EXPRESSION AND SECRETION OF DEFINED CUTINASE VARIANTS BY ASPERGILLUS-AWAMORI

Several cutinase variants derived by molecular modelling and site-dire cted mutagenesis of a cutinase gene from Fusarium solani pisi are poor ly secreted by Saccharomyces cerevisiae. The majority of these variant s are successfully produced by the filamentous fungus Aspergillus awam ori. However, the L51S and T179Y mutations caused reductions in the le vels of extracellular production of two cutinase variants by A. awamor i. Metabolic labelling studies were performed to analyze the bottlenec k in enzyme production by the fungus in detail. These studies showed t hat because of the single L51S substitution, rapid extracellular degra dation of cutinase occurred. The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases, Presumably, the delay in the extracellular accumulation of this cutinase variant is ca used by the enhanced hydrophobicity of the molecule. Overexpression of the A. awamori gene encoding the chaperone BiP in the cutinase-produc ing A. awamori strains had no significant effect on the secretion effi ciency of the cutinases. A cutinase variant with the amino acid change s G28A, A85F, V184I, A185L, and L189F that was known to aggregate in t he endoplasmic reticulum of S. cerevisiae, resulting ire low extracell ular protein levels, was successfully produced by A. awamori. An initi al bottleneck in secretion occurred before or during translocation int o the endoplasmic reticulum but was rapidly overcome by the fungus.