Overview of Experimental Design
We applied 13
CMFA to characterize the metabolic response of G. sulfurreducens
to variations in growth conditions. G. sulfurreducens
was cultured either in chemostats (E1, E3, and E4) or in batch mode (E2) with different combinations of electron donors and acceptors (Table ). In all experiments, 30% [U-13
] acetate was provided as the 13
C carbon source which also served as the electron donor. In both E1 and E2, Fe(III)citrate served as the electron acceptor. In contrast to E1 where 5 mM acetate served as the electron donor in the chemostat, hydrogen and 1 mM acetate were supplemented under batch culture conditions. In E3 and E4, fumarate was provided as the electron acceptor at different fumarate to acetate ratios to examine the resulting metabolic phenotypes under donor-limiting (E3) and acceptor-limiting (E4) conditions. Selected acetate and fumarate concentrations were based on a previous chemostat study [36
] and in silico
simulations with the G. sulfurreducens
genome-scale metabolic model which suggested the optimal ratio of fumarate to acetate to be 3.01 for the complete oxidization of acetate [2
Cultivation conditions for Geobacter sulfurreducens to study intracellular metabolism with different electron donor/acceptor conditions.
Steady-State Effluxes in Chemostat Cultures
Extracellular yield coefficients for the three sets of chemostat cultures (E1, E3, E4) were determined from steady-state rate measurements. The complete oxidation of acetate to CO2 releases of 8 electrons, which is sufficient to convert 8 moles of Fe(III) to Fe(II) or 4 moles of fumarate to succinate. For E1 (5 mM acetate + 55 mM Fe(III)citrate), the molar yield of Fe(II) per mole of acetate consumed was 7.1 ± 0.5, suggesting that only 11.3% of the acetate in the growth medium was utilized for biomass synthesis. When fumarate was used as the electron acceptor, the molar yield of succinate per mole of acetate consumed was 2.9 ± 0.2 for E3 (5 mM acetate + 28 mM fumarate) and 2.5 ± 0.2 for E4 (10 mM acetate + 20 mM fumarate), indicating that a higher percentage of acetate, 27.5% to 37.5%, was utilized for biomass synthesis. The molar yield of fumarate uptake on acetate was 2.8 ± 0.1 and 2.5 ± 0.1 in E3 and E4, respectively. The biomass yield coefficient on acetate (YX/DON = gram dry cell weight produced per mmol acetate uptake) of the Fe(III)citrate culture (E1) was 2.8 × 10-3 ± 2 × 10-4 g/mmol, about 4-fold lower than those of the fumarate cultures, 0.013 ± 2 × 10-3 and 0.012 ± 2 × 10-3 for E3 and E4, respectively. Extracellular concentration measurements can be associated with larger uncertainties than mass spectrometric carbon labeling measurements. Thus, the effluxes were set as free parameters to be estimated from 13C labeling measurements during 13CMFA or calculated later from in vivo flux estimates.
Metabolic Fate of Citrate & Fumarate Carbons
In order to assess metabolic flux distributions in G. sulfurreducens
grown with Fe(III)citrate as the electron acceptor, we needed to determine whether G. sulfurreducens
is able to metabolize citrate in addition to acetate. We also investigated whether G. sulfurreducens
can utilize fumarate as a carbon source since previous studies have suggested that fumarate is not utilized as a carbon source [13
]. To this end, cells were grown in a batch culture containing 10 mM non-labeled acetate with 55 mM Fe(III)citrate (4.2% [U-13
]Fe(III)citrate) and 10 mM non-labeled acetate with 27.5 mM fumarate (30% [2,3-13
]fumarate), respectively. The carbon mass isotopomer distributions (MIDAA
) of alanine, glycine, valine, leucine, isoleucine, phenylalanine, serine, aspartate, and glutamate were compared to those obtained from an identical culture containing only non-labeled compounds.
