THE EFFECT OF PRE-SEQUENCES AND PRO-SEQUENCES AND MULTICOPY INTEGRATION ON HETEROLOGOUS EXPRESSION OF THE FUSARIUM-SOLANI-PISI CUTINASE GENE IN ASPERGILLUS-AWAMORI

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi wa s expressed in Aspergillus awamori using the A. awamori endoxylanase I I (eslA) promoter and terminator. The influence of the origin of the p re-sequence and the presence of a pro-sequence on the efficiency of ex tracellular cutinase production was analysed in single-copy transforma nts containing an expression cassette integrated at the pyrG locus. Tr ansformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold hig her cutinase production level compared to strains containing construct s with an additional cutinase pro-peptide. The effect of multicopy int egration of the expression cassette on cutinase production was analyse d in strains with different numbers of a cutinase construct containing its own pre-pro-sequence. The multicopy strains showed a 6- to 12-fol d increased production of extracellular cutinase relative to the singl e-copy strains. No linear dose response relation to the number of expr ession cassettes present in the strains was observed. The amount of ac tive enzyme produced by the strains correlated with the amount of cuti nase-specific mRNA, suggesting that cutinase overproduction is not lim ited at the level of translation or secretion.