Expression of Barley Endopeptidase B in Trichoderma reesei

Abstract

The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei. Trichoderma reesei is exploited in industry as a producer of hydrolytic enzymes (4). In high-cellulase-producing mutants, the major T. reesei cellulase, cellobiohydrolase I (CBHI), is secreted into the culture medium in the tens of grams per liter range under cellulase-inducing conditions (3). T. reesei has successfully been used as a host for heterologous fungal proteins (7, 13, 14, 18). Recently, T. reesei has also been shown to be able to produce immunologically active antibody derivatives of murine origin (17). The production level of 150 mg of CBHI-Fab fusion antibody per liter in a fermentor cultivation corresponds to 60 mg of Fab fragment per liter (17). The starchy endosperm of barley grain is surrounded by a living aleurone layer which secretes hydrolytic enzymes to the endosperm, allowing mobilization of the reserve materials from their storage place to the growing seedling during germination. The cysteine proteinases endopeptidases A (EPA [37 kDa]) and B (EPB [30 kDa]) are secreted from isolated aleurone layers when they are incubated with gibberellic acid (10, 12) and appear to play an important role in breaking down the water-insoluble storage proteins during germination. EPB is reported to be a nonglycosylated protein and to exist as two closely related isozymes (12). Efficient production of these papain-type proteinases in an alternative host would pave the way for their use in various applications requiring proteolysis, such as in the food processing industry. We have employed two different host strains, T. reesei Rut-C30 (ATCC 56765) (15) and ALKO2221, for the expression of EPB. T. reesei ALKO2221 is a low-protease mutant derived from strain VTT-D-79125 (2) by UV mutagenesis and by screening of the mutagenized conidia on minimal plates (19) containing 1% skim milk and 1% glycerol (12a). The host strains were transformed with the 8.9-kb EPB expression cassette of vector pALK584 (16) as described by Karhunen et al. (8). In pALK584, the 1.15-kb PvuI fragment from a barley epb cDNA clone, pHVEP4, harboring the secretion signals and coding sequences for pro-and mature forms of the protein (11), was ligated to XhoI-linearized plasmid pALK493 (20a). The 2.2-kb StuI-SacII cbh1 promoter fragment from T. reesei VTT-D-80133 (22) and the 1.7-kb BamHI-EcoRI cbh1 3Ј flanking region from T. reesei ALKO2466 (6) were used to

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

“…A signal for the natural-size cbh1 mRNA, about 2.2 kb, was also obtained ( Fig. 2B), because integration of the ebp construct had not occurred in the endogenous cbh1 locus (33). The host strain, T. reesei Rut-C30, did not produce any epb mRNA ( Fig.…”

“…In the expression plasmid pALK584 ( Fig. 1), described by Saarelainen et al (33), the fragment from the EPB cDNA clone was linked between the cbh1 promoter and terminator sequences. The epb cDNA accommodates the signal, pro-, and mature sequences of the proteinase (12).…”

“…The epb cDNA accommodates the signal, pro-, and mature sequences of the proteinase (12). The T. reesei transformant ALKO3713 has only one copy of the gene construct at an ectopic locus and produces CBHI and EPB as described by Saarelainen et al (33). The transformation host, T. reesei Rut-C30 (21), was used as a reference for CBHI production.…”

“…1. Physical map of the EPB expression vector pALK584 used to transform the epb cDNA in T. reesei as described by Saarelainen et al (33). The acetamidase gene amdS is used as a marker for transformation.…”

“…To increase our understanding of protein processing and excretion of heterologous proteins in fungi, a cDNA of a barley (Hordeum vulgare) cysteine endopeptidase B (EPB) has been introduced into T. reesei (33). In barley, the secretion of EPB is fast and is apparently a constitutive process in a tissue surrounding a biosynthetically inactive storage compartment of the grain.…”