Expression of the Piromyces sp. E2 XylA gene under control of a strong, con­stitutive TPI1 promoter on a 2^-based plasmid (pAKX002) in the haploid laboratory strain S. cerevisiae CEN. PK resulted in XI activities ranging from 0.33 to 1.1 ^mol (mgprotein)-1 min-1 in cell extracts [42]. These activities are similar to those of key enzymes of alcoholic fermentation in glucose — fermenting cultures [68]. Apparently, conditions in the cytosol of S. cerevisiae do not preclude accurate folding of the fungal XI, as has previously been re­ported for the Streptomyces rubiginosus XI [21]. In addition, in contrast to the previously expressed XI from T. thermophilus, the Piromyces XI yielded the above-mentioned activities at a temperature of 30 °C.

Although the high XI activities found in XylA-expressing S. cerevisiae strains provided an excellent starting point for further strain development, they did not as such enable a high specific rate of D-xylose fermentation. In fact, the specific growth rate in aerobic cultures on 20 g L-1 D-xylose as the sole carbon source was only 0.005 h-1 (Fig. 4). A similar very low spe­cific growth rate was found in earlier engineered S. cerevisiae strains that expressed the P. stipitis xylose reductase and xylitol dehydrogenase genes [38, 39]. The low rate of D-xylose conversion in strains with a high XI activity sug­gested that D-xylose consumption was either controlled by D-xylose transport or by reactions downstream from D-xylulose.

Since the low specific growth rates of the Piromyces XylA-expressing strains on D-xylose complicated studies in batch cultures, initial studies on D-xylose consumption kinetics and product formation were performed in anaerobic chemostat cultures grown on glucose-xylose mixtures. Anaero­bic chemostat cultivations on glucose alone demonstrated that expression of a heterologous XI did not interfere with product formation during growth on glucose [42]. However, when D-xylose was also included in the medium of the anaerobic glucose-limited chemostat cultures, a significant effect of XylA expression was observed. With 20% of the added D-xylose being consumed, a significant increase of the ethanol yield on consumed glucose was observed (from 0.40 g g-1 to 0.44 g g-1). Although no labeling studies were performed, it stands to reason that this ethanol was produced from the consumed D-xylose.

Interestingly, these anaerobic chemostat cultures of the XylA-expressing strains excreted significant amounts of D-xylulose. At a specific D-xylose con­sumption rate of 0.73 mmol (gbiomass)-1 h-1 this yeast excreted D-xylulose at a rate of 0.20 mmol (gbiomass)-1 h-1 (corresponding to 30% of consumed D-xylose), which suggested that reactions downstream of D-xylulose were rate-controlling. Moreover, small amounts of xylitol were produced in these cultivations, suggesting involvement of a non-specific aldose reductase such as encoded by GRE3 [66]. This information on D-xylulose and xylitol produc­tion was used in subsequent metabolic engineering attempts to improve the D-xylose consumption rate and to minimise xylitol formation.

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