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Adenosine-5'-triphosphate (ATP) is consumed as a biological energy source by many intracellular reactions. Thus, the intracellular ATP supply is required to maintain cellular homeostasis. The dependence on the intracellular ATP supply is a critical factor in bioproduction by cell factories. Recent studies have shown that changing the ATP supply is critical for improving product yields. In this review, we summarize the recent challenges faced by researchers engaged in the development of engineered cell factories, including the maintenance of a large ATP supply and the production of cell factories. The strategies used to enhance ATP supply are categorized as follows: addition of energy substrates, controlling pH, metabolic engineering of ATP-generating or ATP-consuming pathways, and controlling reactions of the respiratory chain. An enhanced ATP supply generated using these strategies improves target production through increases in resource uptake, cell growth, biosynthesis, export of products, and tolerance to toxic compounds.

Background: Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis., Results: To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low- then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae., Conclusions: Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H(+) transporter) and TF(0)F(1)-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.

There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene deletion strains that had over 150% higher total cellular ATP synthetic activity relative to the parental strain. Most of them also showed higher ATP-driven glutathione synthesis. The deleted genes of the identified strains that showed increased efficiency of ATP regeneration for glutathione production could be divided into the following four groups: (1) glycolytic pathway-related genes, (2) genes related to degradation of ATP or adenosine, (3) global regulatory genes, and (4) genes whose contribution to the ATP regeneration is unknown. Furthermore, the high glutathione productivity of DeltanlpD, the highest glutathione-producing mutant strain, was due to its reduced sensitivity to the externally added ATP for ATP regeneration. This study showed that the Permeable Cell Assay was useful for improving the ATP-regenerating activity of E. coli for practical applications in various ATP-driven bioprocesses, much as that of glutathione production.

Adenosine 5'-triphosphate (ATP) generation is an essential biological reaction for all living cells. Recently, we developed a Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity, mainly resulting from glycolysis [Hara, K.Y., Mori, H., 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. J. Biomol. Screen. 11, 310-317]. By using this method, we determined the cellular ATP synthetic activity in the stationary phase of a complete set of single-gene deletion strains of Escherichia coli. Their activities ranged from a minimum of 2% to a maximum of 445%, relative to parental strains. Deletions of metabolism-related genes frequently caused an increase in the rate of ATP synthetic activity, while activity was reduced by deletions of a variety of functional genes, including many poorly characterized genes. We also demonstrated that the deletion of the ptsG gene doubled ATP-driven glutathione synthesis and increased cellular ATP synthetic activity. Our study also indicated that it should be easy to obtain active strains for ATP synthesis from deletion strains. Overall, the data set of this study may be useful to improve E. coli strains for ATP-dependent industrial processes and, therefore, may be important for the design of so-called cell factories.

"Permeable Cell Assay" is an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source for biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this assay, bioluminescence from the luciferin-luciferase reaction is used for the quantitative measurement. Under the assay condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure cellular ATP synthetic activity, combination of osmotic shock and detergent, Triton X-100 treatment is used to make bacterial cells permeable. ATP is discharged from permeable cells and reacted with external luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence is increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity is calculated from the slope of linearly increasing bioluminescence.

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha 3 beta 3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, interim dwell, and rapid 40 degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (alpha 3 beta 3 gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATP gamma S (adenosine 5'-[gamma-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP gamma S hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.

Background: Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. Results: To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low- then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae. Conclusions: Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening. [ABSTRACT FROM AUTHOR]

Reports that the rotation of rotary motor enzyme F1-adenosine triphosphatase (ATPase) spontaneously lapses into long 30 second pauses during steady-state catalysis. Effects of adenosine diphosphate-magnesium and mutation on the pauses; Pausing position of F1-ATPase.