Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens.

Penicillium decumbens is an important industrial filamentous fungus and has been widely used in biorefinery due to its high production of cellulase and hemicellulase. However, molecular engineering has still rarely been applied for strain improvement in P. decumbens. It has been proven that gene targeting manipulation in many filamentous fungi is hampered by nonhomologous end-joining (NHEJ) pathway. To improve gene targeting efficiency in P. decumbens, the putative pku70 encoding the Ku70 homologue involved in the NHEJ pathway was identified and deleted. The Deltapku70 strain showed no apparent defect in vegetative growth, conidiation, and cellulase production, and displayed similar sensitivity to chemical agents of hygromycin B, ethyl methane sulfonate, and H2O2 at different concentrations compared with the wild-type strain. The effect of the absence of pku70 on gene targeting was tested by disruption of creA encoding a putative carbon catabolite repressor and xlnR encoding a putative transcriptional activator. Efficiency of gene targeting for both genes was 100% in the Deltapku70 strain, compared with the low efficiency in the wild-type recipient. Furthermore, the integration types for three single targeting cassettes and the cotransformation of two independent targeting cassettes were primarily investigated in P. decumbens. The highly efficient gene targeting system established in this study will open the way to large-scale functional genomic analysis in P. decumbens and contribute to the study of the mechanism of lignocellulose degradation by P. decumbens.

Identification and characterization of a novel gene, TrCCD1, and its possible function in hyphal growth and conidiospore development of Trichoderma reesei.

To investigate genes with essential functions during hyphal growth or sporulation in the asexual filamentous fungus Trichoderma reesei, we screened a collection of T-DNA insertion mutants and identified the genomic integration events. Two mutants with abnormal phenotypes, named as ccdO and ccdP, were found to have independent T-DNA insertions into a putative TrCCD1 gene locus, the product of which has significant homology to carotenoid cleavage dioxygenases (CCDs). Compared to the parental strain, both mutants tended to produce slow-growing hyphae and had a more than 50% reduction in colony growth rate. Simultaneously, the hyphae of the growing mutants formed wilting tip while the parental strain elongated straightly. To the effect of the TrCCD1 mutation on the conidiospore development, less spores were formed in the mutants than in the parental strain. In addition, disruption of TrCCD1 resulted in another phenotype characterized by a remarkable enhancement in the total carotenoid content. When the wild-type TrCCD1 gene was reintroduced into the ccd mutants, the abnormal phenotypes were rescued. These results suggest that TrCCD1 is involved in carotenoid metabolism and likely required for hyphal growth and conidiospore development in filamentous fungi T. reesei.

Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis.

Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.