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

In Aspergillus nidulans L-rhamnose is catabolised to pyruvate and L-lactaldehyde, and the latter ultimately to L-lactate, via the non-phosphorylated pathway (LRA) encoded by the genes lraA-D, and aldA that encodes a broad substrate range aldehyde dehydrogenase (ALDH) that also functions in ethanol utilisation. LRA pathway expression requires both the pathway-specific transcriptional activator RhaR (rhaR is expressed constitutively) and the presence of L-rhamnose. The deletion of lraA severely impairs growth when L-rhamnose is the sole source of carbon and in addition it abolishes the induction of genes that respond to L-rhamnose/RhaR, indicating that an intermediate of the LRA pathway is the physiological inducer likely required to activate RhaR. The loss-of-function mutation aldA67 also has a severe negative impact on growth on L-rhamnose but, in contrast to the deletion of lraA, the expression levels of L-rhamnose/RhaR-responsive genes under inducing conditions are substantially up-regulated and the production of α-L-rhamnosidase activity is greatly increased compared to the aldA+ control. These findings are consistent with accumulation of the physiological inducer as a consequence of the loss of ALDH activity. Our observations suggest that aldA loss-of-function mutants could be biotechnologically relevant candidates for the over-production of α-L-rhamnosidase activity or the expression of heterologous genes driven by RhaR-responsive promoters.

Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers.

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.

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.

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.

Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization.

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5'-CTGGGGTCTGGGG-3' CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

Catabolism of L-rhamnose in A. nidulans proceeds via the non-phosphorylated pathway and is glucose repressed by a CreA-independent mechanism.

L-rhamnose (6-deoxy-mannose) occurs in nature mainly as a component of certain plant structural polysaccharides and bioactive metabolites but has also been found in some microorganisms and animals. The release of L-rhamnose from these substrates is catalysed by extracellular enzymes including α-L-rhamnosidases, the production of which is induced in its presence. The free sugar enters cells via specific uptake systems where it can be metabolized. Of two L-rhamnose catabolic pathways currently known in microorganisms a non-phosphorylated pathway has been identified in fungi and some bacteria but little is known of the regulatory mechanisms governing it in fungi. In this study two genes (lraA and lraB) are predicted to be involved in the catabolism of L-rhamnose, along with lraC, in the filamentous fungus Aspergillus nidulans. Transcription of all three is co-regulated with that of the genes encoding α-L-rhamnosidases, i.e. induction mediated by the L-rhamnose-responsive transcription factor RhaR and repression of induction in the presence of glucose via a CreA-independent mechanism. The participation of lraA/AN4186 (encoding L-rhamnose dehydrogenase) in L-rhamnose catabolism was revealed by the phenotypes of knock-out mutants and their complemented strains. lraA deletion negatively affects both growth on L-rhamnose and the synthesis of α-L-rhamnosidases, indicating not only the indispensability of this pathway for L-rhamnose utilization but also that a metabolite derived from this sugar is the true physiological inducer.

Recombinant expression of a GH12 ß-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.

The ß-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 ß-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

De novo production of six key grape aroma monoterpenes by a geraniol synthase-engineered S. cerevisiae wine strain.

BACKGROUND: Monoterpenes are important contributors to grape and wine aroma. Moreover, certain monoterpenes have been shown to display health benefits with antimicrobial, anti-inflammatory, anticancer or hypotensive properties amongst others. The aim of this study was to construct self-aromatizing wine yeasts to overproduce de novo these plant metabolites in wines. RESULTS: Expression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain substantially changed the terpene profile of wine produced from a non-aromatic grape variety. Under microvinification conditions, and without compromising other fermentative traits, the recombinant yeast excreted geraniol de novo at an amount (~750 µg/L) well exceeding (>10-fold) its threshold for olfactory perception and also exceeding the quantities present in wines obtained from highly aromatic Muscat grapes. Interestingly, geraniol was further metabolized by yeast enzymes to additional monoterpenes and esters: citronellol, linalool, nerol, citronellyl acetate and geranyl acetate, resulting in a total monoterpene concentration (~1,558 µg/L) 230-fold greater than that of the control. We also found that monoterpene profiles of wines derived from mixed fermentations were found to be determined by the composition of the initial yeast inocula suggesting the feasibility of producing 'à la carte' wines having predetermined monoterpene contents. CONCLUSIONS: Geraniol synthase-engineered yeasts demonstrate potential in the development of monoterpene enhanced wines.

The Aspergillus nidulans Zn(II)2Cys6 transcription factor AN5673/RhaR mediates L-rhamnose utilization and the production of α-L-rhamnosidases.

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)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 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-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 α-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 α-L-rhamnosidase production and L-rhamnose utilization in fungi and could eventually have implications in fungal pathogenesis and pectin biotechnology.

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.

BACKGROUND: 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) 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 α-L-rhamnosidase gene, (AN10277/rhaA). RESULTS: Amino acid sequence data for the oenological α-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 α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-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 α-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 α-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.

Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae.

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T(73) was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.

CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans.

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine.

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.

Regulatory elements mediating expression of xylanase genes in Fusarium oxysporum.

The role of DNA regulatory elements mediating activation of the xylanase-encoding gene xyl4 by the transcription factor XlnR in the fungal pathogen Fusarium oxysporum, was studied by in vitro and in vivo functional analysis of the xyl4 promoter. Recombinant XlnR protein specifically bound the sequence GGCTAA in electrophoretic mobility shift assays. Experiments with xyl4 promoter fusions with the lacZ reporter gene showed that the GGCTAA sequence is required for xylan-induced transcriptional activation of xyl4 in F. oxysporum. The results support a model in which the interaction between the transcriptional activator XlnR and an unknown constitutive repressor regulates xylanase gene expression in F. oxysporum.

Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an alpha-L-rhamnosidase of enological interest.

The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a beta-glucosidase, show increased content mainly of the aromatic compound linalool.

Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenase.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.