Linear regression of the two sets of MIDAA
measured in the acetate/Fe(III)citrate culture gave a slope of 1.003 with R2
of 1.000 and an intercept of 0.000. In addition, the root mean square (RMS) [38
] of the difference between the two MIDAA
sets was 0.0006, which was significantly less than the average MIDAA
determined measurement error (ca. 0.003) [27
]. This result indicates that the citrate carbons were not utilized by G. sulfurreducens
cells, Fe(III)citrate served exclusively as an electron acceptor, and that acetate was the sole carbon source in experiments E1 and E2.
In contrast, the acetate/fumarate culture gave a distinct difference from the natural abundance (slope = 1.151, intercept = 0.030, R2 = 0.9326) with an RMS of 0.011. For instance, MIDAA of aspartate gave the largest discrepancy from the natural abundance (RMS = 0.08), followed by glycine (RMS = 0.054), glutamate (RMS = 0.049), serine (RMS = 0.044), phenylalanine (RMS = 0.032), isoleucine (RMS = 0.012), alanine, leucine, and valine (RMS = 0.004). Our results demonstrate that fumarate carbons can be metabolized into different amino acid biosynthesis precursors in the central metabolic pathways. Cellular fumarate utilization was investigated more quantitatively in terms of 13CMFA.
Assessment of Biological Reproducibility
The consistency between biological replicates was evaluated by comparing the MIDAA of replicates. The squared correlation coefficients (R2) between the MIDAA values of the biological replicates (n = 3) were higher than 0.9990 in all the chemostat experiments (E1, E3, E4), suggesting a high consistency between the replicates. Thus, the experimental data were considered to be unbiased by biological variations, and the intracellular metabolic fluxes were computed using the mean values of replicate MIDAA and measured effluxes.
Comparison of Amino Acid Labeling Patterns
13C-labeling patterns of metabolic products including proteinogenic amino acids are not direct measures of the in vivo flux distributions, but information on the flux distributions is recorded in the labeling patterns. Therefore, gross metabolic discrepancies or similarities induced by the different electron donor/acceptor conditions can be visualized by directly comparing the MIDAA values obtained from these conditions. As shown in Figure , the comparison of MIDAA values suggested that electron acceptor variation (Figure : Fe(III) vs. fumarate) resulted in a larger discrepancy in 13C labeling patterns than electron donor variation (Figure : acetate vs. hydrogen). In terms of the Euclidian distance, the measure of the difference of two sets of data; the MIDAA values from E3 to E1 (Figure ) or E4 to E1 (Figure ) was larger than that from E2 to E1 (Figure ). The distance from E2 to E3 or E4 (Figure ) was smaller than that from E1 to E3 or E4, however larger than the distance between E1 and E2. The most similar amino acid 13C labeling patterns were found between E3 and E4, in which only donor and acceptor concentrations were varied (Figure ). Nevertheless, detectable differences of MIDAA between E3 and E4 indicate that the physiological state of G. sulfurreducens is altered in response to varying donor and acceptor concentrations.
Comparisons of amino acid carbon mass isotopomer distributions between the experiments listed in Table 1. The straight line corresponds to a one-to-one (y = x) line. R: correlation coefficient; D: Euclidian distance.
Overview of in vivo Fluxes
To assess in vivo flux distributions from the four cultures of G. sulfurreducens, 500 stochastic numerical optimization runs were performed by generating random numbers of measurements (from the normal distributions estimated using experimentally determined means and standard deviations) and starting points (from a continuous uniform distribution between the upper and lower bound of each parameter). The details enzyme reactions comprised in the metabolic network are listed in Table , and the 95% confidence intervals for the resulting flux estimates are depicted in Figure . The fluxes were normalized to acetate uptake and can be understood as molar yield coefficients on acetate. For each experiment, the experimental measurements and the corresponding model-predicted values gave a high degree of consistency; their correlation coefficients (R) were higher than 0.9970 at the optimum with the slope and y-axis intercept close to 1 and 0, respectively.
