Lactose induction of cellulase formation in Trichoderma

Lactose (1,4-0-ß-D-galactopyranosyl-D-glucose) is produced to around 1.2 million tons worldwide, primary as a by-product from cheese manufacture or from whey processing industries, but utilized only to a low degree and mostly not very profitable. Thus, lactose is one of the largest renewable substrates available today which is primary used as a fermentation substrate, as a filler or sweetener, and in small amounts in infant and dietetic foods, coffee whiteners and in feed or in non-food products. Lactose itself has limited application in foods mainly due to intolerance problems in part of the population. The ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) is used industrially to produce cellulolytic and hemicellulolytic enzymes, which have received considerable attention because of their potential applications in food, feed, textile, and pulp and paper industries. Several applications, such as bleaching of pulp by xylanolytic enzymes, clarification of juices with pectinases and biobleaching of textiles with cellulases are currently in use. Cellulases and hemicellulase formation can be induced by several mono- and disaccharides, e.g. sophorose, xylobiose, lactose, D-xylose, L-sorbose, most of which are too expensive for industrial fermentations. For cellulase and heterologous protein production with cellulase promoters, lactose is virtually the only carbon source that can be used. However, lactose metabolism is slow and the cellulase yields produced on lactose are lower compared to cellulose, thus warranting a deeper understanding of lactose metabolism towards its targeted improvement. Recent work in the laboratory of the proposer has revelaed that the induction of cellulase transcription by lactose requires the simultaneous degradation of the D-galactose moiety of lactose via the Leloir pathway and the alternative reductive galactose catabolic pathway. At the same time, collaborators of this proposal have identified that the intracellular concentration of oligogalactosides, consisting of galactose and galactitol, correlates with cellulase induction on lactose. Based on these findings, this project aims at testing the hypothesis that (a) galactosyltransferase(s) forms the inducer of cellulases during growth on lactose, which will be pursued by genomic, reverse genetic and metabolomic methods. The verification of this hypothesis, and the identification of respective galactosyltransferase would for the first time allow a targeted improvement of cellulase induction by lactose.

The filamentous fungus T. reesei is today a paradigm for the commercial scale production of different plant cell wall degrading enzymes mainly cellulases and hemicellulases. Its enzymes have a long history of safe use in industry and well established applications are found within the pulp, paper, food, feed or textile processing industries. However, when these enzymes are to be used for the saccharification of cellulosic plant biomass to simple sugars which can be further converted to biofuels or other biorefinery products, and thus compete with chemicals produced from fossile sources, additional efforts are needed to reduce costs and maximize yield and efficiency of the produced enzyme mixtures. One approach to this end is the use of genetic engineering to manipulate the biochemical and regulatory pathways that operate during enzyme production and control enzyme yield. In the course of this project, we have attempted to identify the inducer formed from lactose. While trisaccharides were identified whose accumulation correlated with cellulase formation, the enzymes forming them could not be found. Using a systems biology approach, we found that indeed cellulase formation occurs even if lactose cannot enter the cell which lead to a complete rethinking about the induction mechanism.