Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

• Published in: Applied and Environmental Microbiology Vol. 77; no. 3; pp. 732 - 738
• Main Authors: Zelle, Rintze M, Harrison, Jacob C, Pronk, Jack T, van Maris, Antonius J.A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.2011; American Society for Microbiology (ASM)
• Subjects: Anaerobiosis; ATP; Biochemistry; Biological and medical sciences; Biotechnology - methods; Carbon Dioxide - metabolism; Catalysis; Cytosol - enzymology; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genetic Engineering - methods; Glucose - metabolism; Malate Dehydrogenase - genetics; Malate Dehydrogenase - metabolism; Malates - metabolism; Microbiology; Mitochondria; Physiology; Point Mutation; Pyruvic Acid - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Yeast; Ascomycota; Fungi; Yeast; Enzyme; Saccharomyces cerevisiae
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02132-10

Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO₂. The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylase-negative (Pyc⁻) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc⁻ S. cerevisiae background. When PDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO₂ atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h⁻¹). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2Δ pdc2Δ strain grown under a CO₂ atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C₄-dicarboxylic acid production.