Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction.
Lactose is at present the only soluble carbon source which can be used economically for the production by Hypocrea jecorina (= Trichoderma reesei) of cellulases or heterologous proteins under the control of cellulase expression signals. However, the mechanism by which lactose triggers the formation of cellulases is unknown. To enhance our understanding of lactose metabolism and its relationship to cellulase formation, we have cloned and characterized the gal7 gene (for galactose-1-phosphate uridylyltransferase) of H. jecorina. The gene encodes a polypeptide of 43.8 kDa, the sequence of which exhibits a moderate level of identity (about 50%) to that of the Gal7 proteins of Saccharomyces cerevisiae and Kluyveromyces lactis, and contains an active-site signature typical for galactose-1-phosphate uridylyltransferase family 1. H. jecorina gal7 is not clustered with other genes of galactose metabolism. A single 1.7-kb transcript is synthesized constitutively during the rapid growth phase and accumulated to twice this level during incubation in the presence of D-galactose and L-arabinose and the corresponding polyols (dulcitol, arabitol). A gal7 deletion mutant, constructed by replacing the gal7 reading frame by the H. jecorina pyr4 gene, was unable to grow on D-galactose between pH 4.5 and 7.5, thus proving that in H. jecorina gal7 is essential for metabolism of D-galactose, whereas the growth rate of the mutant on lactose was only reduced by about 50%. The rate of formation of cellobiohydrolase Cel7A and the abundance of the corresponding (cbh1) transcript during growth on lactose was only slightly lower in the absence of gal7, but a significant delay in decay of the cbh1 transcript was noted during later stages of growth. The results suggest that H. jecorina uses only the Leloir pathway for metabolism of D-galactose and lactose. Furthermore, we conclude that metabolism of lactose past the galactose-1-phosphate step is not essential for cellulase formation.
Seiboth
B
B
Bereich Mikrobielle Biochemie und Gentechnologie, Institut für Biochemische Technologie und Mikrobiologie, Technische Universität Wien, Getreidemarkt 9, A-1060 Vienna, Austria. bseiboth@mail.zserv.tuwien.ac.at
Hofmann
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G
Kubicek
C P
CP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis*; D003001:Cloning, Molecular; D017931:DNA Primers; D005690:Galactose / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007785:Lactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014242:Trichoderma / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D002480:Cellulase; D007785:Lactose; D005690:Galactose