Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

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 · 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 .