Overexpression of malic enzyme in yeasts, molds and bacteria
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Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2. The Saccharomyces cerevisiae MAE1gene 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 CO2atmosphere 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 CO2atmosphere, 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.
Malic enzyme is a ubiquitous enzyme that catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2, using either NAD+ (EC 1.1.1.38-39) or NADP+ (EC 1.1.1.40) as the redox cofactor. In eukaryotes, malic enzyme has been found in the cytosol and/or in mitochondria and, in plants, in the chloroplasts (20, 35). The reaction has a ΔG0′ of ca. −8 kJ mol−1 in the decarboxylating direction (19) and, although both carboxylation and decarboxylation have been observed in vitro, substrate affinities and maximum reaction rates generally appear to favor the latter (5, 14, 34). Despite decades of study, the physiological function of malic enzyme often remains enigmatic. Proposed physiological roles for the decarboxylating reaction include provision of pyruvate, inorganic carbon, reducing power, and pH regulation (20, 26). Carboxylating roles for malic enzyme have also been suggested (25, 30), although in vivo evidence is sparse. The strongest indications for in vivomalic enzyme activity in the carboxylating direction come from experiments with engineered Escherichia coli, in which overexpression of malic enzyme improved succinate titers (17, 27, 28). However, interpretation of these results was complicated by the fact that these strains still expressed phospho-enol-pyruvate carboxylase, the main carboxylating enzyme in glucose-grown wild-type E. coli (2)
Malic enzyme is a ubiquitous enzyme that catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2, using either NAD+ (EC 1.1.1.38-39) or NADP+ (EC 1.1.1.40) as the redox cofactor. In eukaryotes, malic enzyme has been found in the cytosol and/or in mitochondria and, in plants, in the chloroplasts (20, 35). The reaction has a ΔG0′ of ca. −8 kJ mol−1 in the decarboxylating direction (19) and, although both carboxylation and decarboxylation have been observed in vitro, substrate affinities and maximum reaction rates generally appear to favor the latter (5, 14, 34). Despite decades of study, the physiological function of malic enzyme often remains enigmatic. Proposed physiological roles for the decarboxylating reaction include provision of pyruvate, inorganic carbon, reducing power, and pH regulation (20, 26). Carboxylating roles for malic enzyme have also been suggested (25, 30), although in vivo evidence is sparse. The strongest indications for in vivomalic enzyme activity in the carboxylating direction come from experiments with engineered Escherichia coli, in which overexpression of malic enzyme improved succinate titers (17, 27, 28). However, interpretation of these results was complicated by the fact that these strains still expressed phospho-enol-pyruvate carboxylase, the main carboxylating enzyme in glucose-grown wild-type E. coli (2)
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