Use of in vivo C-13 nuclear magnetic resonance Spectroscopy to elucidate L-arabinose metabolism in Yeasts

Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 were shown to grow well on L-arabinose, albeit exhibiting distinct features that justify an in-depth comparative study of their respective pentose catabolism. Carbon-13 labeling experiments coupled with in vivo NMR were used to investigate L-arabinose metabolism in these yeasts, thereby complementing recently reported physiological and enzymatic data. The label supplied in L-[2-(13)C]arabinose to non-growing cells, under aerobic conditions, was found on C1 and C2 of arabitol and ribitol, C2 of xylitol, and on C1, C2 and C3 of trehalose. The detection of labeled arabitol and xylitol constitutes additional evidence for the operation in yeast of the redox catabolic pathway widespread in filamentous fungi. Furthermore, labeling at positions C1 of trehalose and arabitol demonstrates that glucose-6-phosphate is recycled through the oxidative pentose phosphate pathway (PPP). This result was interpreted as a metabolic strategy to regenerate NADPH, the cofactor essential to sustain L-arabinose catabolism at the level of L-arabinose reductase and L-xylulose reductase. Moreover, the observed synthesis of D-arabitol and ribitol provides a route to supply NAD(+) under oxygen-limiting conditions. In P. guilliermondii PYCC 3012, the strong accumulation of L-arabitol (up to 0.4 M, intracellular concentration) during aerobic L-arabinose metabolism denotes the existence of a bottleneck at the level of L-arabitol 4-dehydrogenase. This report provides the first experimental evidence of a link between L-arabinose metabolism in fungi and the oxidative branch of PPP, and suggests rational guidelines for the design of strategies towards the production of new and efficient L-arabinose-fermenting yeasts.