Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MF alpha 1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MF alpha 1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.