Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

Abstract

Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO 2 . 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 carboxylasenegative (Pyc ؊ ) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvatecarboxylating, 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 CO 2 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 pyc1,2⌬ pdc2⌬ strain grown under a CO 2 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 C 4 -dicarboxylic acid production.

Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells

“…In contrast, we observed high transcriptional expression of pyruvate dehydrogenase ( Pdha1 , Figure 3E), which catalyzes formation of acetyl coA. We observed low transcriptional expression of malic enzyme 2 ( Me2 ) (Figure 3E), but in vivo this enzyme preferentially decarboxylates malate to form pyruvate rather than the reverse reaction (Zelle et al, 2011). Thus, we reasoned that pyruvate plays a non-redundant role with LCFAs not necessarily because of the unique anaplerotic reactions it can mediate, but potentially due to different metabolic cues that regulate its formation and oxidation.…”

Manipulation of the ATP pool as a tool for metabolic engineering

“…For example, using either malic enzyme or PEP carboxykinase (acting in the carboxylating and thus opposite direction of the typical decarboxylating activity) instead of pyruvate carboxylase increases the overall ATP yield by one ATP per C3[EPS]C4 carboxylation event. These replacements enable anaerobic malic acid production and increase the ATP yield for redox-neutral succinic acid production significantly [15,[30][31][32][33].…”

Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon

“…As shown by our [ 14 C]-labeling experiments (Figure 3 and Supplementary Tables S5-S8), anaplerotic fixation of CO 2 through its carboxylation is catalyzed by PFOR (HLASF_0492-0495) at the rates 0.04 ± 0.01 fg C per cell per day. During the growth on pyruvate the anaplerotic assimilation of bicarbonate by HSR2 is likely catalyzed by a NADP-malic enzyme (HLASF_1723), working in the direction of carboxylation (Hong and Lee, 2001;Zelle et al, 2011).…”