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The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene crel. In this study, strains were constructed in which the crel gene was either completely removed or replaced by a truncated mutant variant, crel-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of crel had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures~ in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that crel; acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing cónditions. There was no phenotypic difference between the &rel and crel-1 mutant strains in any of the experiments done, indicating that the crel-1 gene is practically a null allele. The results of this work indicate that crel is a valid target gene in strain engineering for improved enzyme production in T. reesei. [ABSTRACT FROM AUTHOR]

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderrnã reesei under the control of an inducible cbhl promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenyl- butyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter-1 purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters ofp-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin. [ABSTRACT FROM AUTHOR]