Abbreviations and EC numbers of the enzymes considered in the central metabolic network of G. sulfurreducens for 13C metabolic flux analysis.
Figure 2 In vivo flux estimates (95% confidence intervals given for the flux mean estimates) for the central metabolic network of G. sulfurreducens, corresponding to E1, E2, E3, and E4 (Table 1). All fluxes are normalized to acetate uptake (ACKr, PTAr). Gray double-headed (more ...)
Under the examined four conditions, the majority of consumed acetate was oxidized through the TCA cycle (Figure ). When comparing fluxes in E1 and E2, the flux through the initial reaction of the TCA cycle (CS/ACONT) in the H2/Fe(III) culture with 1 mM acetate (E2) was significantly less than that obtained from the acetate/Fe(III) culture (E1). The addition of hydrogen increased the flux from acetyl-CoA to pyruvate (POR: pyruvate synthase) as well as other gluconeogenetic fluxes and resulted in a 2-fold higher biomass yield on acetate (cf. YXS values in Figure ). The coupling of Fe(III) reduction to H2 oxidation, an energy generating process resulted in decreased demand for energy generation by the oxidation of acetyl-CoA via the TCA cycle and therefore increased the fluxes of acetate carbons for gluconeogenesis, biomass synthesis, and other anabolic activities.
Surprisingly, when fumarate was provided as the electron acceptor in both E3 and E4, flux through the initial reactions of the TCA cycle (CS/ACONT) was slightly higher than 1, suggesting that acetate was not the sole source of carbon for the TCA cycle. In addition to acetate, fumarate served as carbon source in these cultures and likely introduced the additional carbons into the TCA cycle. The in vivo
flux distribution suggests that when fumarate was provided as the electron acceptor, fumarate was not only reduced to succinate but also converted to malate by fumarase (FUM) and further to oxaloacetate via malate dehydrogenase (MDH). During cell growth with fumarate as the electron acceptor, synthesis of oxaloacetate from fumarate contributed significantly to gluconeogenesis via its conversion to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK/PEPCKG in Figure ). In addition, there was a significant net conversion of phosphoenolpyruvate into acetyl-CoA (via the combined actions of PDH and/or POR) in the fumarate cultures. This flux accounted for the elevation in the TCA cycle relative to the acetate uptake flux. The fumarate uptake was slightly higher than the succinate secretion, indicating the consumption of fumarate. Our 13
CFMA results suggest that fumarate serves not only as an electron acceptor, but also as a carbon source and to a lesser extent, as an electron donor. This finding contrasts with those of previous cultivation and genetic studies [13
] which suggested that fumarate was not utilized as a carbon source or an electron donor.
In contrast to the gluconeogenesis during growth with fumarate, no significant gluconeogenetic flux through PEPCK/PEPCKG was observed during growth with Fe(III) citrate, where the conversion of acetyl-CoA into pyruvate by PDH/POR was the main gluconeogenetic flux (Figure ). In all cases, a small flux through PC was observed that seemed to be necessary for anaplerosis towards oxaloacetate to initiate the TCA cycle. As stated above, PEPCK/PEPCKG also showed anaplerotic function including ATP production and carboxylation in both Fe(III) cultures (E1 and E2), although its contribution to the anaplerosis in the culture with hydrogen (E2) was insignificant.
