Jacques A. E. Benen

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

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.

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides.

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-GalpA-([1→4]-α-D-GalpA)3-(1→4)-D-GalpA, is composed of five galacturonopyranose units (D-GalpA) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

The Active Site Topology of Aspergillus niger Endopolygalacturonase II as Studied by Site-directed Mutagenesis*

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis inAspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K m and V max) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D‐galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L‐arabinose and D‐galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding β‐galactosidase and the L‐arabinose releasing enzymes were not only expressed in the presence of D‐galacturonic acid, but also in the presence of L‐arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D‐galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L‐rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D‐galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L‐arabinose, L‐rhamnose or ferulic acid.

The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.

The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens

The plant cell wall functions as a barrier to biotic and abiotic agents. Plant pathogenic bacteria and fungi produce cell wall degrading enzymes (CWDEs) which are believed to degrade this barrier, thereby facilitating both inter- and intracellular growth and providing nutrients to the invader. A pectate lyase from the bacterium Erwinia chrysanthemi was the first CWDE that was shown to be required for full virulence (Roeder and Colmer 1985). Subsequent molecular genetic studies have shown that many other bacterial CWDEs are virulence factors (reviewed by Hugouvieux-Cotte-Pattat et al. 1996). It took many years before similar evidence was obtained for the involvement of fungal CWDEs in pathogenesis, in spite of several efforts (reviewed by Walton 1994; Annis and Goodwin 1997). Eventually, an endopolygalacturonase from Aspergillus fiavus was shown to play a role in the invasion of cotton bolls (Shieh et al. 1997).

Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger

Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 Å resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well‐defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred.

Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties

We isolated and characterized a new type of endopolygalacturonase (PG)‐encoding gene, pgaD, from Aspergillus niger. The primary structure of PGD differs from that of other A. niger PGs by a 136 amino acid residues long N‐terminal extension. Biochemical analysis demonstrated extreme processive behavior of the enzyme on oligomers longer than five galacturonate units. Furthermore, PGD is the only A. niger PG capable of hydrolyzing di‐galacturonate. It is tentatively concluded that the enzyme is composed of four subsites. The physiological role of PGD is discussed.