Intracellular pH and metabolic activity of long-chain dicar☐ylic acid-producing yeast

The intracellular pH (pHi of α-ω-dicar☐ylic acid producingCandida tropicalis was determined by a fluorescence technique using the pH-sensitive fluorescent probe 5(6)-car☐yfluorescein diacetate. Fermentations withn-tridecane substrate to produce α,ω-tridecanedioic acid were carried out in a 5-l bioreactor in which growth and production were separated. During the growth phase, the measured pHi values were varied from 5.65 to 6.15 in all the experiments performed under different constant pH-operating conditions. The specific rates of growth, sucrose consumption, CO2 production, and O2 consumption were correlated with pHi. Cytochrome P450 monooxygenase (P450), which catalyzesn-alkane hydroxylation, was only slightly expressed during the growth phase. During the first 6 h of the production phase, P450 activity was induced rapidly accompanying higher pHi. A much higher level of P450 activity was observed at pHi of 6.55±0.15 for all the fermentations, with maximum productivity (1.919 g/l/h) occurring when using an optimal pH-control strategy. However, P450 activity, tridecanedioic acid productivity, and pHi decreased progressively during the latter part of the production phase, as a consequence of the metabolic activity changes of the cells. Even though culture pH has only a slight influence on pHi, the metabolic activity ofC. tropicalis is sensitive to the variations in pHi. The measured pHi varied from 6.1 to 6.7 during the production phase for all the fermentations. Thus, both tridecanedioic acid productivity and P450 activity are correlated with pHi or pH gradients across the cell membrane.