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Applied and Environmental Microbiology, March 2008, p. 1845-1855, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.02453-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of In Vivo 13C Nuclear Magnetic Resonance Spectroscopy To Elucidate L-Arabinose Metabolism in Yeasts{triangledown}

César Fonseca,1 Ana Rute Neves,2 Alexandra M. M. Antunes,3 João Paulo Noronha,3 Bärbel Hahn-Hägerdal,4 Helena Santos,2* and Isabel Spencer-Martins1*

Centro de Recursos Microbiológicos, Department of Life Sciences, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal,1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal,2 REQUIMTE, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal,3 Department of Applied Microbiology, Lund University, SE-221 00 Lund, Sweden4

Received 31 October 2007/ Accepted 20 January 2008

Candida arabinofermentans PYCC 5603T 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-[2-13C]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.

* Corresponding author. Mailing address for Helena Santos: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal. Phone: 351 214 469 828. Fax: 351 214 428 766. E-mail: santos{at}itqb.unl.pt. Mailing address for Isabel Spencer-Martins: Centro de Recursos Microbiológicos, Department of Life Sciences, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Phone and fax: 351 212 948 530. E-mail: ism{at}fct.unl.pt

{triangledown} Published ahead of print on 1 February 2008.

Applied and Environmental Microbiology, March 2008, p. 1845-1855, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.02453-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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