title: Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.
abstract: When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.
authors.affiliation: Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Marko
authors.initials: M
authors.lastname: Kuyper
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Winkler
authors.forename: Johannes P
authors.initials: JP
authors.lastname: van Dijken
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics* / Q000378:metabolism; D000693:Anaerobiosis; D003470:Culture Media; D019020:Directed Molecular Evolution; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D020089:Piromyces / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014994:Xylose / Q000378:metabolism*
chemical_list: D003470:Culture Media; D000431:Ethanol; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase