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.

—Yamada et al.

Although lignocellulosic biomass (such as rice straw, which is one of the most abundant lignocellulosic waste materials), is seen a promising starting material for bioethanol production for a number of factors, it is still much more expensive to process than grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis of the biomass, the authors note.

Hydrolysis of cellulose requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and ß-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation, the authors note. In this study, they used a previously developed method (cocktail δ-integration) to optimize cellulase-expression levels in yeast.

In cocktail δ-integration, several kinds of cellulase-expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (that is, expressing the optimum ratio of cellulases) can be easily obtained, they note.

In they study, the cocktail δ-integration method was used to optimize cellulase expression in two yeast strains of opposite mating types. These strains were mated to produce a diploid strain with enhanced cellulase expression, which was then evaluated for its efficiency in converting cellulose to ethanol from PASC and pretreated rice straw.

As expected from the PASC hydrolysis reaction results, high ethanol production and yield from PASC was achieved using the diploid strain prepared in molasses medium. When we compared our results with those from different cellulase-expression systems of S. cerevisiae published previously, our diploid strain clearly achieved the highest ethanol production and yields. In addition to these promising findings, the cellulolytic enzyme-expressing diploid yeast strain was also able to produce ethanol from pretreated rice straw.

Although the ethanol production rate from rice straw was nearly identical to that from PASC, the ethanol yield from rice straw was relatively low. This result suggests that highly crystalline regions of cellulose in rice straw were not effectively degraded, thus reducing these regions by improving the efficiency of pretreatment or further optimizing cellulolytic enzyme-expression ratios in recombinant diploid yeast may lead to improved bioethanol yields from agricultural waste biomass. Using the cocktail δ-integration method, cellulase expression in yeast could be optimized for degradation of rice straw. Furthermore, hemicelluloses and a lignin matrix surrounding cellulose would also prevent effective degradation of lignocellulose; however, these could be removed by pretreatment or with use of additional exogenously expressed enzymes.

—Yamada et al.

Resources

• Yamada et al. (2011) Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnology for Biofuels 4:8 doi: 10.1186/1754-6834-4-8