Direct Conversion of Xylan to Ethanol by Recombinant Saccharomyces cerevisiae Strains Displaying an Engineered Minihemicellulosome

<jats:title>ABSTRACT</jats:title> <jats:p> Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant <jats:named-content content-type="genus-species">Saccharomyces cerevisiae</jats:named-content> strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from <jats:named-content content-type="genus-species">Clostridium thermocellum</jats:named-content> contained one or three cohesin modules and was tethered to the cell surface through the <jats:named-content content-type="genus-species">S. cerevisia</jats:named-content> e a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated <jats:sc>d</jats:sc> -xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed. </jats:p>