Regulation of pyruvate metabolism in chemostat cultures of Kluyveromyces lactis CBS 2359

Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied in glucose-limited, ethan ol-limited and acetate-limited chemostat cultures and during a glucose puls e added to a glucose-limited steady-state culture. Enzyme activity levels o f the pyruvate dehydrogenase complex, pyruvate decarboxylase, alcohol dehyd rogenase, acetyl-CoA synthetase and glucose-6-phosphate dehydrogenase were determined in all steady-state cultures. In addition, the mRNA levels of Kl ADH1-4, KlACS1, KlACS2, KlPDA1, KLPDC1 and RAG1 were monitored under steady -state conditions and during glucose pulses. In K, lactis, as in Saccharomy ces cerevisiae, enzymes involved in glucose utilization (glucose-g-phosphat e dehydrogenase, pyruvate dehydrogenase, pyruvate decarboxylase) showed the highest expression levels on glucose, whereas enzymes required for ethanol or acetate consumption (alcohol dehydrogenase, acetyl-CoA synthetase) show ed the highest enzyme activities on ethanol, In cases where mRNA levels wer e determined, these corresponded well with the corresponding enzyme activit ies, suggesting that regulation is mostly achieved at the transcriptional l evel. Surprisingly, the activity of the K. lactis pyruvate dehydrogenase co mplex appeared to be regulated at the level of KlPDA1 transcription. The co nclusions from the steady-state cultures were corroborated by glucose pulse experiments. Overall, expression of the enzymes of pyruvate metabolism in the Crabtree-negative yeast K. lactis appeared to be regulated in the same way as in Crabtree-positive S. cerevisiae, with one notable exception: the PDA1 gene encoding the E1 alpha. subunit of the pyruvate dehydrogenase comp lex is expressed constitutively in S, cerevisiae, Copyright (C) 2000 John W iley & Sons, Ltd.