Fusarium graminearum A 3/5 as a Novel Host for Heterologous Protein Production

Abstract

We describe a novel fungal expression system which utilizes the Quorn myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of a trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.

Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in chemostat culture

“…Reduction in specific growth rate is generally thought to occur as the result of diversion of the host cell's energy and resources from growth processes to the maintenance and expression of the recombinant DNA (a so-called metabolic load or burden). F. venenatum A3/5 does not naturally secrete many proteins (Royer et al, 1995), so the observed reduction in specific growth rate may reflect the "burden" of GAM production or it may reflect disruptions to the genome which were introduced by the integration of the foreign DNA into the genome.…”

“…Naylor (Marlow Foods, Billingham, UK). F. venenatum JeRS 325 was obtained by transformation of F. venenatum A3/5 with plasmid pJeRS40 (9.9 kb) and contains the A. niger glucoamylase gene (glaA) under the control of a F. oxysporum trypsin-like protease promoter and terminator (Royer et al, 1995). The plasmid used in the transformation also contained a copy of the A. nidulans acetamidase gene (amdS) as a selectable marker.…”

Growth-rate-independent production of recombinant glucoamylase byFusarium venenatum JeRS 325

“…Naylor (Marlow Foods, Billingham). Fusarium venenatum JeRS 325 was obtained by transformation of F. venenatum A3/5 (formerly F. graminearum A3/5; Yoder and Christianson, 1998) with plasmid pJeRS40 and contains the Aspergillus niger glucoamylase gene (glaA) under the control of an F. oxysporum trypsin-like protease promoter and terminator (Royer et al, 1995). The plasmid used in the transformation also contained a copy of the A. nidulans acetamidase gene (amdS) as a selectable marker.…”

“…Other promoters that have been shown to be useful include amylase (amy; Christensen et al, 1988), glyceraldehyde-3-phosphate dehydrogenase (gpdA; Punt et al, 1991), alcohol dehydrogenase (alcA; Smart, 1991), 1,4-␤-endoxylanase (exlA; Gouka et al, 1996aGouka et al, , 1996b, and proteases from Fusarium spp. (Morita et al, 1994(Morita et al, , 1995Royer et al, 1995).…”

“…Recently, the trypsin-like alkaline protease promoter of Fusarium oxysporum has been used to produce heterologous protein in Fusarium venenatum A3/5 (Berka et al, 1998;Royer et al, 1995;Wiebe et al, 1999 (Royer et al, 1995) and production could be regulated by controling culture pH (Wiebe et al, 1999), however, little is known about enzyme production under the control of this promoter. The present study describes production of heterologous glucoamylase in F. venenatum JeRS 325, a transformant which produces A. niger glucoamylase under the control of the F. oxysporum trypsin-like protease promoter, in batch, fed-batch, and glucose-limited chemostat cultures at various dilution rates.…”