Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis.

Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned, and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and ß-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.

Molecular insights into the inhibition of glutamate dehydrogenase by the dicarboxylic acid metabolites.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+ -dependent reversible conversion of α-ketoglutarate (AKG) to l-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+ -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate, and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

Environmental role of aromatic carboxylesterases.

The carboxylesterases (EC 3.1.1.x) are widely distributed and form an important yet diverse group of hydrolases catalysing the ester bond cleavage in a variety of substrates. Besides acting on plant cell wall components like cutin, tannin and feruloyl esters, they are often the first line of defence to metabolize drugs, xenobiotics, pesticides, insecticides and plastic. But for the promiscuity of some carboxylesterases and cutinases, very few enzymes act exclusively on aromatic carboxylic acid esters. Infrequent occurrence of aromatic carboxylesterases suggests that aromatic carboxylesters are inherently more difficult to hydrolyse than the regular carboxylesters because of both steric and polar effects. Naturally occurring aromatic carboxylesters were rare before the anthropogenic activity augmented their environmental presence and diversity. An appraisal of the literature shows that the hydrolysis of aromatic carboxylic esters is a uniquely difficult endeavour and hence deserves special attention. Enzymes to hydrolyse such esters are evolving rapidly in nature. Very few such enzymes are known and they often display much lower catalytic efficiencies. Obviously, the esters of aromatic carboxylic acids, including polyethylene terephthalate waste, pose an environmental challenge. In this review, we highlight the uniqueness of aromatic carboxylesters and then underscore the importance of relevant carboxylesterases.

Protein expression and secretion by filamentous fungi.

In the search for optimal platforms for protein expression and secretion, filamentous fungi in principle provide some of the best microbial cell factories. They are inherently endowed with the ability to secrete proteins. Fungi belonging to Aspergillus and Trichoderma species are well-studied for industrial production of proteins and enzymes. Our understanding of these organisms at the level of transcription, translation, post-translational processing and the secretory pathways has improved significantly in recent years. Despite this, the ability of these fungal secretion platforms has not yet been able to match their intrinsic secretion capacity to produce foreign proteins. Details of the molecular mechanisms of the secretory pathways in filamentous fungi are emerging. This knowledge can be gainfully employed to enhance protein production in filamentous fungi, particularly in the secretion of heterologous proteins of value.

High-Throughput Screening of Dye-Ligands for Chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. The choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices serve as a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose the best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lie in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

Autophagy is important to the acidogenic metabolism of Aspergillus niger.

Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvation in the A. niger Δatg1 strain. The acidogenic growth of the fungus consisted of a brief log phase followed by an extended autophagy-like state throughout the idiophase of fermentation. Mycelia in the idiophase were highly vacuolated and EGFP was localized to the vacuoles but no autolysis was observed. Both autophagy and acidogenesis are compromised in Δatg1 and Δatg8 strains of A. niger. The acidogenic growth of the fungus thus appears to mimic a condition of nutrient limitation and is associated with an extended autophagy-like state. This crucial role of autophagy in acidogenic A. niger physiology could be of value in improving citric acid fermentation.

2-Oxoglutarate cooperativity and biphasic ammonium saturation of Aspergillus niger NADP-glutamate dehydrogenase are structurally coupled.

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized. The C-terminal sequence (residues 315-460, named the D-segment) was implicated in the AnGDH cooperativity. The D-segment residues largely contribute to the monomer-monomer interface of each trimer in the native hexamer and are far removed from the enzyme active site. The D-segment appears to be a part of the allosteric network responsible for 2-oxoglutarate homotropic interactions in AnGDH. AnGDH and its C415S mutant, but not AtGDH, also showed atypical, biphasic ammonium saturation, particularly at sub-saturating 2-oxoglutarate concentrations. We found that the sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response are tightly coupled; the analysis of AnGDH-AtGDH chimeras ascribes the two features to the AnGDH D-segment. The two non-Michaelis-Menten substrate saturations of AnGDH were influenced by ionic strength. Increase in ionic strength reduced the nH of 2-oxoglutarate saturation as well as abolished the biphasic response, suggesting that polar/ionic interactions determine the allosteric, inter-subunit communications. The biochemical analysis in the context of available structural data implicates the D-segment of AnGDH in the allosteric feature of this enzyme. The coupling of sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response could possibly confer growth advantage to A. niger experiencing carbon and/or nitrogen limitation.

