ARTICLE Functional Assembly and Characterization of a Modular Xylanosome for Hemicellulose Hydrolysis in Yeast

ABSTRACT: Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin-tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a b-xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface-expression scaffoldins were modular constructs with or without carbohydrate binding modules from Ther-motoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and en-zyme–substrate proximity, and the effects of binding do-main choice and position on xylan hydrolysis were determined. The scaffoldin-based enzymes (with no binding domain) showed a 1.6-fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1-fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3-fold over free enzymes, with a xylose productivity of 105mg g1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4-fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5-fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes to-ward hemicellulose saccharification in yeast, and the impor-tance of the choice and position of the carbohydrate binding module for enhanced synergy