Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol

Abstract

By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a beta-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of approximately 1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.

Innovative Biological Solutions to Challenges in Sustainable Biofuels Production

“…Compared to the noncomplexed cellulase systems, the cellulosome could provide a "quantum leap" in the development of biofuel technology thanks to its highly ordered structural organization that enables enzyme proximity synergy and enzymesubstrate-microbe complex synergy (Bayer et al, 2007). To date, the trifunctional minicellulosomes have been successfully assembled in vivo in S. cerevisiae, and the resulting recombinant strain could simultaneously hydrolyze and ferment amorphous cellulose to ethanol, providing a relatively convenient engineering platform (Wen et al, 2010).…”

“…Engineering of the N. crassa cellodextrin transport system into S. cerevisiae promotes efficient growth of this yeast on cellodextrins, and the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system in simultaneous fermentation experiments (Galazka et al, 2010). An alternative engineering strategy to construct CBP-enabling yeast species is to endow S. cerevisiae with the ability to utilize cellulose by heterologously expressing a functional cellulase system (Wen et al, 2010). Nature has provided two ways of designing such yeast strains: noncomplexed cellulase systems and complexed cellulase systems (i.e., cellulosomes) (Wen et al, 2010;Chundawat et al, 2011).…”

“…Recently, a promising technology, known as consolidated bioprocessing (CBP), was developed for biofuel production from lignocellulosic biomass. It involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol by combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step (Linger et al, 2010;Wen et al, 2010). Although yeast is utilized to ferment sugars derived from cornstarch or sugarcane into ethanol, it cannot ferment the cellodextrins naturally released from lignocellulosic biomass by cellulases and requires multiple enzymes, including β-glucosidases, to quantitatively produce fermentable glucose (Sun & Cheng, 2002;Galazka et al, 2010;Chundawat et al, 2011).…”

“…Nature has provided two ways of designing such yeast strains: noncomplexed cellulase systems and complexed cellulase systems (i.e., cellulosomes) (Wen et al, 2010;Chundawat et al, 2011). By mimicking the noncomplexed cellulase system, several groups successfully constructed cellulolytic S. cerevisiae strains that directly ferment amorphous cellulose to ethanol, although the titer and yield were relatively low (Fujita et al, 2004;Den Haan et al, 2007;Wen et al, 2010). Compared to the noncomplexed cellulase systems, the cellulosome could provide a "quantum leap" in the development of biofuel technology thanks to its highly ordered structural organization that enables enzyme proximity synergy and enzymesubstrate-microbe complex synergy (Bayer et al, 2007).…”

“…An alternative engineering strategy to construct CBP-enabling yeast species is to endow S. cerevisiae with the ability to utilize cellulose by heterologously expressing a functional cellulase system (Wen et al, 2010). Nature has provided two ways of designing such yeast strains: noncomplexed cellulase systems and complexed cellulase systems (i.e., cellulosomes) (Wen et al, 2010;Chundawat et al, 2011). By mimicking the noncomplexed cellulase system, several groups successfully constructed cellulolytic S. cerevisiae strains that directly ferment amorphous cellulose to ethanol, although the titer and yield were relatively low (Fujita et al, 2004;Den Haan et al, 2007;Wen et al, 2010).…”