Ethanol production from hydrolysate by genetically modified industrial strains of Saccharomyces cerevisiae

The interest in the use of plant hydrolysates for the production of fuel alcohol has grown considerably in recent years, particularly as the need to protect the environment has increased. This interest encouraged first the study of natural xylose fermenting organisms and subsequently the genetic modification of Saccharomyces cerevisiae to enable it to utilise xylose. The addition or induction of the genes for xylose utilisation (xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XKS)) in yeast is not sufficient to enable S. cerevisiae to ferment xylose to ethanol to give a yield close to the theoretical, and further metabolic engineering is necessary if the process is to be efficient. In particular, a redox imbalance is created when NADPH requiring XR is employed with NADH generating XDH. Considerable improvements have been gained through further metabolic engineering of various redox reactions in the strains. Insight of cell responses to xylose utilisation has been gained through transcriptional profiling and proteomics. Much of the research on xylose utilisation by yeast has been carried out on medium containing pure xylose as carbon source, with or without the addition of glucose as a co-substrate, whereas plant hydrolysates contain a mixture of carbon sources and also several toxic compounds. Limited research has shown that these compounds are typically more toxic to the natural xylose-utilising yeast such as Pichia stipitis than to S. cerevisiae, but also that lab strains of S. cerevisiae have similarly low tolerance to growth in hydrolysate. Therefore we have constructed xylose-utilising industrially derived S. cerevisiae strains and studied their performance on hydrolysates. We have systematically compared ethanol production by an industrially derived strain of S. cerevisiae, which has been genetically modified to carry XR and XDH from P. stipitis integrated into its genome under control of the PGK1 and ADH1 promoters, respectively, and a copy of its own XKS1 under control of the TPI or ADH1 promoter integrated at the native XKS1 site, with lab strains of S. cerevisiae carrying similarly integrated copies of the genes, when grown in oxygen-restricted conditions on hydrolysates with a high xylose content. In addition, data on ethanol production by P. stipitis under the same conditions is also shown.