Management of enzyme diversity in high-performance cellulolytic cocktails

Abstract

Background: Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes.

Results: One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail.

Conclusions: These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Keywords: Bioethanol; Cellulolytic cocktail improvement; Enzymatic hydrolysis; Lignocellulosic biomass.

Fig. 1

Graphic representation of enzymes and cocktail performance versus process conditions. Solid line represents the activity profile of the whole cocktail under a range of process conditions ( i.e., pH, temperature, substrate concentration, mixing, etc.). Dotted lines represent the activity profile for individual enzymes in the mixture. At industrially controlled conditions (shadowed in green) contributive enzymes (black) would be capable to act while less-contributive (red) would show a reduced or negligible activity

Fig. 2

Glucose released by GH6 enzymes supplemented over whole cocktail (WC). Control (light gray): 10 mg/g of WC. EG VI (dark gray): 10 mg/g of WC supplemented with 2 mg/g of EG VI. CBH IIa (dark gray): 10 mg/g of WC supplemented with 2 mg/g of CBH IIa. CBH IIb (dark gray): 10 mg/g of WC supplemented with 2 mg/g of CBH IIb. WC (black): 12 mg/g of WC

Fig. 3

Glucose released after 72 h of hydrolysis of PCS by 10 mg/g of cocktails produced by ΔegVI and ΔcbhIIb strains compared with parental strain