Improving initial xylose metabolism in recombinant Saccharomyces cerevisiae

Abstract: The aim of the thesis is to improve the initial steps of xylose metabolism in recombinant Saccharomyces cerevisiae. S. cerevisiae takes up xylose of poor affinity by means of hexose transporters. Metabolic control analysis was used to investigate whether the low xylose utilisation rate is due to inefficient transport. Xylose transport was found to have little control over the aerobic xylose utilisation rate in strain TMB3001, due to its high steady-state intracellular xylose concentration. In strain TMB3260, due to the overexpression of xylose reductase (XR), the intracellular xylose concentration was lower than in TMB3001 and transport had stronger control over the xylose flux at low extracellular xylose concentrations. Candida intermedia PYCC4715 was identified as being a yeast very efficient in utilising xylose. Kinetic characterisation of the xylose transport revealed the presence of a low-affinity facilitated diffusion system and a high-affinity proton symport system. The former appeared to be constitutive, whereas the latter was repressed by glucose and xylose. Both systems were of high capacity compared with known transport systems from other xylose utilising yeasts, which may facilitate cloning in S. cerevisiae. The conversion of xylose to xylulose by XR and xylitol dehydrogenase (XDH) leads to an imbalance in cofactor regeneration. This, in turn, results in the excretion of xylitol by xylose-utilising recombinant S. cerevisiae. Xylitol production can be circumvented by substituting xylose isomerase (XI) for XR and XDH. XI from Streptomyces rubiginosus was expressed inactively in S. cerevisiae due to misfolding of the protein. XI from Thermus thermophilus is actively expressed in S. cerevisiae, but possesses insufficient activity at 30°C. Three mutants were isolated after error-prone PCR and selection in Escherichia coli. Although the catalytic rate constants were 5-9 fold as high for the mutants as for the wild type enzyme, but the catalytic efficiency was not improved significantly. The mutats have lower sensitivity to inhibition by xylitol, which makes them especially suitable for expression in S. cerevisiae.