Evaluation of recombinant auxiliary proteins for biomass pretreatment and saccharification

Biomass pretreatment is essential for enzymatic depolymerization of cellulose and hemi cellulose to generate mono sugars required for ethanol fermentation by yeast. While several environmental friendly and carbon–neutral pretreatment methods have been under development, they are yet to achieve commercial viability. The major limitation is the restricted access and binding of enzymes to cellulose and hemicellulose fibres. Accessory proteins such as expansins, swollenins and lytic polysaccharide mono oxygenases (LPMO) are known for loosening the cellulose and hemicellulose fibres facilitating higher binding of cellulases. Herein, we present the production of expansins, swollenins, and lytic polysaccharide monooxygenases in a recombinant host, Pichia pastoris, and investigate their synergistic effects with cellulase enzymes. Out of four swollenins, two expansins and two LPMOs, we achieved high expression titers (~ 2.0—5.0 g/L) for an expansin and one LPMO. These two auxiliary proteins were tested along with cellulases for improved cellulose to glucose conversion efficiencies in presence and absence of lignin modifying enzymes (LME) from fungal cultures. LME independent biomass pretreatment is evaluated for cost efficient saccharification aided by auxiliary proteins (Scheme 1). Our findings underscore the potential utility of these auxiliary proteins for scaling up biomass pretreatment and commercial ethanol production trials.