The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori

Abstract

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence. The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.

The Secretion Pathway in Filamentous Fungi: A Biotechnological View

“…It is generally believed that signal sequences do not account for major differences in the production yields of the recombinant proteins that they precede, although not many studies in which this issue has been addressed in detail are available. The level of the F. solani pisi cutinase produced in A. awamori was not greatly affected by the replacement of the cutinase presequence by the leader peptide of the endogenous Aspergillus endoxylanase (van Gemeren et al, 1996). In contrast, the extracellular level of the A. restrictus restrictocin produced in both A. nidulans and A. niger was significantly higher when the restrictocin, rather than the glucoamylase signal sequence, was used (Brandhorst and Kenealy, 1995).…”

“…Hence, in their work with restrictocin, Brandhorst and Kenealy (1995) also observed a markedly positive effect of the restrictocin prosequence on the secretion of this protein. However, addition of the glucoamylase prosequence at the N-terminus of cutinase (van Gemeren et al, 1996) and chymosin (van Hartingsveldt et al, 1990) reduced the secretion levels of these two heterologous proteins in Aspergillus.…”

Cutinase: From molecular level to bioprocess development

“…Fusions of the cutinase gene to the endoxylanase II promoter with four different pre-pro-or pre-sequences were constructed from synthetic oligonucleotides. The multicopy strains showed a 6-to 12-fold-increased production of extracellular cutinase relative to the single-copy strains (van Gemeren et al, 1995(van Gemeren et al, , 1996.…”

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

“…Cutinases are natural extracellular enzymes in their native organisms. Previously, secretory expression of cutinases in different hosts was mediated by a secretion signal peptide (9)(10)(11)(12)(13)(14). In the present study, cutinase activity in the culture medium from a 3-liter fermentor of T. fusca cutinase, heterologously expressed in E. coli without a signal peptide, reached 1,063.5 U/ml (2,380.8 mg/ liter) after 24 h. This represents a 4.4-fold increase over the highest previously reported yield (546 mg/liter) (12).…”

“…Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11,12), and Pichia pastoris (13). The highest yield of extracellular protein, 546 mg/liter, was obtained in a 5-liter bioreactor utilizing an engineered S. cerevisiae cellular system (12).…”