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.

Summary The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol‐producing host plant, but Bclcc2 replacement mutants were as virulent as the wild‐type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant–pathogen interactions and the phytoalexin concept.

The Wlamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of -amylase, glucoamylase and -glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions.We have identiWed 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identiWed enzymes, a putative -glucosidase (AgdB) and an -amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identiWed was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative -glucan modifying processes are discussed.

Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the ␣-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel ␣-glucanotransferase enzyme with disproportionating (cleaving ␣134 and synthesizing ␣136 and ␣134 glycosidic linkages) and ␣136 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-␣-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional 1 H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more ␣136 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (␤/␣) 8 barrel that use sucrose to synthesize ␣-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 ␣-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of ␣136 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.Glucansucrase (GS) (or glucosyltransferase [GTF]) enzymes (EC 2.4.1.5) of lactic acid bacteria (LAB) use sucrose to synthesize a diversity of ␣-glucans with ␣136 (dextran; found mainly in Leuconostoc), ␣133 (mutan; found mainly in Streptococcus), alternating ␣133 and ␣136 (alternan; reported only in Leuconostoc mesenteroides), and ␣134 (reuteran; synthesized by GTFA and GTFO from Lactobacillus reuteri strains) glycosidic bonds (1,14,16,23,34).The first glycoside hydrolase 70 (GH70) family 3-dimensional (3D) structures, recently elucidated (9, 38), showed that the catalytic domains of GS enzymes possess a (␤/␣) 8 barrel structure similar to that of members of the GH13 family, confirming earlier secondary-structure predictions (4, 21). The core of the proteins belonging to the GH13 family comprises 8 ␤-sheets alternated with 8 ␣-helices. In GS enzymes, however, this (␤/␣) 8 barrel structure is circularly permuted (21). Also, the four conserved regions (regions I to IV) identified in members of the ␣-amylase family GH13 (31) are present in glucansucrases. However, as a consequence of the circular per...

c 4,6-␣-Glucanotransferase (4,6-␣-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some ␣1¡4 linkages but mostly (relatively long) linear chains with ␣1¡6 linkages and are soluble dietary starch fibers. 4,6-␣-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-⌬N (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-␣-GTase enzymes. The data show that GTFB-⌬N is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-⌬N were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Starch is the second-most-abundant carbohydrate on earth and a major dietary carbohydrate for humans; as a storage carbohydrate it is present in seeds, roots, and tubers of plants (1). It consists of ␣-glucan polymers with ␣1¡4 linkages and a low percentage of ␣1¡6 linkages, in the form of amylose and branched amylopectin (2). Starches are applied in various industrial products such as food, paper, and textiles, often after processing by physical, chemical, or enzymatic treatment (3-6).Dietary fibers and low-glycemic-index (low-GI) food are considered healthy food contributing to our long-term well-being (7,8). Of all the nutritional types of starch, slowly digestible starch with low GI has drawn the strongest interest. Annealing/heatmoisture treatment, recrystallization, and enzymatic treatment are recognized approaches to obtain slowly digestible starch (9-11). Slowly digestible starch materials prepared by physical processing suffer losses upon boiling; therefore, structural modifications through enzymatic treatment of starch are more desirable. In the human digestive system, the ␣1¡6 linkages in starch are hydrolyzed at a lower rate than ␣1¡4 linkages (12, 13). Branching enzymes, alone or in combination with ␤-amylase, are used to increase the percentage of ␣1¡4,6 branches in starches (12)(13)(14).The 4,6-␣-glucanotransferase (4,6-␣-GTase) enzymes, such as GTFB, GTFW, and GTFML4, of Lactobacillus reuteri strains constitute a subfamily of glycoside hydrolase family 70 (GH70); GH70 ...