Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiaewith optimized cellulase expression

Abstract

BackgroundHydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.ResultsThe engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.ConclusionsWe have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

“…In view of these limitations, consolidated bioprocessing (CBP), which involves enzyme production, saccharification, and fermentation in a single step, has been proposed as a promising platform for cellulosic biofuel production (3,4). Several studies have investigated the heterologous expression of cellulases in ethanol-producing organisms (5)(6)(7)(8)(9)(10). In particular, the expression of cellulases in Saccharomyces cerevisiae has been widely examined (5)(6)(7)(8)(9)(10)(11).…”

“…Several studies have investigated the heterologous expression of cellulases in ethanol-producing organisms (5)(6)(7)(8)(9)(10). In particular, the expression of cellulases in Saccharomyces cerevisiae has been widely examined (5)(6)(7)(8)(9)(10)(11). The cellulases that are commonly expressed in S. cerevisiae include endoglucanases (EGs), cellobiohydrolases (CBHs), and ␤-glucosidases (BGLs).…”

Recombinant Cellulase and Cellulosome Systems

“…These results strongly argue for a successful optimization of cellulase ratio. The activity of the ratio-optimized strain was further improved by making it diploid [19]. The optimized diploid showed an ability to produce ethanol directly from PASC (7.6 g/L in 72 hours) or pretreated rice straw (7.5 g/L) in yeast peptone (YP) medium without addition of exogenous enzymes.…”

“…The optimized diploid showed an ability to produce ethanol directly from PASC (7.6 g/L in 72 hours) or pretreated rice straw (7.5 g/L) in yeast peptone (YP) medium without addition of exogenous enzymes. This was the first report of direct conversion to ethanol of agricultural waste residue without exogenous enzyme addition by recombinant cellulase-expressing yeast [19]. Other strategies used to incorporate enzymes at specific ratios into artificial cellulosomes using yeast consortia are discussed later in this chapter (Section 3.4.1).…”

De-Oiled Rice Bran as a Source of Bioethanol

“…Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation (Yamada et al, 2011).…”