The flux distributions in acetate/fumarate chemostats E3 and E4 revealed that growing G. sulfurreducens under donor-limiting conditions (E3) versus acceptor-limiting conditions (E4) resulted in differences in fumarate uptake and succinate secretion effluxes, as well as in the utilization of the fumarate carbon source. In both cases, the net fumarate uptake flux was higher than the sum of succinate secretion, further suggesting that fumarate was utilized as an additional carbon source. The reductive flux of SUCOAS/ATO/FRD5 was higher in the donor-limited chemostat than that in the acceptor-limited conditions. Fumarate uptake and succinate secretion effluxes were also larger in E3 than in E4. In addition, the oxidative TCA fluxes (CS/ACONT, AKGD/OOR, FUM, MDH) in E3 were slightly higher than those in E4. Our results suggest that excessive fumarate in E3 resulted in elevation of the reductive flux of succinate dehydrogenase in conjunction with increased oxidative TCA fluxes. Furthermore, excessive fumarate present in E3 elevated the initial flux of gluconeogenesis carried out by PEPCK/PEPCKG. A more detailed investigation of the fumarate cultures is provided in a later section entitled 'Fumarate Utilization'.
Branch Point Flux Distributions
Based on a one-way ANOVA conducted at a significance level of 0.05, none of the relative flux estimates were equal in all four experiments, with the exception of the anaplerotic flux of PC. Likewise, comparison of relative fluxes for all possible pair-wise distributions revealed only a few instances in which fluxes were identical. Although many of the flux discrepancies between experiments may be attributable to differences in acetate uptake and/or biomass formation, some fluxes were not expected to vary. For instance, the fluxes from phosphoglycerate to glucose 6-phosphate in gluconeogenesis and pentose phosphate pathway fluxes should be proportional to the anabolic demand under all four conditions. To gain detailed understanding of the metabolic response of G. sulfurreducens under varying growth conditions, relative flux distributions at key branch points in gluconeogenesis, anaplerosis, the TCA cycle, and amino acid metabolism were examined in detail (Figure ).
Relative flux distributions (95% confidence interval) at key branch points of the central metabolic network for different experimental conditions of E1, E2, E3, and E4 (Table 1).
The synthesis of phosphoenolpyruvate is one key branch point of gluconeogenesis. As shown in Figure , the major pathway for phosphoenolpyruvate synthesis for gluconeogenesis varied with electron acceptors. When Fe(III) was the electron acceptor (E1 & E2), phosphoenolpyruvate for gluconeogenesis was generated almost exclusively (89 - 99%) from pyruvate by PYK/PPDK/PPS, and phosphoenolpyruvate synthesis via the decarboxylation of oxaloacetate (PEPCK/PEPCKG) did not contribute significantly to gluconeogenesis. In contrast, when fumarate was provided as the electron acceptor (E3 & E4), the decarboxylation of oxaloacetate (PEPCK/PEPCKG) was the main route for phosphoenolpyruvate biosynthesis for gluconeogenesis. In the acceptor-limited culture (E4), phosphoenolpyruvate for gluconeogenesis was synthesized almost exclusively from oxaloacetate (ca. 93%), whereas in the donor-limited culture ca. 60% of phosphoenolpyruvate for gluconeogenesis was generated from oxaloacetate by PEPCK/PEPCKG by consuming ATP/GTP.
Conversion of pyruvate to oxaloacetate by pyruvate carboxylase (PC) plays a key role in anaplerosis. As shown in Figure this branch point of anaplerosis was less sensitive towards electron acceptor variation than gluconeogenesis. In the both Fe(III) cultures, ca. 9% of pyruvate was converted into oxaloacetate for anaplerosis, whereas the amount of pyruvate that was diverted to anaplerosis varied from 4 to 15% during growth with fumarate. This variation appeared to reflect the degree to which oxaloacetate was diverted to gluconeogenesis. In the acceptor-limited culture (E4), where 90-95% of the oxaloacetate synthesized from fumarate was used for gluconeogenesis, about 15% of pyruvate was utilized for anaplerosis. In contrast, in the donor-limited culture (E3), 57-61% of oxaloacetate was consumed for gluconeogenesis and 4% of pyruvate was diverted to anaplerosis.
