Ethanol production from inulin by recombinant Saccharomyces cerevisiae with inulinase gene from Kluyveromyces marxianus

Abstract

In recent years, efforts to produce biofuels from renewable biomass have been done for overcoming the problems of uncertain fuel supply and carbon dioxide emissions. One of the most representative biofuels is bioethanol because it has greater octane booster properties, is not toxic and does not contaminate water sources. Inulin consisting of linear β-2,1-linked polyfructose chains terminated by a glucose residue (C6nH10n+2O5n+1) is a polyfructan which is present in plants such as Jerusalem artichoke, chicory and dahlia. It can be converted to fructose by inulinase, then fructose can be utilized easily by microorganisms such as Saccharomyces cerevisiae. However, S. cerevisiae cannot consume inulin itself because it does not have inulin-degrading enzymes. This thesis was carried out to produce ethanol from inulin by recombinant S. cerevisiae with the inulinase gene from Kluyveromyces marxianus by metabolic engineering approach. Three types of promoters (GPD, PGK1, truncated HXT7) and signal sequences (KmINU, MFα1, SUC2) of an expression cassette were compared to select the optimized expression system when the inulinase gene from Kluyveromyces marxianus (KmINU) was introduced into S. cerevisiae D452-2 used as a host. Nine plasmids having different combinations of promoter and signal sequence were constructed and introduced into S. cerevisiae D452-2. The recombinant S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) not only had the highest KmINU activity per dry cell weight, but also exhibited the most outstanding fermentation properties among the nine recombinant S. cerevisiae strains. It was observed that KmINU accumulated in the medium and the specific activity (U/mg dry cell weight) increased with cultivation time. The fermentation performances of a wild type strain grown in an acid-hydrolyzed inulin solution were compared with those of the recombinant S. cerevisiae grown in the same amount of inulin. Ethanol production based on inulin needs three steps including acid hydrolysis, neutralization and fermentation. In aspects of the cost and efficiency, the use of the S. cerevisiae with the inulinase gene is competitive because the recombinant yeast strain allows inulin hydrolysis and ethanol production simultaneously. Finally, a batch fermentation of S. cerevisiae D452-2/p426PM in a bioreactor with 200 g/L inulin was performed and resulted in 0.47 g ethanol/g inulin of ethanol yield and 1.02 g/L∙h of ethanol productivity.