Physiological Engineering of Xylose Utilisation by Recombinant Saccharomyces cerevisiae

Xylitol production by recombinant, <i>XYL1</i>-expressing <i>Saccharomyces cerevisiae</i> was investigated in fed-batch fermentation using different cosubstrates for growth, and generation of reduced cofactors and maintenance energy. Xylose was converted into xylitol with 1:1 yield. Using ethanol as cosubstrate, the yield of xylitol on ethanol and the specific xylitol productivity decreased with increasing aeration, whereas with decreasing aeration acetic acid accumulated, causing cell death. An increase in pH from 4.5 to 6.5 removed the toxic effect of 10 g l-1 acetic acid, and a xylitol yield on ethanol, specific productivity and volumetric productivity of 2.4 g g-1, 0.07 g g-1 h-1 and 1.0 g l-1 h-1, respectively, were obtained under oxygen-limited conditions in a rich medium. Using glucose as cosubstrate enabled anaerobic xylitol production, but the glucose feed rate required careful control because glucose accumulation inhibited xylose conversion. In a mineral medium the xylitol yield on glucose, specific productivity and volumetric productivity were 1.2 g g-1, 0.19 g g-1 h-1 and 0.42 g l-1 h-1, respectively. Among fermentable sugars, galactose was the most efficient cosubstrate, yielding 6.1 g xylitol g-1 galactose in fed-batch fermentation. In continuous xylitol production with immobilised cells in a packed-bed reactor using glucose as cosubstrate, the xylitol yield on glucose, specific productivity and volumetric productivity were 0.44 g g-1, 0.10 g g-1 h-1 and 5.6 g l-1 h-1, respectively.

The uptake of xylose, XR activity or supply rate of reduced cofactors control the rate of xylose conversion to xylitol. The uptake of xylose was not rate-limiting under cosubstrate-limited conditions, but many cosubstrates inhibited xylose conversion when present in high concentration, due to competitive and non-competitive inhibition of transport, rendering the transport step rate-controlling. The XR activity had very low control over the xylose conversion rate, as a 20-fold increase in specific activity increased the specific xylitol productivity less than 2-fold. In glucose-limited chemostat cultivation, xylose conversion affected the redox balance, as indicated by an increase in acetate yield and pentose phosphate pathway activity, and a decrease in glycerol yield. A 2-fold increase in glucose flux was accompanied by a 1.3-fold increase in xylose conversion rate, indicating that the supply rate of reduced cofactors was a rate-controlling factor.

In a <i>XYL1</i>- and <i>XYL2</i>-expressing strain, ten-fold overexpression of transaldolase enhanced growth but not ethanol production in anaerobic batch fermentations of xylose/glucose mixtures. Simultaneous glucose metabolism enhanced the xylose conversion rate and decreased xylitol excretion compared with xylose metabolism alone, but did not enhance ethanol production.

In the recombinant strains, high expression levels of plasmid-encoded genes led to high protein burden and marked growth disadvantage compared with revertants, which caused a rapid decline in recombinant enzyme activity during chemostat cultivation in a selective medium. The excretion of amino acids into the medium, destroying its selectivity, structural rearrangements within the plasmid and the effect of cultivation conditions on the regulation of the expression promoters contributed to the instability.