Ethanol Fermentation From Jerusalem Artichoke Tubers By The Genetically-modified Saccharomyces Cerevisiae Strains Secreting Inulinase

Jerusalem artichoke tubers with inulin as major component are potential feedstock for fuel ethanol production. Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value. In the fed-batch ethanol fermentation, the ethanol concentration were 74.5 and 77.5 g/L respectively after 48 h and the inulinase activities ranged from 10 to 25 U/mL. Another fed-batch fermentation was conducted in a 3 L fermentor at 30℃under anaerobic conditions. After 60 h, the ethanol concentration were nearly up to 80 g/L and the total sugar in the end were about 25 g/L, and the reducing sugar were always 5 g/L among the whole process.Integration vector pFA6a-rDNA-pgk-inu was constructed by cloning inulinase gene (inu), which originated from Kluyveromyces marxianus, pgk promoter and rDNA gene into the vector pFA6a. After digested by SphI, the pFA6a-rDNA-pgk-inu fragment was transformed into the industrial strain Saccharomyces cerevisiae 6525 and the G418 resistance gene KanMX acted as a dominant selectable marker. Among all the positive clones,6525/rDNA-14 and 6525/rDNA-13 were selected and their inulinase activities were 23 and 20 U/mL, which were both higher than the wild type. In the fed-batch ethanol fermentation, the ethanol concentrations were up to 72.5 and 69.5 g/L.