Downloaded from www.microbiologyresearch.org by IP: 54.224.87.118 On: Wed, 08 Jun 2016 11:27:53 D-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates Levente Karaffa, 1 Erzse ´ bet Fekete, 1 Christian Gamauf, 2 Attila Szentirmai, 1 Christian P. Kubicek 2 and Bernhard Seiboth 2 Correspondence Bernhard Seiboth bseiboth@mail.zserv.tuwien.ac.at 1 Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, H-4010, PO Box 63, Debrecen, Hungary 2 Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/1665, A-1060 Wien, Austria Received 27 November 2005 Revised 31 January 2006 Accepted 6 February 2006 Lactose (1,4-O-b-D-galactopyranosyl-D-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei ). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on D-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that D-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with D-galactose, lactose, galactitol and D-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that D-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0?015 h ”1 ) but not at higher dilution rates. At the same dilution rate, growth on D-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on D-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0?015 h ”1 than D-galactose and induced cellulases even at growth rates up to 0?042 h ”1 . Results of chemostats with an equimolar mixture of D-galactose and D-glucose essentially mimicked the behaviour on D-galactose alone, whereas an equimolar mixture of D-galactose and galactitol, the first intermediate of a recently described second pathway of D-galactose catabolism, led to cellulase induction at D=0?030 h ”1 . It is concluded that D-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified. INTRODUCTION The pantropical ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) is used industrially to produce various extracellular enzymes including cellulases and hemicellu- lases. The major extracellular protein produced is cellobio- hydrolase I (Cel7A) and therefore its promoter is also used to drive heterologous protein production (Penttila ¨, 1998). Cellulases are usually formed on insoluble cellulose- containing materials, but their formation can also be induced by several mono- and disaccharides such as L-sorbose, sophorose and lactose (Aro et al., 2005). Lactose (1,4-O-b- D-galactopyranosyl-D-glucose) is a rather unusual inducer for cellulases as it is not expected to occur in the natural environment of H. jecorina. However, as a by-product of the dairy industry it represents an attractive renewable carbon source for industrial enzyme production with H. jecorina, but its slow metabolism and the fact that cellulase yields are lower than on cellulose still limit its use (Andreotti et al., 1980). Elucidating the mechanism by which lactose induces cellulase formation would therefore be helpful to improve its industrial use. Lactose metabolism in H. jecorina is initiated by extra- cellular hydrolysis by b-galactosidases (Seiboth et al., 2005). The resulting D-glucose enters the glycolytic pathway directly whereas the D-galactose moiety can either be phos- phorylated by galactokinase to galactose 1-phosphate and enter the Leloir pathway (Frey, 1996) or be reduced by an Abbreviations: cbh1, Cel7A (cellobiohydrolase I)-encoding gene; cbh2, Cel6A (cellobiohydrolase II)-encoding gene; cre1, Cre1 (carbon catabolite repressor)-encoding gene; D, dilution rate. 0002-8719 G 2006 SGM Printed in Great Britain 1507 Microbiology (2006), 152, 1507–1514 DOI 10.1099/mic.0.28719-0