Role of the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in cellulase induction by lactose

Abstract

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.

FIG. 1.

Presence of consensus binding sites for known fungal transcription factors in the bga1 upstream noncoding region. The positions are relative to the position of the translation start codon (ATG). The positions above the lines are the sites of the consensus sequences on the coding strand, and the positions below the lines are the sites on the noncoding strand. Symbols: ⋄, Cre1; ▪, Hap2/3/5; ▿, Ace1; ○, AmyR; ★, Msn2/4.

FIG. 2.

Regulation of bga1 gene transcription during growth on various carbon sources of QM9414 (A) and RUT-C30 (B). Samples were obtained after 3 and 6 h of growth after transfer to different carbon sources (1%, wt/vol). In the case of lactose a sample was taken after 24 h of batch growth (conidial inoculum). Abbreviations: Gly, glycerol; Glc, d-glucose; Gal, d-galactose; Ara, l-arabinose; Xyl, d-xylose; Gol, galactitol; Aol, l-arabinitol; Xol, xylitol; Lac, lactose. 18S RNA served as a loading control.

FIG. 3.

Effect of bga1 gene deletion on growth and enzyme formation. (A) Southern analysis of parental strain QM9414 and two bga1 deletion mutants, ΔBGA1-1 and ΔBGA1-12. Genomic DNA was digested with HindIII and probed with a 5-kb HindIII fragment of the bga1 gene. Homologous insertion of the bga1 deletion cassette into the endogenous bga1 locus resulted in an increase (about 500 kb) in the size of the hybridizing band from 5 kb in the parent strain to 5.5 kb in the bga1 deletion mutants. (B) Growth of QM9414 (♦) and strain ΔBGA1 (▵) in shake flasks with peptone (0.1%, wt/vol) and lactose (1%, wt/vol) as carbon sources. The error bars indicate standard deviations for three different experiments. (C) Northern analysis of cbh1 and cbh2 transcript levels during growth on lactose. 18S RNA served as a loading control.

FIG. 4.

bga1 overexpression improves lactose metabolism but impairs cellobiohydrolase expression. (A) Growth of H. jecorina QM9414 (♦) and strains PKI-BGA3 (○), PKI-BGA7 (⋄), and PKI-BGA13 (□) on lactose in shake flasks. The error bars indicate standard deviations for three different experiments. (B) Effect of bga1 overexpression on cbh1 and cbh2 accumulation during growth on lactose.