Yang et al. Microbial Cell Factories 2010, 9:90 http://www.microbialcellfactories.com/content/9/1/90

RESEARCH Open Access Metabolic response of towards electron donor/acceptor variation Tae Hoon Yang1*†, Maddalena V Coppi2†, Derek R Lovley2, Jun Sun1*

Abstract Background: Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via 13C-based metabolic flux analysis. We examined the 13C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with 13C-acetate. Results: Using 13C-based metabolic flux analysis, we observed that donor and acceptor variations gave rise to differences in gluconeogenetic initiation, tricarboxylic acid cycle activity, and amino acid biosynthesis pathways. Culturing G. sulfurreducens cells with Fe(III) as the electron acceptor and acetate as the electron donor resulted in pyruvate as the primary carbon source for gluconeogenesis. When fumarate was provided as the electron acceptor and acetate as the electron donor, the flux analysis suggested that fumarate served as both an electron acceptor and, in conjunction with acetate, a carbon source. Growth on fumarate and acetate resulted in the initiation of gluconeogenesis by phosphoenolpyruvate carboxykinase and a slightly elevated flux through the oxidative tricarboxylic acid cycle as compared to growth with Fe(III) as the electron acceptor. In addition, the direction of net flux between acetyl-CoA and pyruvate was reversed during growth on fumarate relative to Fe(III), while growth in the presence of Fe(III) and acetate which provided hydrogen as an electron donor, resulted in decreased flux through the tricarboxylic acid cycle. Conclusions: We gained detailed insight into the of G. sulfurreducens cells under various electron donor/acceptor conditions using 13C-based metabolic flux analysis. Our results can be used for the development of G. sulfurreducens as a chassis for a variety of applications including and renewable biofuel production.

Background groundwater and understanding their physiology is Geobacter conserve energy for growth by cou- crucial for optimizing such applications. pling the complete oxidation of organic compounds to G. sulfurreducens hasservedasamodelorganismfor the reduction of Fe(III) and a variety of toxic and radio- Geobacter species as it is amenable to genetic manipula- active metals [1-4]. Geobacter sulfurreducens produce tion and was the first Geobacter species to have its gen- electrically conductive pili that function as nano-wires ome fully sequenced [11,12]. Constraint-based in silico to promote electron transfer to insoluble electron accep- modeling studies have been applied to understand meta- tors such as Fe(III) oxide and electrodes [5,6]. As a bolic characteristics of Geobacter species [2,4,13]. The result of these properties, Geobacter species are utilized in silico study of G. sulfurreducens metabolism has to harvest electricity from waste organic matter [7,8] enabled prediction of the metabolic response of Geobac- and have served as biocatalysts in ter species to a variety of genetic and culture perturba- applications [9,10]. Geobacter species have great bio- tions in terms of a genome-scale metabolic flux balance technological potential for contaminant removal from analysis. In general, constraint-based in silico modeling approaches necessitate a biological objective function * Correspondence: [email protected]; [email protected] such as specific growth rate that can be minimized or † Contributed equally maximized in order to predict steady-state metabolic flux 1Genomatica, Inc., 10520 Wateridge Circle, San Diego, CA 92121, USA distributions. Based upon the overall stoichiometry of the Full list of author information is available at the end of the article

© 2010 Yang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yang et al. Microbial Cell Factories 2010, 9:90 Page 2 of 15 http://www.microbialcellfactories.com/content/9/1/90

