(107d) Characterization of Modular Designer Xylanosomes for Enhanced Hemicellulose Hydrolysis in Yeast

In nature, cellulosomes are displayed on the surface of anaerobes as highly ordered enzyme-complexes that enhance the catalytic efficiency based on enzyme-substrate-microbe synergy. The goal of this research was to functionally assemble and characterize five modular, designer “xylanosomes” on the surface of Saccharomyces cerevisiae for the enhanced saccharification of birchwood xylan. An endoxylanase (Thermomyces lanuginosus), a β-xylosidase (Aspergillus niger), and an acetylxylan esterase (Aspergillus awamori) were successfully displayed on the yeast cell surface from scaffoldins that were designed with or without carbohydrate binding modules (family 22 or family 3 CBMs). The combinatorial effects of enzyme-enzyme synergy (1.6-fold) within the scaffoldin and enzyme-substrate synergy (2.1-fold) on xylan hydrolysis were decoupled, and the influence of choice and position of the CBM was also investigated.  The addition of a xylan binding domain from Thermotoga maritima improved hydrolysis by 3.3-fold over free enzymes, with a xylose productivity of 105 mg g^-1 substrate after 72 hours hydrolysis. The results of this study demonstrate the applicability of designer xylanosomes towards hemicellulose saccharification in yeast, and the importance of choice and position of the CBM in these modular constructs. In addition, recent work on similarly constructed cellulosomes for cellulose saccharification, for the production of ethanol and biorenewable chemical precursors will be presented.