The second generation biofuel by lignocellulosic biomass consists of lignin, hemicellulose, cellulose, pectin and other components. Cellulose and hemicellulose is the principle carbon source of lignocellulosic biomass. The minicellulosome complexes via assembled cohesin-dockerin interaction which purpose to use their synergistic activity. We focused on functional enzyme complexes for effective ethanol fermentation using pretreated substrate such as cellulose and hemicellulose. In contrast with Saccharomyces cerevisiae, Pichia pastoris can utilize pentose without genetic manipulation. Simple ethanol fermentation strategy is developed for high-yield secretory expression of heterogeneous protein via cellulase, xylanase, and minicellulosome. P. pastoris was engineered for expression of recombinant scaffolding protein (mCbpA) xylanase (XynB) and chimeric endoglucanase (cCelE). Chimeric endoglucanase is fused with dockerin module and endoglucanase using fusion PCR protocol. The mCbpA, XynB and cCelE with gene coding for the secretion signal sequence of the α-mating factor were highly expressed in the yeast P. pastoris under the control of the ADH2 Promoter, which is activated under O2 limit condition. Transcriptional activator for expression of enzymes under ADH2 promoter could guide the regulation of protein express without any inducer or handling. This enzyme complex have synergistic activity for degrading polysaccharides into pentose and hexose. When it is in stationary phase with closed vial or flask, regulation is became ideal for industrial protein expression condition. Finally, we have developed the activity of conversion to ethanol 2.1-folds production than wild type. In this study, we describe a clear result for efficient fermentation methods for ethanol production using enzyme complex and its application from biomass to useful product such as FAEEs (fatty acid ethyl esters) or bioplastics.
[This research was supported by Creative Allied Project (CAP) of the Korea Research Council of Fundamental Science and Technology(KRCF)/Korea Institute of Science and Technology (KIST) (Project No. 2E24832)]