The condensation of acetyl-CoA and oxaloacetate (CS/ACONT) is the initiation step of the TCA cycle. As shown in Figure , the relative amount of acetyl-CoA (between 92% and 97%) entering the TCA cycle was comparable for all cultivation conditions. The relative amount of acetyl-CoA entering the TCA cycle was nearly identical for the two fumarate cultures (E3 and E4). In the Fe(III) cultures, the percentage of acetyl-CoA entering the TCA cycle in the culture with acetate as the sole electron donor (97%) was higher than that in the presence of both hydrogen and acetate as the electron donors (92%). Examination of oxaloacetate utilization (Figure ) revealed that nearly all of the oxaloacetate produced during growth via Fe(III) reduction (96 - 99%) was utilized to initiate the TCA cycle, whereas only 82% to 85% of oxaloacetate utilized to initiate the TCA cycle in the two fumarate cases. This difference can be attributed to the utilization of fumarate as an additional carbon source for gluconeogenesis and amino acid biosynthesis.
A recent study [3
] indicated that G. sulfurreducens
has two pathways for isoleucine biosynthesis: the precursor 2-oxobutanoate can be generated (i
) by the deamination of threonine and (ii
) from pyruvate and acetyl-CoA through the citramalate pathway. Risso et al. (2008) [3
] observed that the citramalate pathway accounted for the majority of isoleucine biosynthesis during growth with fumarate. This was also the case in the present study where 88 to 93% of 2-oxobutanoate was synthesized from pyruvate and acetyl-CoA in the two fumarate cultures (Figure ). A smaller fraction of 2-oxobutanoate, ca. 70%, was produced via the citramalate pathway in the acetate Fe(III) culture (E1). In contrast, the 2-oxobutanoate was synthesized almost exclusively from threonine (98 - 99%) in E2 culture containing hydrogen, Fe(III), and 1 mM acetate. During growth in the presence of hydrogen, decreased demand for energy production by the TCA cycle may result in TCA cycle intermediates such as oxaloacetate (main precursor for aspartate and threonine), more available for other uses such as amino acid biosynthesis. In fact, a complete TCA cycle is not required during growth on hydrogen [13
G. sulfurreducens also has two pathways for the biosynthesis of glycine: through the conversion of threonine to glycine and acetaldehyde by threonine aldolase (THRLAD) and from the conversion of serine to glycine by glycine hydroxymethyltransferase (GHMT). 13CMFA indicated that the percentages of threonine converted into glycine and acetaldehyde varied under different conditions (Figure ). In E1, 99% of threonine was converted to glycine and acetaldehyde. The acetaldehyde was further converted to acetyl-CoA (generating NADH) and the acetyl-CoA was then recycled into the central metabolic pathways.
Another interesting observation which has not been previously described in the literature was that a large portion of 3-phosphoglycerate (75 - 89%) was converted to serine (Figure ). At this metabolic branch point, the flux into glyceraldehyde 3-phosphate for gluconeogenesis was kept at a relatively low level corresponding to anabolic demands. Serine was subsequently converted to pyruvate by serine deaminase. As shown in Figure , there were significant fluxes through serine. During growth with Fe(III) as the electron acceptor (E1, E2), there was a ATP-consuming futile cycle consisting of phosphoenolpyruvate, phosphoglycerate, serine, and pyruvate, where phosphoenolpyruvate synthase (PPS) and/or pyruvate phosphate dikinase (PPDK) consume ATP when converting pyruvate to phosphoenolpyruvate.
In spite of its small contribution to 2-oxobutanoate biosynthesis, a high level of threonine deaminase activity (25-higher than that of citramalate synthase) has been previously detected in soluble extracts of G. sulfurreducens
growing on fumarate [3
]. The threonine deaminase of G. sulfurreducens
is also capable of deaminating serine to pyruvate [3
], and is likely the enzyme deaminating serine in G. sulfurreducens
, as this species does not contain the Fe(II)-dependent and oxygen sensitive serine deaminases gene found in Clostridia
and plants. Moreover, knocking out the threonine deaminase eliminated all detectable serine deaminase activity in soluble extracts prepared under aerobic conditions [3
]. Given the presence of relatively high levels of serine deaminase activity in G. sulfurreducens
extracts, the relatively high flux through serine deaminase that observed by 13
CMFA is plausible.
