Preparation of Mutants Resistant to Catabolite Repression ofTrichoderma reesei

Abstract

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth's cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-lO and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% AviceI alone. These results suggest that KDD-lO and DGD-16 are comparatively derepressed by glucose foe cellulase production. 2875The industrial use of cellulase for the conversion of cellulose to glucose has beep handicapped by the relatively high cost of cellulase. 1) Therefore, it seemed useful to obtain microorganisms with high cellulase producing abilities. Among cellulolytic microorganisms, T. reesei is known to be the highest producer of cellulase, and many reports have appeared on T. reesei mutants capable of hyperproduction of cellulase.2 '" 14)We also began to improve the cellulase producing ability of T. reesei KY 746, which was derived from QW 9414 by monocolony isolation, and developed a semiquantitative plate assay method for selecting fungal mutants showing hyperproduction of cellulases. 15 ) In order to lower the cost of cellulase, inexpensive carbon sources are also necessary for cellulase production. We think that molasses, extensively used in the fermentation industry, and agricultural wastes, such as t Production of Ethanol from Biomasses. Part II. whey, rice straw and bagasse, may be available for cellulase production. Glucose and other easily metabolizable sugars inhibit the formation of cellulase by the mechanism of catabolite repression. 16 ) When a large amount of molasses, which contains high concentrations of glucose and fructose, is used for cellulase production, catabolite repression will occur. To overcome these problems, cellulase mutants resistant to catabolite repression have been sought, and among those found MCG-77 (Natick)4) and C-30 (Rutgers)6) have been well investigated. When these mutants were used for cellulase· production in liquid cultures, the addition of only 1% glucose to the cellulose medium 4 ) or to the 1% cellobiose medium instead of 1%cellulose was made. 12 ) But, there has been no report on the cellulase production in a liquid culture under the conditions of a high concentration of glucose such as in the case of the addition of 3% glucose to acel-

Identification of Major Facilitator Transporters Involved in Cellulase Production during Lactose Culture ofTrichoderma reeseiPC-3-7

“…[20][21][22] This mutant was developed following several rounds of random mutagenesis. 23,24) During screening for hyper-cellulolytic strains, we found that this strain exhibited an absence of carbon catabolite repression, and that it produced high amounts of cellulases during induction by cellulose, sophorose, and the monosaccharide L-sorbose. [23][24][25] To understand the genetic basis for these phenotypes better, recently we sequenced the genome of PC-3-7 using Next Generation Sequencing (NGS), and identified a total of 154 SNPs.…”

Preparation and Application ofTrichoderma reeseiMutants with Enhancedβ-Glucosidase

“…T. reesei strain KY 746, which was selected by monocolony isolation from QM 9414 (Natick Laboratories), was used as a starting strain for mutation work. Many mutants obtained previously3, 4,12) and succeeding mutants derived from them by N-methyl-N'-nitro-N-nitrosoguanidine treatment were used. The genealogy of these mutants is shown in Fig.…”

“…We improved endo-J1-glucanase and cellobiohydrolase production in T. reesep, 4) and obtained new mutants which increased the production of J1-glucosidase. This paper describes a screening method for these mutants and an effective enzymatic saccharification of cellulose to glucose by their cellulolytic systems.…”

Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy- d -Glucose

“…Both genes share a common intergenic upstream activation sequence (UAS) region whose activation results in the coordinate expression of MALT and MALS (39). Thus, glucose plays two roles in maltose utilization: it interferes with induction of the MAL transcriptional activator by maltose and it represses the expression of the permease and the maltase (12,14,20).2-Deoxy-D-glucose (DOG), a toxic glucose analog, has frequently been used to isolate glucose-deregulated mutants (19,21,22,38,40). Mutants isolated in galactose and DOG from industrial S. cerevisiae strains ferment equimolar mixtures of glucose and galactose to ethanol rapidly and completely (2) and have altered sugar transport activity (29,32).…”

“…2-Deoxy-D-glucose (DOG), a toxic glucose analog, has frequently been used to isolate glucose-deregulated mutants (19,21,22,38,40). Mutants isolated in galactose and DOG from industrial S. cerevisiae strains ferment equimolar mixtures of glucose and galactose to ethanol rapidly and completely (2) and have altered sugar transport activity (29,32).…”