Functional Display of Complex Cellulosomes on the Yeast Surface via Adaptive Assembly

Abstract

A new adaptive strategy was developed for the ex vivo assembly of a functional tetravalent designer cellulosome on the yeast cell surface. The design is based on the use of (1) a surface-bound anchoring scaffoldin composed of two divergent cohesin domains, (2) two dockerin-tagged adaptor scaffoldins to amplify the number of enzyme loading sites based on the specific dockerin−cohesin interaction with the anchoring scaffoldin, and (3) two dockerin-tagged enzymatic subunits (the endoglucanse Gt and the β-glucosidase Bglf) for cellulose hydrolysis. Cells displaying the tetravalent cellulosome on the surface exhibited a 4.2-fold enhancement in the hydrolysis of phosphoric acid swollen cellulose (PASC) compared with free enzymes. More importantly, cells displaying the tetravalent celluosome also exhibited an ∼2-fold increase in ethanol production compared with cells displaying a divalent cellulosome using a similar enzyme loading. These results clearly indicate the more crucial role of enzyme proximity than just simply increasing the enzyme loading on the overall cellulosomal synergy. To the best of our knowledge, this is the first report that exploits the natural adaptive assembly strategy in creating artificial cellulosome structures. The unique feature of the anchoring and the adaptor scaffoldin strategy to amplify the number of enzymatic subunits can be easily extended to more complex cellulosomal structures to achieve an even higher level of enzyme synergy.

Thermostable Glycoside Hydrolases in Biorefinery Technologies

“…Hypothetical highly engineered cell for one-pot biotransformation of lignocellulosic feedstock into liquid fuels [83]. The cellulosome scheme is based in the recently published functional display of complex cellulosomes on the yeast surface [89]. The furfural resistance by reducing cofactors was taken from the model proposed for the engineered E. coli tolerant to furfural [90].…”

Cell surface engineering of industrial microorganisms for biorefining applications

“…Using this approach, ethanol production was increased by up to three-fold (Goyal et al, 2011). This laboratory subsequently constructed a tetravalent cellulosome using a natural adaptive assembly strategy (Tsai et al, 2013). Yeast cells displaying the tetravalent celluosome demonstrated a 2-fold increase in ethanol production compared with cells displaying a divalent cellulosome using a similar enzyme loading level (Tsai et al, 2013).…”

“…This laboratory subsequently constructed a tetravalent cellulosome using a natural adaptive assembly strategy (Tsai et al, 2013). Yeast cells displaying the tetravalent celluosome demonstrated a 2-fold increase in ethanol production compared with cells displaying a divalent cellulosome using a similar enzyme loading level (Tsai et al, 2013). These results suggested that enzyme proximity was more important for cellulosomal synergy than simply increasing the amount of enzyme loaded on the cell surface.…”

Bioprospecting thermostable cellulosomes for efficient biofuel production from lignocellulosic biomass

“…Tsai et al (2010) optimized the mixing ratio of different populations and constructed a synthetic consortium expressing different components of cellulosomes for the conversion of amorphous (phosphoric Table 1 Fierobe et al (2001) acid-swollen) cellulose to ethanol with 93 % of maximum theoretical yield. A 4.2-fold increase in the hydrolysis of phosphoric acid-swollen cellulose (PASC) was reported with a tetravalent cellulosome expressed on the surface of the yeast cell as compared to the free enzymes (Tsai et al 2013). Ito et al (2009) developed recombinant yeast having a designer minicellulosome system displayed on its surface by assembling different components of the cellulosomal system C. cellulovorans, T. reesei and A. aculeatus for the hydrolysis of β-glucan.…”