Inactivation of the Kluyveromyces lactis KIPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobicalcoholic fermentation

The KIPDA1 gene, encoding the E1 alpha subunit of the mitochondrial pyruvat e-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis geno mic library by screening with a 1.1 kb internal fragment of the Saccharomyc es cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KIPDA 1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell e xtracts. The maximum specific growth rate on glucose of null mutants was 3. 5-fold lower than that of the wild-type, whereas growth on ethanol was unaf fected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype , i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic KIpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KIPDA1 gene but n ot, as previously found for S. cerevisiae pda1 mutants, by cultivation in t he presence of leucine, The occurrence of aerobic fermentation and slow gro wth of the KIpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and a cetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypas s completely replace the BDH complex, thus allowing fully respiratory growt h. This resulted in a lower biomass yield [g biomass (g glucose)(-1)] than in the wild-type due to a higher consumption of ATP in the PDH bypass compa red to the formation of acetyl-CoA via the PDH complex.