Trypsin-like protease (TLP) production in Fusarium oxysporum and Fusarium venenatum and use of the TLP promoter for recombinant protein (glucoamylase) production

Abstract

The production of native trypsin-like protease (TLP) in wild type strains of Fusarium oxysporum (214) and F. venenatum (A3/5) was assessed and compared with the expression of recombinant glucoamylase (GAM) under the F. oxysporum TLP promoter in F. venenatum JeRS 325. In the two non-recombinant strains, TLP was only detected in the supernatants of batch cultures after the onset of stationary phase and TLP production was highest in the presence of a proteinaceous nitrogen source at pH 7.5. In chemostat cultures of F. oxysporum, the specific TLP production rate was negatively correlated with specific growth rate ( μ=0.03–0.09 h −1). In F. venenatum, A3/5 at dilution rates between 0.06 and 0.15 h −1, specific TLP production was also negatively correlated with specific growth rate. The F. oxysporum TLP promoter regulates GAM production in F. venenatum JeRS 325, but the specific GAM production rate is known to be constant between 0.05 and 0.19 h −1, showing that regulation of the promoter in the recombinant host differs from that in the native strain. Western blot analysis demonstrated that GAM production began in batch cultures of F. venenatum JeRS 325 during the decelerating growth phase, and that de novo synthesis of GAM occurred during stationary phase.