High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure.
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 approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.
Paloheimo
Marja
M
Roal Oy, FIN-05201 Rajamäki, Finland. marja.paloheimo@roal.fi
Mäntylä
Arja
A
Kallio
Jarno
J
Suominen
Pirkko
P
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D044902:beta-Mannosidase; D043364:Endo-1,4-beta Xylanases; D043366:Cellulose 1,4-beta-Cellobiosidase