Recombinant production of an inulinase in a saccharomyces cerevisiae gal80 strain

Abstract

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 was overexpressed by using the GAL10 promotor in a Δgal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to a mating factor α signal sequence for secretory expression. Use of the Δgal80 strain allowed for the galactose-free induction of inulinase expression using a glucose-only medium. Shake-flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the Δgal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both aerobic and anaerobic conditions by about 2.9- and 1.7- fold, respectively. A 5-l fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at the OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5-l scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and with the Ph controlled at 5.0. The temperature was maintained at 30°C and 37°C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/l, respectively.