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.

Trehalose‐6‐phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose‐6‐phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose‐6‐phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose‐6‐phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.Sugar transporters are integral plasma membrane proteins that catalyze the first rate-limiting step of glycolysis in Saccharomyces cerevisiae (11). Several strategies are used by this organism to adjust the activities of these proteins to different environmental conditions (22). One of these strategies is the irreversible inactivation of the transporters, which occurs when protein synthesis is impaired upon exhaustion of a nitrogen source in the medium (3-5, 9, 23, 27) and which results in dramatic physiological effects (20,21,23). This inactivation, known as catabolite inactivation (17), affects mainly the V max of the transporters, follows first-order kinetics, and is an energy-dependent process stimulated by fermentable substrates (3,4,9). By using polyclonal antibodies against a recombinant maltose transporter protein, it has been shown that catabolite inactivation is due to proteolysis (26). The experiments reported here attempt to establish the location and mechanism of the maltose transporter degradation. We investigated the inactivation of the maltose transporter by measuring the rate of maltose uptake with radioactive sugar as well as by determining the cellular content of the transporter with polyclonal antibodies. Possible loci investigated are the plasma membrane, which is the locus of transporter action, the cytoplasm, and the vacuole after internalization of the transporter by endocytosis. In this study, we used strains defective in the internalization step of endocytosis as well as strains that show a defect either in the ''chymotrypsin-like'' activity of the proteasome complex or in the two main vacuolar endopeptidases.

The maltose transport capacity of fermenting Succharomyces cerevisiue rapidly decreases when protein synthesis is impaired. Using polyclonal antibodies against a recombinant maltose transporter-protein we measured the cellular content of the transporter along this inactivation process. Loss of transport capacity was paralleled by a decrease of cross-reacting material which suggests degradation of the transporter. We also show that in ammonium-starved cells the half-life of the maltose transporter is 1.3 h during catabolism of glucose and > 15 h during catabolism of ethanol.

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter ؊1 , i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 ؋ 10 6 M ؊1 s ؊1 toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.