The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding β-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.
Keywords: Unified enzymatic saccharification and fermentation (USF) system; bioethanol; neoagarobiose hydrolase; recombinant yeast; β-agarase.