Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain

Abstract

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed 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 △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 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 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 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.