It has been hypothesized that futile cycles in cellular metabolism are involved in the regulation of biochemical pathways. Quian and Beard (2006) [39
] suggested that futile cycles actively shift the effective equilibrium by expending energy where the magnitude of changes in effective equilibria and sensitivities is a function of the amount of energy used by a futile cycle. Also, it has been suggested that cellular regulation of the ATP/ADP ratio depends on both the specific growth rate and the environmental conditions [40
]. Tang et al. (2007) [31
] has found futile cycle activity in Geobacter metallireducens
, however metabolic function of futile cycles is yet fully understood. The observed futile cycle might be related to an important metabolic function, e.g., in regulating substrate utilization for gluconeogenesis and the oxidative TCA cycle.
Specific Metabolic Fluxes
In addition to the relative branch point flux distributions, we compared the specific metabolic fluxes, which are quantities scaled by cell growth (see Methods Eq. 4), to understand the growth-associated metabolic activities. As shown in Table , the specific fluxes from phosphoglycerate to hexose 6-phosphate of the lower gluconeogenesis and pentose phosphate pathways were invariant under different culture conditions. These results suggest that these pathways operated purely to satisfy anabolic demand (Table ) and that the differences in the relative fluxes through these pathways shown in Figure were due to different biomass yields on acetate under different conditions. The specific fluxes of the upper gluconeogenesis pathway from pyruvate to phosphoenolpyruvate were similar to those from phosphoenolpyruvate to phosphoglycerate during growth with Fe(III) (E1 & E2). In comparison, these specific fluxes were considerably lower than those from phosphoenolpyruvate to phosphoglycerate when fumarate was the electron acceptor. This appeared to be due to the substantial contribution of the phosphoenolpyruvate carboxykinase to gluconeogenesis by converting oxaloacetate to phosphoenolpyruvate.
Specific fluxes (fluxes normalized by the biomass yield estimate: (mmolmetablite/h)(gbiomass/h)-1) in the central metabolic pathways of the experiments listed in Table 1.
Precursor demand of G. sulfurreducens from the intermediary metabolites involved in the central metabolic pathways in Figure 2.
As indicated in Figure , the initial step in gluconeogenesis was the generation of pyruvate from acetyl-CoA (POR) in the Fe(III) cultures and phosphoenolpyruvate formation from oxaloacetate by PEPCK/PEPCKG in the fumarate cultures. As shown in Table , the specific fluxes of POR were identical in the two Fe(III) cultures, and the specific fluxes of PEPCK/PEPCKG in the fumarate cultures were similar. Thus, the initial gluconeogenetic flux per unit biomass synthesis remained constant when the same electron acceptor was utilized.
Also shown in Table , the specific flux through the TCA cycle during growth with Fe(III) was approximately 2.4-fold larger in E1 (5 mM acetate) than E2 (H2
+ 1 mM acetate). This is due to the direct energy production via hydrogen oxidation which decreased the energy production demand for the TCA cycle. However, energy generation via hydrogen oxidation did not appear to completely replace energy generation via acetate oxidation by the TCA cycle. During growth in the presence of hydrogen and 1 mM acetate, flux through the TCA cycle was higher than what was required for the anabolic demand for α-ketoglutarate and oxaloacetate, and a significant amount of acetate was oxidized via the TCA cycle. One reason of the energy production via acetate oxidation in the presence of hydrogen might be the relatively poor solubility of H2
in water. The Henry's constant of H2
in water is only 7.8 × 10-4
at 298°K and therefore the dissolved H2
concentration at 0.60 atm H2
is approximately 0.5 mM. This can also be due to other metabolic factors such as hydrogenase activity [41
] or cell preference for generating reducing power through the oxidative TCA cycle.
