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

Abstract

Background: 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. 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.

Results: The 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.

Conclusions: We 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.

Figure 1

Time course of ethanol production from PASC by haploid strains. Triangles = MT8-1; squares = MT8-1/IBEC; diamonds = MT8-1/cocδBEC3. Data are averages from three independent experiments (error bars represent SE).

Figure 2

Transcription levels of cellulolytic enzymes of haploid strain MT8-1/cocδBEC3, 1440/cocδBEC3 and diploid strain MNII/cocδBEC3. Gray bar = β-glucosidase; white bar = endoglucanase; black bar = cellobiohydrolase. Data are averages from six independent experiments (error bars represent SE).

Figure 3

Time course of ethanol production from PASC by diploid strain MNII/cocδBEC3. Triangles = precultured using YPD; diamonds = precultured using molasses medium. Data are averages from three independent experiments (error bars represent SE).

Figure 4

Time course of ethanol production from pretreated rice straw by diploid strain MNII/cocδBEC3 precultured in molasses medium. Data are averages from three independent experiments (error bars represent SE).