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

Abstract

Bioverfa hr ungstechn i k,University o f Stuttgart, Allmandring 31, D70569 S t u t t g a rt, Germ a n y 2 Kluyver Institute of 3 lnstitut fur The KlPDA1 gene, encoding the E l a subunit of the mitochondria1 pyruvatedehydrogenase (PDH) complex was isolated from a Kluyweromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerewisiae PDA 1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerewisiae counterpart. Disruption of KlPDAI resulted in complete absence of PDH activity in cell extracts. 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 unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtreenegative 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 Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA7 gene but not, as previously found for S. cerewisiae pdal mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the KlpdaI null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only a t relatively low growth rates (D = 010 h-') in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-l] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

“…The altered balance between these activities, as shown with the adh°mutant strain, leads to the presence of two G6PDH activity bands. This pattern can be explained by the activation of the KlAld6-dependent PDH bypass, which conveys the glucose flux toward NAD(P)H accumulation-dependent acetaldehyde and acetate production (42) (Fig. 1B).…”

“…To test the influence of fermentative and respiratory metabolism on the presence of the two activities, we analyzed three isogenic mutants impaired for those metabolisms. Klpda1⌬, one of these mutants, lacks the E1a subunit of the pyruvate dehydrogenase complex and, therefore, is unable to dissimilate pyruvate through mitochondria (36,42). This respiratory mutant showed a G6PDH activity pattern identical to that of the wild type (Fig.…”

Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene

“…The deleted strains PMI and MW341-5/Klpdc1⌬ were obtained from wild-type strains PM6-7A and MW341-5, respectively, by disruption of KlPDC1 with a deletion cassette containing the marker gene URA3 from S. cerevisiae (3,6). The deleted strains GG1993 (20) and PM6-7A/Klpda1⌬ were obtained from wild-type strains CBS2359 and PM6-7A, respectively, by disruption of KlPDA1 with the bacterial Tn5BLE gene from plasmid pUT322 (7). Double-deleted strains were constructed as follows.…”

“…In this yeast, pyruvate is largely channeled into the tricarboxylic acid cycle by the pyruvate dehydrogenase (PDH) complex. On the other hand, K. lactis strains with no PDH activity have a vigorous fermentative metabolism on glucose in aerobic batch cultures, and only in glucose-limited aerobic chemostat conditions can the Klpda1⌬ strains metabolize glucose exclusively through the cytoplasmic acetyl-CoA pathway (20).…”

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

“…Slow consumption was probably related to disruption of KlPDA1, because this disruption in the wild-type strain resulted in poor growth in glucose medium. 17) The industrially important yeast Candida utilis, classified as a Crabtree-negative yeast, is currently used to produce several valuable chemicals, such as glutathione and RNA. [18][19][20] C. utilis can grow on inexpensive substrates, e.g., pulping-waste liquors from the paper industry, while most other yeasts cannot.…”