In the fumarate cultures, the specific acetate uptake and TCA cycle activity was dramatically lower than that in the Fe(III) cultures (Table ). The specific acetate uptake in the two fumarate cultures was 65 to 66 mmol acetyl-CoA per gram biomass synthesis as compared to 166 to 369 mmol of acetyl-CoA per gram biomass synthesis for the Fe(III) cultures (Table ). The standard redox potential of the half reaction Fe3+
° = 771 mV) is higher than that of fumarate2-
° = 31 mV). Thus, it is expected that a higher electromotive force (ΔE
°) would be generated by the acetate/Fe(III) pair than the acetate/fumarate pair. However, the fumarate cultures consumed less acetate per unit biomass formation than the Fe(III) cultures, suggesting that the cells in the fumarate cultures utilize acetate more efficiently than in the Fe(III) cultures. Higher biomass yields on acetate in the fumarate cultures relative to Fe(III) have been previously reported [2
] and is likely due to the fact that Fe(III) is reduced on the outside of the cells which results in an additional energetic cost for exporting electrons.
The 13CMFA results suggest that G. sulfurreducens utilizes fumarate not only as the electron acceptor for acetate oxidation but also as an additional carbon source. Based on efflux measurements by HPLC, fumarate uptake and succinate secretion were found to be very close in both fumarate cultures (cf. 'Steady-State Effluxes in Chemostat Cultures' section). However, the optimal solutions found by the stochastic 13CMFA runs resulted in larger fumarate uptake than succinate secretion (Figure ) and the efflux estimates were within the statistical range given by the means and standard deviations determined experimentally (95% confidence interval). As a result, the small percentage (< 10%) of fumarate consumed as a carbon source in the fumarate cultures, which could not be determined by HPLC efflux measurements was discovered by 13CMFA with more accurate GC/MS labeling measurements.
As shown in Table , the uptake of fumarate increased the specific oxidative TCA fluxes of fumarase (FUM) and malate dehydrogenase (MDH) compared to that of α-ketoglutarate dehydrogenase (AKGD/OOR). Consequently, phosphoenolpyruvate carboxykinase (PEPCK/PEPCKG) was the enzyme responsible for initiating gluconeogenesis in the fumarate cultures, while pyruvate synthase (POR) initiated gluconeogenesis in the Fe(III) cultures. In the fumarate cultures, acetyl-CoA was replenished from pyruvate (PDH/POR) which was produced by phosphoenolpyruvate through 3-phosphoglycerate and serine. Between the two fumarate cultures, cells in the donor-limited condition E3 had higher net fumarate reduction, succinate secretion, oxidative TCA activities, and gluconeogenesis activities than those in the acceptor-limited condition E4 (Table ).
To gain insight into fumarate utilization, we compared relative fluxes around fumarate and succinate, which were normalized by fumarate uptake (Figure ). Overall, relative fumarate utilization including net fumarate reduction, fumarase flux, succinate secretion, etc., was similar in both cultures (Figure ). A notable difference between the two fumarate cultures was found in the reversibility of FRD5. FRD5 of G. sulfurreducens
has a dual function in fumarate reduction and succinate oxidation [32
]. Both reductive (ν2
) and oxidative (ν3
) fluxes between fumarate and succinate were smaller in the donor-limited culture (E3) than in the acceptor-limited culture (E4). In particular, the flux through the oxidative direction was only 2 to 7% per unit fumarate uptake in E3 with the corresponding reductive flux of 60 to 65%, as compared to the oxidative flux of 29 to 37% with the reductive flux of 85 to 94% under the donor-limiting condition.
95% confidence intervals given for key TCA fluxes (νi) in the fumarate cultures (E3, E4), normalized by fumarate uptake. νnet = ν2 - ν3.
