Expression of Genes Encoding F ₁ -ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

Abstract

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F 1 domain of the membrane-bound (F 1 F 0 ) H ؉ -ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F 1 -ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F 1 -ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.Lactic acid bacteria are used extensively in the dairy industry, where the production of lactic acid is important for texture, flavor, and preservation purposes. In addition, lactic acid bacteria are also used for industrial lactate production, which has numerous applications, such as cosmetics, cleaning agents, and biodegradable polylactic acid polymers. From an industrial point of view there is great interest in improving the performance of these organisms with respect to both the rate and the yield of lactate production.In spite of the importance of glycolysis for fermentation purposes, it is still not known what controls the glycolytic flux in microbial bioreactors. It has been suggested that the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a high level of control (estimated to be 90% of the control) over the glycolytic flux in nonproliferating cells of Lactococcus lactis (33). However, it has recently been shown that GAPDH has no control over the glycolytic flux in steadily growing L. lactis cells (Solem, Koebmann, and Jensen, unpublished data). The control over the glycolytic flux exerted by lactate dehydrogenase was also reported to be close to zero (2).According to metabolic control theory (16, 25), flux control can reside in any of the steps in a system; i.e., it can reside in the numerous processes that consume the ATP generated in glycolysis (8,17). Indeed, we have recently shown that at least 75% of the control over glycolysis in aerobic Escherichia coli cultures occurs in the ATP-consuming reactions (26). This result was obtained by overexpression of genes encoding part of the F 1 unit of the (F 1 F 0 ) H ϩ -ATPase, which resulted in uncoupling of glycolysis from biomass production and a 70% increase in the glycolytic flux.In this paper we show that expression of genes encoding...

Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

“…In Escherichia coli, the glycolytic flux appears to be almost exclusively controlled by the processes that consume ATP (23). A similar study with L. lactis MG1363 showed that ATP consumption controls the glycolytic flux in nongrowing cells but not during fast growth (24). This result implies that the control of glycolytic flux in fast-growing cells probably resides in the glycolytic reactions themselves, which would fit with the observations of Poolman et al (33).…”

“…A plasmid harboring the atpA, atpG, and atpD genes, encoding F 1 -ATPase in L. lactis (24), was introduced into L. lactis CS811 by standard transformation procedures.…”

Bifidobacterium lactisDSM 10140: Identification of theatp(atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny

“…For many bacteria it has been shown that treatment with acid and a subsequent decrease in the intracellular pH are accompanied by an increase in the amount of the F 1 F 0 -ATPase (2,19,21,30,31). This phenomenon has been studied most extensively in S. faecalis (2,17), in L. lactis (18,19), and in L. acidophilus (21), in which the cytoplasmic pH is maintained by the amount and activity of the H ϩ -ATPase, which catalyzes the ATP-driven translocation of protons from the cytoplasm. In S. faecalis it has been demonstrated that the transcriptional activity of the atp operon is not pH regulated and that the increase in the enzyme level is pH regulated at the enzyme assembly step.…”

“…Analysis of the GϩC contents of these organisms and the bias of their codon usage support the hypothesis that there was probably horizontal transfer of the atpD genes. Notably, these organisms showed a reshuffling of the atp operon; in fact, the F 0 gene order of Lactococcus and Streptococcus is atpEBF, compared to the most common order, atpBEF (18,20). The atpDand 16S rRNA gene-based trees were generated from the same set of strains with only two exceptions for the 42 strains.…”

“…Conversely, in bacteria that lack a respiratory chain, its role is to create a proton gradient, and this process is then driven by ATP hydrolysis. This is the case for streptococci (3,20), E. faecalis (17), L. acidophilus (21), and Lactococcus lactis (18,19). In all these bacteria the activity of the F 1 F 0 -ATPase increases as the pH of the growth media decreases.…”