Transcription of the genes for sulfur assimilation and methionine biosynthesis in Saccharomyces cerevisiae is regulated by the size of the intracellular pool of an organic sulfur compound. The identity of this compound is not clear, but suggestions include S-adenosylmethionine (SAM) and cysteine. By studying the repression of selected sulfur assimilation (MET) genes, we found that the ability to form cysteine from homocysteine is crucial for methionine-mediated repression to take place. The transcription of MET14 and MET25 could not be repressed by methionine in strains in which either STR4 (which encodes cystathionine beta-synthase) or STR1 (cystathionine gamma-lyase) was disrupted, whereas the repression was independent of GSH1 (which encodes the enzyme responsible for the first step in glutathione biosynthesis from cysteine). In contrast, cysteine could repress the MET genes in all of these strains. Two genes that presumably encode cystathionine gamma-synthase and cystathionine beta-lyase were identified by genetic disruption (ORFs YJR130c and YGL184c), yielding yeast strains that cannot convert cysteine into homocysteine. Repression by cysteine was possible in either disruptant, suggesting a role in repression for cysteine alone. While some repression of MET genes could be accomplished by homocysteine in a strain that cannot form SAM from methionine, a low intracellular level of SAM seems to be necessary for full cysteine-mediated repression to take place.

BackgroundThe 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.ResultsIn 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%.ConclusionsThe 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.

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.

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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.