We further calculated the net production of redox equivalents (NAD(P)H and menaquinol) and carbon dioxide from the in vivo
flux estimates. Because G. sulfurreducens
can utilize both NADH and NADPH for Fe(III) and fumarate reduction [43
]; NADH and NADPH are pooled for redox balance. NAD(P)H is produced in the central metabolic network by the reactions ICDHy (isocitrate → α-ketoglutarate), AKGD/OOR (α-ketoglutarate → succinyl-CoA), MDH (malate → oxaloacetate), ME1/ME2 (malate → pyruvate), PDH/POR (pyruvate → acetyl-CoA), GAPD (glyceraldehyde 3-phosphate → 1,3-bisphosphoglycerate), and ADA (acetaldehyde → acetyl-CoA). In addition, cells consume 14.2 mmol NADPH and produce 2.32 mmol NADH per gram biomass synthesis based on the genome-scale model [2
]. 3-phosphoglycerate dehydrogenase involved in serine biosynthesis produces NADH, yet the next reaction carried out by 3-phosphoserine aminotransferase involves conversion of glutamate to 2-oxoglutrate for transamination. Without 2-oxoglutamate secretion, glutamate has to be regenerated from 2-oxoglutarate which consumes one redox equivalent. Hence, there is no net production of redox equivalent in this case. In G. sulfurreducens
, FRD5 reduces menaquinone to menaquinol when succinate is oxidized to fumarate and vice versa for the fumarate reduction. The fluxes related to decarboxylation/carboxylation are ICDHy, AKGD/OOR, PC, ME1/ME2, PEPCK/PECKG, PDH/POR, and other biosynthetic pathways. In addition, cells produce about 1.36 mmol CO2
per gram biomass synthesis. Details of anabolic NADPH net consumption, NADH net production, and net decarboxylation are listed in additional file 1
In the acetate/Fe(III) culture (E1), the molar yields of redox equivalents and CO2 net production on acetate were 3.97 ± 0.08 and 1.97 ± 0.05, respectively. These corresponded to a molar yield of Fe(III) reduction on acetate of 7.94 ± 0.16 and a ratio of Fe(II) to CO2 production of 4.03 ± 0.13. Comparing the stoichiometry of complete acetate oxidation with Fe(III), i.e., CH3COOH + 8Fe3+ + 10H2O → 2CO2 + 8Fe2+ + 8H3O+, the Fe(III) reduction and CO2 production drawn from the in vivo flux estimates for E1 are consistent with the theoretical stoichiometry.
The direct oxidation of hydrogen coupled with Fe(III) reduction is H2
O → 2Fe2+
]. Despite hydrogen utilization, cells in the hydrogen/Fe(III) culture (E2) still produced 3.56 ± 0.05 moles of redox equivalents with 1.80 ± 0.04 moles of CO2
per mole of acetate taken up. These results corresponded to a molar yield of Fe(III) reduction on acetate of 7.12 ± 0.10 and a ratio of Fe(II) to CO2
production of 3.96 ± 0.10. Thus, Fe(III) reduction together with CO2
production coincides with the theoretical stoichiometry of Fe(III) reduction on acetate. In contrast to E1, cells oxidized less acetate via the TCA cycle (smaller yield of Fe(III) reduction and reduced TCA fluxes) and consequently doubled biomass yield on acetate (Figure & Table ).
The complete acetate oxidation with fumarate follows CH3COOH + 4C4H4O4 + 2H2O → 2CO2 + 4C4H6O4. The net conversion of fumarate to succinate per acetate was 2.9 ± 0.1 in the donor-limited culture (E3) and 2.5 ± 0.2 in the acceptor-limited (E4). The molar yield of CO2 production on acetate was comparable with the theoretical stoichiometry: it was 2.0 ± 0.5 and 1.8 ± 0.3 in E3 and E4, respectively. These results may be due to additional decarboxylation of fumarate carbons, e.g., gluconeogenesis initiated from oxaloacetate by PEPCK/PEPCKG and acetyl-CoA replenishment from pyruvate by PDH. In addition, two fumarate carbons can potentially be released through these reactions.