Characterization of 4-guanidinobutyrase from Aspergillus niger.

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn2+ enzyme from the same fungus, purified GBase protein is associated with Zn2+ ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.

Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in Pseudomonas sp. Strain C5pp.

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth's biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate Pseudomonas sp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles.IMPORTANCE The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family. Pseudomonas sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior. We determined the first high-resolution crystal structures of glutamate dehydrogenase from the fungus Aspergillus niger (AnGDH), a unique NADP+-dependent allosteric enzyme that is forward-inhibited by the formation of mixed disulfide. We determined the structures of the active enzyme in its apo form and in binary/ternary complexes with bound substrate (α-ketoglutarate), inhibitor (isophthalate), coenzyme (NADPH), or two reaction intermediates (α-iminoglutarate and 2-amino-2-hydroxyglutarate). The structure of the forward-inhibited enzyme (fiAnGDH) was also determined. The hexameric AnGDH had three open subunits at one side and three partially closed protomers at the other, a configuration not previously reported. The AnGDH hexamers having subunits with different conformations indicated that its α-ketoglutarate-dependent homotropic cooperativity follows the Monod-Wyman-Changeux (MWC) model. Moreover, the position of the water attached to Asp-154 and Gly-153 defined the previously unresolved ammonium ion-binding pocket, and the binding site for the 2'-phosphate group of the coenzyme was also better defined by our structural data. Additional structural and mutagenesis experiments identified the residues essential for coenzyme recognition. This study reveals the structural features responsible for positioning α-ketoglutarate, NADPH, ammonium ion, and the reaction intermediates in the GDH active site.

Novel Route for Agmatine Catabolism in Aspergillus niger: 4-Guanidinobutyrase Assay.

The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4-aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald's method. However, the low sensitivity of this method often renders it impractical to perform fine kinetic analysis. To overcome this limitation, a high sensitive method for detecting GABA is exploited that can even detect 1 µM of GABA in the assay mixture. The samples are deproteinized by perchloric acid (PCA) and potassium hydroxide treatment prior to HPLC analysis of GABA. The method involves a pre-column derivatization with o-phthalaldehyde (OPA) in combination with the thiol 3-mercaptopropionic acid (MPA). The fluorescent GABA derivative is then detected after reversed phase high performance liquid chromatography (RP-HPLC) using isocratic elution. The protocols described here are broadly applicable to other biological samples involving urea and GABA as metabolites.

Contribution of arginase to manganese metabolism of Aspergillus niger.

Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (ΔXCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain < parent strain < ΔXCA-29 strain. While the soluble fraction forms 60% of the total mycelial Mn[II] content, arginase accounted for a significant fraction of this soluble Mn[II] pool. Changes in the arginase levels affected the absolute mycelial Mn[II] content but not its distribution in the various mycelial fractions. The A. niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production.

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min(-1) mg(-1), using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase from Aspergillus niger.

Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study, Aspergillus niger NADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Šresolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space group R32, with unit-cell parameters a=b=173.8, c=241.5 Å, α=ß=90, γ=120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress.

Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.

High-throughput screening of dye-ligands for chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

A novel selectable marker based on Aspergillus niger arginase expression.

Selectable markers are valuable tools in transforming asexual fungi like Aspergillus niger. An arginase (agaA) expression vector and a suitable arginase-disrupted host would define a novel nutritional marker/selection for transformation. The development of such a marker was successfully achieved in two steps. The single genomic copy of A. niger arginase gene was disrupted by homologous integration of the bar marker. The agaA disruptant was subsequently complemented by transforming it with agaA expression vectors. Both citA and trpC promoters were able to drive the expression of arginase cDNA. Such agaA+ transformants displayed arginase expression pattern distinct from that of the parent strain. The results are also consistent with a single catabolic route for arginine in this fungus. A simple yet novel arginine-based selection for filamentous fungal transformation is thus described.

Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.