High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

Abstract

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only ϳ10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

“…In shake-flasks, the highest production level was 230 mg l 21 from the non-fusion construct, whereas the activity levels from the fusion construct were about five times lower. Fusion to a secreted host protein has improved the heterologous production of, for example, murine Fab fragments (Nyyssönen et al, 1993) and a bacterial xylanase (Paloheimo et al, 2003) in T. reesei, but in our case this effect was not observed. On the other hand, this was the first time that HFBI was used as a production carrier protein.…”

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

“…It is well established that expression of foreign proteins fused to CBMs results, for the most part, in high expression levels (24,52,101,105,147,151,160,162,166,(181)(182)(183). As a result, expression vectors (pET34 to pET38) incorporating CBMs as fusion tags were developed (143).…”

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

“…The corresponding cel7 genes, including their own signal sequences, were exactly fused to the T. reesei cel7A promoter using PCR. Transcription termination was ensured by the T. reesei cel7A terminator, and the Aspergillus nidulans amdS marker gene was used for selection of the transformants as described in detail in Karhunen et al (1993), Paloheimo et al (2003b) and Haakana et al (2004). The linear expression cassettes were transformed into T. reesei industrial production strains that lack the genes encoding the major cellulases Cel7A, Cel6A, Cel7B, and Cel5A .…”

“…For that purpose, the transformants were grown for 2 days at 308C on Trichoderma minimal medium (Penttilä et al, 1987) containing 2% proteose peptone (Difco), and genomic DNA was isolated from the fungal mycelia. Shake flask and laboratory scale bioreactor cultivations of the heterologous Cel7 producing strains were performed as described in Paloheimo et al (2003b).…”