA function for growth on l‐rhamnose and α‐l‐rhamnosidase production. In the present work, two genes encoding the subsequent steps catalysed by l‐2‐keto‐3‐deoxyrhamnonate (l‐KDR) aldolase (AN9425) and l‐lactaldehyde dehydrogenase (AN0554) are identified. Loss‐of‐function mutations cause adverse growth effects on l‐rhamnose. Akin to genes lraA‐C and those encoding rhamnosidases (rhaA, rhaE), their expression is induced on l‐rhamnose via the transcriptional activator RhaR. Interestingly, the aldolase belongs to the ftablamily of bacterial l‐KDR aldolases (PF03328/COG3836) and not that of yeasts (PF00701/COG0329). In addition, AN0554 corresponds to the previously characterized aldA gene (encodes aldehyde dehydrogenase involved in ethanol utilization) thus revealing a previously unknown role for this gene in the catabolism of l‐rhamnose.

The Loss-of-Function Mutation aldA67 Leads to Enhanced α-L-Rhamnosidase Production by Aspergillus nidulans

“…Using a genetic approach, we recently demonstrated a novel and crucial role for the alc regulon gene aldA in the LRA pathway for L-rhamnose catabolism in which its gene product (ALDH) catalyses the conversion of L-lactaldehyde to lactate [14]. Expression of the alc genes in the presence of ethanol is mediated by the transcriptional activator AlcR, and acetaldehyde is its co-inducer.…”

“…The latter is derived from ethanol via the activity of the alcohol dehydrogenase ADHI and then converted by ALDH to acetate, which is subsequently assimilated into central metabolism as acetyl-CoA. We recently reported genetic evidence for a novel and essential role for the aldA gene product in Lrhamnose catabolism [14]-the conversion of L-lactaldehyde to lactate-and in this regard it is noteworthy that earlier biochemical studies of ALDH showed it to be an enzyme of broad rather than narrow aldehyde substrate specificity [24,29]. We also demonstrated previously that the physiological inducer of rhamnose utilisation is an intermediate of the LRA pathway [13].…”

“…The products of the lra genes yield L-pyruvate and L-lactaldehyde, and subsequently ALDH catalyses the conversion of Llactaldehyde to L-lactate (Figure 1A-see Section 3.1). Deletion of lraA resulted in the inability to grow on L-rhamnose and loss of the production of α-L-rhamnosidases, thus demonstrating not only the indispensability of this pathway for L-rhamnose utilization but also establishing that the true physiological inducer of the genes involved in L-rhamnose metabolism is a catabolite derived from this sugar and not the sugar itself [13,14]. Induction of the expression of the catabolic genes and the genes encoding the two characterised α-L-rhamnosidases (RhaA and RhaE) is mediated by the pathway-specific transcriptional activator RhaR in the presence of L-rhamnose [13][14][15][16][17][18].…”

“…Deletion of lraA resulted in the inability to grow on L-rhamnose and loss of the production of α-L-rhamnosidases, thus demonstrating not only the indispensability of this pathway for L-rhamnose utilization but also establishing that the true physiological inducer of the genes involved in L-rhamnose metabolism is a catabolite derived from this sugar and not the sugar itself [13,14]. Induction of the expression of the catabolic genes and the genes encoding the two characterised α-L-rhamnosidases (RhaA and RhaE) is mediated by the pathway-specific transcriptional activator RhaR in the presence of L-rhamnose [13][14][15][16][17][18]. The observation that rhaR is expressed in the absence of L-rhamnose [18] suggests that the physiological inducer activates extant RhaR, a situation not dissimilar to that seen for xylanase gene induction in which the presence of xylose is required for transcriptional activation mediated by XlnR [19].…”

“…Our studies in A. nidulans concerning the metabolic fate of the L-lactaldehyde generated from the reaction catalysed by LraD provided strong evidence for the involvement of an L-lactaldehyde dehydrogenase (LADH) activity that is encoded by the well-studied ethanol/alc-regulon gene aldA (AN0554; aldehyde dehydrogenase-ALDH, [23]). RhaRdependent induction of aldA was observed when L-rhamnose was present as the sole source of carbon [14], and growth of an aldA67 loss-of-function mutant [24,25] on rhamnose was seen to be very severely impaired compared to that of the wild type, whereas growth of both strains on glucose was identical. In the present study, we have examined the molecular background of the crucial role played by aldA in L-rhamnose metabolism and its potential biotechnological relevance.…”