Use of In Vivo 13 C Nuclear Magnetic Resonance Spectroscopy To Elucidate l -Arabinose Metabolism in Yeasts

Abstract

ABSTRACT 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 nuclear magnetic resonance (NMR) spectroscopy 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 nongrowing cells, under aerobic conditions, was found on C-1 and C-2 of arabitol and ribitol, on C-2 of xylitol, and on C-1, C-2, and C-3 of trehalose. The detection of labeled arabitol and xylitol constitutes additional evidence for the operation in yeast of the redox catabolic pathway, which is widespread among filamentous fungi. Furthermore, labeling at position C-1 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 for sustaining 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 with which to supply NAD + under oxygen-limiting conditions. In P. guilliermondii PYCC 3012, the strong accumulation of l -arabitol (intracellular concentration of up to 0.4 M) during aerobic l -arabinose metabolism indicates the existence of a bottleneck at the level of l -arabitol 4-dehydrogenase. This report provides the first experimental evidence for a link between l -arabinose metabolism in fungi and the oxidative branch of the PPP and suggests rational guidelines for the design of strategies for the production of new and efficient l -arabinose-fermenting yeasts.