Optimization of Displayed Cellulases in Yeast Suitable for Ionic Liquid Pretreated Biomass by Cocktail δ Integration Methodology

With problems such as exhaustion of fossil resources, the bioethanol production from abundance rich and renewable lignocellulosic biomass has recently become an attractive technology. The structure of lignocellulosic biomass is too rigid due to the high density of hydrogen bond within its structure, and thus the several pre-treatment methods had been proposed for deconstructing of biomass. Among them, biomass pre-treatment with ionic liquid has a lot of attention because this method is environmentally benign methodology with comparable low energy consumption. By using ionic liquid as a solvent for biomass suspension, we had achieved well the construction of bio-ethanol production system combined with our developed arming yeast previously [1]. In common sense, the degradation process of pre-treated biomass is usually required by enzymatic degradation with cellulase enzyme(s). Our developed system combined with arming yeast and ionic liquid biomass pretreatment could deduce the loading of cellulase regent for efficient degradation. In addition, recently, the biomass based ionic liquid for biomass pretreatment was discovered, and we also achieved the efficient pretreatment of Bagasse from sugarcane with this ionic liquid, cholinium ionic liquid [2].

For efficient degradation of biomass with cellulases, there are optimal combination ratio between these cellulases including Endoglucanase (EG), Cellobiohydrolase (CBH I and CBH II), and β-glucosidase (BGL). In our laboratory, we have been also developed the optimizing methodology of displaying several cellulases on the yeast cell surface by using cocktail δ-sequence [3]. By using this method, we could be established the adequate yeast strain depended on target biomass.

In this study, the combination of cocktail δ-integration method for cellulases displaying and cholinium ionic liquid biomass pretreatment was evaluated by simultaneous scarification and fermentation (SSF) with constructed arming yeast. As a result, we successfully stablished an engineered yeast, and could produce 0.8 g/L ethanol from 5 g/L bagasse without any addition of cellulose enzymes.

[1] K. Nakashima, K, Yamaguchi, N. Taniguchi, S. Arai, R. Yamada, S. Katahira, N. Ishida, H. Takahashi, C. Ogino, A. Kondo. Direct bioethanol production from cellulose by the combination of cellulase displaying yeast and ionic liquid pretreatment. Green Chem., 13(10), 2948-2953 (2011)
[2] K. Ninomiya, S. Omote, C. Ogino, K. Kuroda, M. Noguchi, T. Endo, R. Kakuchi, N. Shimizu, K. Takahashi. Saccharification and ethanol fermentation from cholinium ionic liquid-pretreated bagasse with a different number of post-pretreatment washings. Bioresour. Technol., 189, 203-209 (2015)
[3] R. Yamada, N. Taniguchi,T. Tanaka, C. Ogino, H. Fukuda, A. Kondo. Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb. Cell Fact., 9; 32 (2010)

Keywords
Celluase display; cocktail δ integration; ionic liquid; lignocellulosic biomass pretreatment