Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus β-glucosidase genes.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and β-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley β-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and β-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and β-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley β-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.