Distinct effects of sorbic acid and acetic acid on the electrophysiology and metabolism of Bacillus subtilis.

Sorbic acid and acetic acid are among the weak organic acid preservatives most commonly used to improve the microbiological stability of foods. They have similar pKa values, but sorbic acid is a far more potent preservative. Weak organic acids are most effective at low pH. Under these circumstances, they are assumed to diffuse across the membrane as neutral undissociated acids. We show here that the level of initial intracellular acidification depends on the concentration of undissociated acid and less on the nature of the acid. Recovery of the internal pH depends on the presence of an energy source, but acidification of the cytosol causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the membrane potential than acetic acid. Together these effects may also slow the rate of ATP synthesis significantly and may thus (partially) explain sorbic acid's effectiveness.

Alternative routes to biofuels: light-driven biofuel formation from CO2 and water based on the 'photanol' approach.

For a sustainable energy future, the development of efficient biofuel production systems is an important prerequisite. Here we describe an approach in which basic reactions from phototrophy are combined in single organisms with key metabolic routes from chemotrophic organisms, with C(3) sugars as Glyceraldehyde-3-phosphate as the central linking intermediate. Because various metabolic routes that lead to the formation of a range of short-chain alcohols can be used in this approach, we refer to it as the photanol approach. Various strategies can be explored to optimize this biofuel production strategy.

Respiration of Escherichia coli can be fully uncoupled via the nonelectrogenic terminal cytochrome bd-II oxidase.

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

Control of specific growth rate in Saccharomyces cerevisiae.

In this contribution we resolve the long-standing dispute whether or not the Monod constant (K(S)), describing the overall affinity of an organism for its growth-limiting substrate, can be related to the affinity of the transporter for that substrate (K(M)). We show how this can be done via the control of the transporter on the specific growth rate; they are identical if the transport step has full control. The analysis leads to the counter-intuitive result that the affinity of an organism for its substrate is expected to be higher than the affinity of the enzyme that facilitates its transport. Experimentally, we show this indeed to be the case for the yeast Saccharomyces cerevisiae, for which we determined a K(M) value for glucose more than two times higher than the K(S) value in glucose-limited chemostat cultures. Moreover, we calculated that at glucose concentrations of 0.03 and 0.29 mM, the transport step controls the specific growth rate at 78 and 49 %, respectively.

Protein production by Escherichia coli wild-type and DeltaptsG mutant strains with IPTG induction at the onset.

During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate.

Changes in the redox state and composition of the quinone pool of Escherichia coli during aerobic batch-culture growth.

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

Reducing the glucose uptake rate in Escherichia coli affects growth rate but not protein production.

Although glucose is an inexpensive substrate widely used as a carbon source in Escherichia coli recombinant fermentation technology, 10-30% of the carbon supply is wasted by excreting acetate. In addition to the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. Because glucose can enter the cell via several transport systems, isogenic strains defective in one or two of these transport systems were constructed. The effects of changes in the glucose uptake capacity on the in vivo flux distribution to a desired end product (beta-galactosidase) and to acetate were studied. The lack of one of the components (IICB(Glc) protein) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced the growth rate significantly. The maintenance of a low-copy plasmid in this strain resulted in further arrest of the growth rate. However, beta-galactosidase production had no effect on growth rate. This strain directed more carbon into biomass and carbon dioxide, and less into acetate. Beta-galactosidase was produced in amounts not significantly different from the wild-type strain from half the amount of glucose. An explanation for the experimental results is given, making use of published results on metabolic regulation.

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans.

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.

Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae.

The tendency of Saccharomyces cerevisiae to favor alcoholic fermentation over respiration is a complication in aerobic, biomass-directed applications of this yeast. Overproduction of Hap4p, a positive transcriptional regulator of genes involved in respiratory metabolism, has been reported to positively affect the balance between respiration and fermentation in aerobic glucose-grown batch cultures. In this study, the effects of HAP4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN.PK 113-7D under a variety of growth conditions. In aerobic glucose-limited chemostat cultures, overexpression of HAP4 increased the specific growth rate at which aerobic fermentation set in by about 10% relative to the isogenic wild-type. Upon relief of glucose-limited conditions, the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain. The effect of Hap4p overproduction was most drastic in aerobic, glucose-grown chemostat cultures in which ammonium was limiting. In such cultures, the biomass yield on glucose was double that of the wild-type.

Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli.

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a delta ArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis.

Redirection of the respiro-fermentative flux distribution in Saccharomyces cerevisiae by overexpression of the transcription factor Hap4p.

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.

Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae.

The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h-1 under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h-1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8-HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zero-trans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.

The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli.

Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT

Production of actinorhodin by Streptomyces coelicolor A3(2) grown in chemostat culture.

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.

Bioenergetic consequences of microbial adaptation to low-nutrient environments.

A striking property of many prokaryotes is their enormous metabolic flexibility with respect not only to catabolic and anabolic substrates but also with respect to the continuously changing availability of nutrients. The phenotypic responses to low-nutrient growth conditions involve structural changes in the cellular make-up, changes in the specific capacity of the enzyme system(s) involved in uptake and/or assimilation of the limiting nutrient and changes in the affinity of these enzymes. Here the responses of some members of the Enterobacteriaceae to potassium-, ammonium- and energy source-limited conditions will be reviewed. The focus will be on the energetic consequences of these adaptations as reflected by the growth yield value for the energy source (Y energy source). It will be illustrated that Y energy source values can be dramatically lowered as a result of incomplete oxidation of the energy source (overflow metabolism), bypassing potential sites of energy conservation (uncoupling) or catabolic cycles that have no other apparent effect than the hydrolysis of ATP (futile cycles). Thus, it is concluded that adaptation to low nutrient conditions aims at maintaining high metabolic fluxes at low nutrient concentrations at the cost of a loss in the energetic efficiency of the overall metabolism.

Pyrroloquinoline quinone, a chemotactic attractant for Escherichia coli.

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.

The physiological function of periplasmic glucose oxidation in phosphate-limited chemostat cultures of Klebsiella pneumoniae NCTC 418.

Periplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen- and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)