Lactose regulation of cellulase gene transcription

Lactose, the principal carbohydrate present of whey, a byproduct of cheese manufacture, is a renewable and cheap carbon source for several industrial fermentations. For Hypocrea jecorina (anamorph Trichoderma reesei) it is virtually the only soluble carbon source for economic production of cellulases or heterologous proteins under cellulase promoters. Unfortunately, the use of lactose is limited by slower growth rates of the fungus and by lower cellulase yields. Improvements are to be expected once the limiting reactions in lactose metabolism and the mechanism of promotion of cellulase formation are understood. Towards this goal, we have already obtained evidence that (a) the galactose moiety of lactose is catabolized via the Leloir pathway, (b) that an impairment of the galactokinase reaction strongly reduces stimulation of cellulase gene expression by lactose, and (c) that the induction of cellulase gene expression by lactose works via a different mechanism than induction by cellulose. In this project, we consequently aim at an identification of the nucleotide motifs in the cellulase promoter, which specify induction by lactose, and cloning of the gene(s) encoding the respective DNA-binding protein(s). The cbh2 (cellobiohydrolase II) promoter - rather than the cbh1 promoter - shall be used as a target of investigation, as it has a less complex architecture as the cbh1 promoter, and we have all components for turning-on in hand. In addition, the role of the galactokinase step in then induction by lactose shall be investigated in detail as this could be a limiting step in cellulase formation on lactose.

The filamentous fungus H. jecorina is a potent producer of enzymes and recombinant proteins on a technical scale. Industrial strains of H. jecorina are able to produce up to 100 grams per litre of cellulases which are used by various industries. The disaccharide lactose induces cellulase formation in H. jecorina and accumulates as a cheap and renewable by-product of the dairy industry. It is therefore used for enzyme and recombinant protein production. We have therefore started a systematic approach to identify the main enzymes and regulators of the lactose pathway with the aim of producing tailor-made strains for industrial enzyme production. Lactose and D-galactose degradation was extensively studied in the two yeasts Saccharomyces cerevisiae and Kluyveromyces lactis. The GAL(LAC) regulon has become a paradigm for transcriptional control in lower eukaryotes, and a model system for gene regulation. Our results illustrate that the regulatory system may be restricted to yeasts and it seems that it is not representative for fungi in general. In H. jecorina lactose is cleaved by an extracellular ß-galactosidase into its two monomers D-glucose and D-galactose. D-glucose is degraded via the glycolytic pathway while D-Galactose is either degraded by the classical Leloir pathway or a second novel pathway discovered in this study. The second novel pathway for D-galactose which we discovered in this project is composed of different enzymes of the L-arabinose and D-xylose catabolic pathway. In filamentous fungi the Leloir pathway genes are in general non-clustered and one of its components lacks the important aldose 1-epimerase domain. The Leloir pathway genes are constitutively expressed during growth, induced not only by D-galactose but also by L-arabinose. Galactokinase, the first enzyme of the D-galactose pathway, is in yeast a bifunctional protein responsible for D-galactose phosphorylation and transcriptional induction of the Leloir genes. In H. jecorina the galactokinase is dispensable for the induction of the Leloir pathway genes. This and the fact that in filamentous fungi no orthologues of transcriptional activator GAL4/LAC9 could be detected support our findings that the Leloir pathway is controlled in a different way. The transcriptional activator for cellulase induction is not related to the yeasts Gal4/Lac9 activators. Finally, cellulase induction during growth on lactose depends on the action of the first enzymes of both D-galactose degrading pathways and is regulated via a low level of ß-galactosidase.