A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).

BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering. RESULTS: In this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the T. reesei genome ensuring that enzyme production from different transformants can be directly compared. The ade2 locus was selected as integration site since ade2 mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a tku70 disruption to increase gene targeting efficiency and a new bidirectional marker, pyr2, for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%. CONCLUSIONS: The new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of T. reesei and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with ade2 as integration site was confirmed by expression of a Thermomyces lanuginosus lipase.

Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system.

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.

Differential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid.

The allopolyploid yeast Saccharomyces carlsbergensis appears to be a relatively newly formed species hybrid, and therefore constitutes a good model for studying early steps in hybrid speciation. Using reverse transcription-coupled polymerase chain reaction to monitor derepression of the S. carlsbergensis homologues of the sulfur assimilation genes MET14 and MET2, we found that both homologues of these genes are regulated in the same pathway-specific manner, but surprisingly, with different kinetics, as the genes derived from one of the parent species (the non-Saccharomyces cerevisiae-like) are alleviated from repression much faster than the genes from the other parent (the S. cerevisiae-like). This probably reflects differing physiological adaptation of the parent species, and the finding may contribute to the general understanding of hybrid speciation.