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 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. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.

Cell surface engineering of industrial microorganisms for biorefining applications

“…The construction of a minihemicellulosome and/or xylanosome for hemicellulose hydrolysis also has been reported (Sun et al, 2012;Srikrishnan et al, 2013). Arabinoxylan is a heteropolymeric chain of a beta-1,4-linked xylose backbone substituted with arabinose residues, and represents a principal component of plant cell walls.…”

The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing

“…It has been demonstrated that the application of yeast surface display to the enzymes (i.e., cellulases, hemicellulases) relevant to lignocellulosic bioethanol production has exhibited improved hydrolytic activities with respect to their free enzyme counterparts. In addition, enhancement in the degree of synergy was observed when mixtures of the said surface displayed enzymes (i.e., cellulases, hemicellulases) were utilized for substrate hydrolysis [42][43][44][45][46].…”

“…Though the application of the surface display adaptor assembly for hemicellulosic ethanol is limited, it is quite relevant that this type of surface display strategy was explored for this type of substrate. Srikrishnan, et al [44] demonstrated the assembly of a minixylanosome (a counterpart of the minicellulosome) on yeast. This assembly consists of a trivalent tri-functional adaptor subunit that is attached on the surface of yeast where three hemicellulases fused with different dockerin domains: the Thermomyces lanuginosus endoxylanases (XynA), Aspergillus niger β-xylosidase (XylA), and the Aspergillus awamori acetylxylan esterase (AXE) assemble and display onto.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…Xylanase activity was increase up to 3.3 times more than free enzymes after 72 h hydrolysis Srikrishnan et al, 2013 Cellobiose or CMC (100 g/l) K. marxianus Thermoascus aurantiacus cellulase genes, including cellobiohydrolase 1 (cel7A), endoglucanase 1 (cel5A) and β-glucosidase (bgl3A) genes Secretion 43.4 g/l ethanol Hong et al 2007 .…”

“…The strain displaying a bi-functional (xylanase II and β-xylosidase) hemicellulosome could produce 1 g/l ethanol after 80 h (ethanol yield: 0.31 g ethanol/g birchwood xylan consumed). Srikrishnan et al (2013) successfully assembled five trimeric xylanosomes on the cell surface of S.cerevisiae. To enhance synergistic hydrolysis of birchwood xylan, three dockerin-tagged fungal enzymes, including endoxylanase (XynAc), β-xylosidase (XlnDt) and acetylxylan esterase (AwAXEf) were displayed.…”