Assembly of New Minicellulosomes through Disulfide Bonds Using Yeast a-Agglutinin on Saccharomyces Cerevisiae Surface for Cellulosic Ethanol Production

Saccharomyces cerevisiae is an ideal candidate for combining cellulases production, cellulose hydrolysis and ethanol fermentation into a single step to achieve consolidated bioprocessing (CBP). Previous reports generally assembled minicellulosomes on yeast cell surface through cohesion and dockerin interaction and these heterologous minicellulosomes expression often leaded to low display and assembly efficiency. In this study, we designed a new assembled pattern of minicellulosome on S. cerevisiae surface through disulfide bonds using yeast a-agglutinin system. N-terminal 149 amino acids of yeast a-agglutinin Aga1p is used as the cohesin domain and repeated cohesin domains with a cellulose-binding domain (multi-aga1) are synthesized for the scaffodin construction. The display efficiency of multi-aga1 scaffodin was 4 times higher than the traditional CipA3 scaffodin from Clostridium thermocellum. Cellulases, including a cellobihydrolase, an endoglucanase and a β-glucosidase, are fused with N-terminal Aga2p as dockerin.  The recombinant cellulases can functionally interact with N-terminal of yeast a-agglutinin Aga1p through disulfide bonds. Cell surface assembly of minicellulosome is confirmed by the immunofluorescence microscopy and flow cytometric analysis (FASC). The newly developed recombinant yeast strain with self-surface assembly of minicellulosome can utilize phosphoric acid-swollen cellulose (PASC) and produce 720 mg/L bioenthanol. The assembly of minicellulosomes through disulfide bonds using yeast a-agglutinin obtained much higher display efficiency and reduced the cellular burden to expression heterologous proteins. This system is also readily to be used for assembly of cellulosomes with two miniscaffoldins.