Triggering of cellulase biosynthesis by cellulose in Trichoderma reesei. Involvement of a constitutive, sophorose-inducible, glucose-inhibited beta-diglucoside permease.

We prepared [U-14C]cellobiose by cultivating Acetobacter pasteurianus in the presence of [U-14C]glucose and hydrolyzing the [U-14C]cellulose formed with beta-glucosidase-free cellulase from Trichoderma reesei. This 14C-labeled cellobiose was used to investigate the presence of an uptake system for cellobiose in T. reesei. Evidence was obtained for the presence of a high affinity (Km for cellobiose 0.3 microM) but low activity (2.5 milliunits/mg fungal dry weight) cellobiose permease. The permease is formed constitutively, but higher levels are formed after addition of sophorose (glucosyl-beta-1,2-diglucoside), a reputed cellulase inducer. The permease appears to be specific for beta-diglucosides, as the uptake of [U-14C]cellobiose is inhibited by sophorose, gentiobiose (glucosyl-beta-1,3-glucoside), and cellobiose. Under these conditions, cellooligodextrines (n, 4-7; final concentration, 1 mM) are not inhibitors. Glucose, but no other monosaccharides, inhibits the permease. The hypersecretory mutant T. reesei RUT C-30 exhibits elevated permease activities, whereas in T. reesei QM 9979, a mutant strain defective in the induction of cellulases by cellulose or sophorose, strongly reduced permease activities were demonstrated. The results stress a hitherto not recognized point of control in the induction of cellulases by T. reesei at the level of uptake of cellulose oligosaccharides.

The Trichoderma cellulase regulatory puzzle: from the interior life of a secretory fungus.

Novel applications for cellulases have reinitiated interest in the regulation of production of these enzymes by the soft rot fungus Trichoderma reesei and related species. This paper reviews the current state of knowledge concerning the question "How can insoluble molecules like cellulose initiate their own breakdown by a microorganism?" The evidence available--based on biochemical as well as molecular biological approaches--favors a model in which conidial bound cellobiohydrolases carry out a first exo-exo-wise attack on the cellulose molecule. The disaccharides so formed (cellobiose, alpha-cellobiono-1,5-lactone) are then taken up by the mycelia and promote further cellulase biosynthesis. Evidence available suggests that they are further metabolized to, rather than being, the "true" inducer. Speculations on the nature of the inducer are presented. The roles of the beta-glucosidases of Trichoderma in this process are discussed. The pathway of cellulase secretion is discussed on the basis of electron microscopical as well as gene sequence information.

Subcellular compartmentation of penicillin biosynthesis in Penicillium chrysogenum. The amino acid precursors are derived from the vacuole.

The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.

Characterization of commercial Trichoderma reesei cellulase preparations by denaturing electrophoresis (SDS-PAGE) and immunostaining using monoclonal antibodies.

Fifteen different cellulase preparations from Trichoderma reesei, obtained either commercially or from pilot plants, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies against two cellobiohydrolases (CBH I, CBH II), an endoglucanase (EG I), and beta-glucosidase. The staining patterns were compared with the activities of the preparations against filter paper (FPU), carboxymethylcellulose (CMC-ase), cellobiose (beta-glucosidase), and azocasein (protease). Variable amounts of proteolytic degradation products of CBH I, CBH II, and EG I were seen in most samples, and only half of them contained intact beta-glucosidase. The degree of proteolysis did not correlate with any significant difference in the respective activities of these preparations against filter paper cellulose or carboxymethylcellulose. In more than 50% of all cases a decreased beta-glucosidase activity and the absence of intact beta-glucosidase protein in Western blots was observed in preparations displaying high proteolytic activity.

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains.

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and beta-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but - unlike in extracellular culture fluids from Trichoderma - CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Changes in the concentration of fructose 2,6-bisphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentration.

The presence of fructose 2,6-bisphosphate (Fru-2,6-P2) and phosphofructokinase 2 (PFK 2) were established in the citric-acid-producing filamentous fungus Aspergillus niger. Fru-2,6-P2 levels were around 3.0 (+/- 0.8) nmol per g dry weight during growth on sucrose, and half of this in mycelia grown on citrate as a carbon source. PFK 2 was detected with a specific activity of 150 mU/mg protein and a Km for fructose 6-phosphate of 40 microM. Induction of citric acid accumulation (acidogenesis) in A. niger by cultivation on high concentrations of sucrose, or replacement on 14% (w/v) sucrose correlated with an increase in the intracellular concentration of Fru-2,6-P2. A similar correlation was obtained when A. niger was cultivated on different carbon sources, which induced different rates of acidogenesis. The increase in Fru-2,6-P2 during transfer to 14% (w/v) sucrose was not correlated with the behaviour of mycelial concentrations of cyclic AMP, a potential regulator of Fru-2,6-P2 formation in other organisms, nor with that of Fru-6-P and ATP, the precursors of its formation. The extracellular addition of cyclic AMP and theophylline, an inhibitor of cellular cyclic AMP breakdown, increased both Fru-2,6-P2 concentration and acidogenesis in mycelia cultivated in 1% (w/v) sucrose medium. It is concluded that Fru-2,6-P2 controls citric acid accumulation by enabling increased rates of glucolysis, a prerequisite to acidogenesis.

D-amino acid oxidase from the yeast Trigonopsis variabilis.

D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme.

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.