530 1993 t reesei alko4468 is an endoglucanase i and ii negative strain it was constructed from the strain alko3620 by further replacing the endoglucanase 1 gene cel7b egl1 penttil u00e4 et al gene 45 253 263 1986 by the e coli hygromycin b phosphotransferase gene hph gritz et al gene 45 179 188 1983 conferring resistance to hygromycin b the 1 7 kb noti nsii fragment from the plasmid prlm ex 30 mach et al curr genet 25 567 570 1994 was used in which of the hph gene is expressed from the t reesei pyruvate kinase pki promoter and the transcription is terminated using the cel6a terminator sequences the plasmid prlm ex 30 was kindly provided by prof christian p kubicek institut f u00fcr biochemische technologie tu wien austria the cel7b flanking regions were as described in suominen et al mol gen genet 241 523 530 1993 the single copy replacements of the cel5a and cel7b genes by the marker genes in t reesei alko3620 and alko4468 were verified by southern blot analysis as described in suominen et al mol gen genet 241 523 530 1993 t reesei strains were sporulated on pd agar slants potato dextrose broth difco detroit mich the transformants were selected on trichoderma minimal medium containing acetamide as a nitrogen source penttil u00e4 et al gene 61 155 164 1987 the fungal mycelia for dna isolations were obtained after growing the strains for two days on trichoderma minimal medium containing 2 proteose peptone difco complex lactose based cellulase inducing media joutsjoki et al curr genet 24 223 228 1993 were used for enzyme production.