Although S. cerevisiae cannot grow on D-xylose as the sole carbon source, its genome does contain genes that code for a non-specific NADH-dependent al­dose reductase (GRE3) and for a xylitol dehydrogenase (XYL2). It has been shown that over-expression of these native S. cerevisiae genes using endoge­nous promoters enabled a specific growth rate of 0.01 h-1 on D-xylose in shake flasks [64]. However, in these shake-flask cultures this engineered yeast strain converted D-xylose into xylitol with a yield of 55%. Under anaero­bic conditions, precluding respiratory NAD+ regeneration, the strain over­expressing the endogenous enzymes was unable to utilise D-xylose [64].

In addition to this metabolic engineering approach, the presence of en­dogenous genes for D-xylose-converting enzymes has been used in recent experiments by Attfield and Bell (2006), describing a non-recombinant S. cere- visiae strain that grows on D-xylose as the sole carbon source in aerobic shake flask cultures. In their study a combination of population genetics and evolutionary engineering [5,60] resulted in an increase in growth rate from extremely low, barely measurable growth rates to a specific growth rate of around 0.12 h-1 (a doubling time of less than 6 h) over a period of 1400 days. Apparently, this S. cerevisiae strain had evolved in such a way that the very low “background” xylose reductase and xylitol dehydrogenase activities, which were previously described as insufficient for growth on D-xylose [8], increased to levels that did enable growth. Indeed, subsequent analysis of the evolved strain showed that xylose reductase activity had increased fourfold and the xylitol dehydrogenase activity 80-fold relative to the parental strain. The ac­tual genes that underwent mutation have not yet been characterised. Although this very interesting study underlines the tremendous potential of evolution­ary approaches, the selection procedure inevitably resulted in a yeast strain displaying the characteristics of redox imbalance, such as xylitol production.

1.4