Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast.
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 β-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 Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme-enzyme and enzyme-substrate proximity, and the effects of binding domain 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 105 mg g(-1) 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 toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy.
Department of Chemical Engineering and Materials Science, The Henry Samueli School of Engineering, University of California, Irvine, California, USA.
Sneha
S
Srikrishnan
Wilfred
W
Chen
Nancy A
NA
Da Silva
D000115:Acetylesterase / Q000235:genetics / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
D002241:Carbohydrates; D005656:Fungal Proteins; D011134:Polysaccharides; C007916:hemicellulose; D000115:Acetylesterase; C081851:acetylxylan esterase; D014995:Xylosidases