The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

SummaryFive Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V max,app ranged from 10 to 900 U mg )1 , while the K m,app ranged from 0.16 to 0.6 mg ml )1 . Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel ␤-helix with 10 complete turns. The ␤-helix is composed of four parallel ␤-sheets, and has one very small ␣-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the ␤-helix. The plant cell wall consists of a network of complex carbohydrates like cellulose, hemicellulose, and pectin. The latter is the most complex of these carbohydrates. It contains so-called "smooth regions" and "hairy regions." The smooth regions, also known as homogalacturonan, consist of ␣(1,4)-linked D-galacturonic acid residues, whereas the hairy regions, or rhamnogalacturonan I, are characterized by stretches of alternating ␣(1,2)-linked D-galacturonic acid and L-rhamnose (1). The rhamnose residues can be substituted at their O4 atoms by arabinose or galactose (2). Throughout the pectin molecule, the galacturonic acid residues can be methylated at O6 and/or acetylated at O2 and/or O3 (3). Due to its complex structure, modification of pectin by plants or complete breakdown by microorganisms requires many different enzymes.In microorganisms several classes of pectinases have been identified. These classes comprise pectate-, pectin-, and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases, and polygalacturonases, which all depolymerize the main chain; and pectin methylesterases and pectin-and rhamnogalacturonan acetylesterases, which act on the substituents of the main chain. Crystal structures are known of members of several classes of main chain depolymerizing pectinases. These include pectate lyases from Erwinia chrysanthemi and Bacillus subtilis (4 -6), pectin lyases from Aspergillus niger (7,8), and rhamnogalacturonase A from Aspergillus aculeatus (9). Recently, the crystal structure of endopolygalacturonase from the bacterium Erwinia carotovora was solved (10). The lyases cleave the substrate by ␤-elimination, whereas rhamnogalacturonases and polygalacturonases use acid/base-catalyzed hydrolysis (11,12). Despite their completely different reaction mechanisms, and their groupings in different sequence homology families, the x-ray structures of pectate lyase, pectin lyase, and rhamnogalacturonase reveal a similar unique right-handed parallel ␤-helix topology (13,14).

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The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.