metabolic model, which is typically an underdetermined chemostat, hydrogen and 1 mM acetate were supple- system, optimal solutions of steady-state fluxes are mented under batch culture conditions. In E3 and E4, computed by minimizing/maximizing the selected objec- fumarate was provided as the electron acceptor at differ- tive. However, such optimal solutions are objective- ent fumarate to acetate ratios to examine the resulting dependent, and a proper choice of an objective is metabolic phenotypes under donor-limiting (E3) and non-trivial and can be condition-specific [14]. Therefore, acceptor-limiting (E4) conditions. Selected acetate and the information predicted by a constraint-based in silico fumarate concentrations were based on a previous che- approach needs to be interpreted carefully in the context mostat study [36] and in silico simulations with the of the actual in vivo functional objective of the system. G. sulfurreducens genome-scale metabolic model which Metabolic flux analysis using isotopic labeling techni- suggested the optimal ratio of fumarate to acetate to be ques provide a means to study metabolic pathway activ- 3.01 for the complete oxidization of acetate [2]. ities in vivo without the bias of the selection of a particular biological objective function. Amongst avail- Steady-State Effluxes in Chemostat Cultures able techniques, 13C-based metabolic flux analysis Extracellular yield coefficients for the three sets of che- (13CMFA) has proven to be the most advanced tool for mostat cultures (E1, E3, E4) were determined from quantifying in vivo intracellular pathway activities steady-state rate measurements. The complete oxidation [15-18]. Modeling and experimental/analytical techni- of acetate to CO2 releases of 8 electrons, which is suffi- ques for 13CMFA are well-established, and the method cient to convert 8 moles of Fe(III) to Fe(II) or 4 moles has been utilized to gain a quantitative understanding of of fumarate to succinate. For E1 (5 mM acetate + a variety of biological systems [15-30]. Recently, 55 mM Fe(III)citrate), the molar yield of Fe(II) per mole 13CMFA has also been applied to Geobacter to unveil of acetate consumed was 7.1 ± 0.5, suggesting that only central pathway fluxes in G. metallireducens [31] and to 11.3% of the acetate in the growth medium was utilized elucidate an alternative isoleucine biosynthetic pathway for biomass synthesis. When fumarate was used as the in G. sulfurreducens [3]. electron acceptor, the molar yield of succinate per mole G. sulfurreducens can utilize acetate and hydrogen as of acetate consumed was 2.9 ± 0.2 for E3 (5 mM acetate electron donors, Fe(III) and fumarate as electron accep- + 28 mM fumarate) and 2.5 ± 0.2 for E4 (10 mM acet- tors [32-34] and has a single bifunctional enzyme that ate + 20 mM fumarate), indicating that a higher percen- catalyzes both fumarate reduction and succinate oxida- tage of acetate, 27.5% to 37.5%, was utilized for biomass tion [32]. Results from previous results suggest that synthesis. The molar yield of fumarate uptake on acetate G. sulfurreducens does not utilize fumarate as a carbon was 2.8 ± 0.1 and 2.5 ± 0.1 in E3 and E4, respectively. source [13,33,35]. However, the intracellular fate of The biomass yield coefficient on acetate (YX/DON = gram fumarate carbons has not been investigated. The goal of dry cell weight produced per mmol acetate uptake) of this study was to use 13CMFA to quantitatively charac- the Fe(III)citrate culture (E1) was 2.8 × 10-3 ±2×10-4 terize the in vivo intracellular metabolic flux response of g/mmol, about 4-fold lower than those of the fumarate G. sulfurreducens cultured with different electron donors cultures, 0.013 ± 2 × 10-3 and0.012±2×10-3 for E3 (acetate and hydrogen) and acceptors (Fe(III) and fuma- and E4, respectively. Extracellular concentration mea- rate). Results from the present work are important surements can be associated with larger uncertainties for the understanding of the central metabolism of than mass spectrometric carbon labeling measurements. G. sulfurreducens and the optimization of bioremedia- Thus, the effluxes were set as free parameters to be esti- tion processes mediated by Geobacter species. mated from 13C labeling measurements during 13CMFA or calculated later from in vivo flux estimates. Results and Discussion Overview of Experimental Design Metabolic Fate of Citrate & Fumarate Carbons We applied 13CMFA to characterize the metabolic In order to assess metabolic flux distributions in response of G. sulfurreducens to variations in growth G. sulfurreducens grown with Fe(III)citrate as the elec- conditions. G. sulfurreducens was cultured either in che- tron acceptor, we needed to determine whether G. sul- mostats (E1, E3, and E4) or in batch mode (E2) with dif- furreducens is able to metabolize citrate in addition to ferent combinations of electron donors and acceptors acetate. We also investigated whether G. sulfurreducens 13 (Table 1). In all experiments, 30% [U- C2] acetate was can utilize fumarate as a carbon source since previous provided as the 13C carbon source which also served as studies have suggested that fumarate is not utilized as the electron donor. In both E1 and E2, Fe(III)citrate acarbonsource[13,33,35,37].Tothisend,cellswere served as the electron acceptor. In contrast to E1 where grown in a batch culture containing 10 mM non- 5 mM acetate served as the electron donor in the labeled acetate with 55 mM Fe(III)citrate (4.2% Yang et al. Microbial Cell Factories 2010, 9:90 Page 3 of 15 http://www.microbialcellfactories.com/content/9/1/90

Table 1 Cultivation conditions for Geobacter sulfurreducens to study intracellular metabolism with different electron donor/acceptor conditions ID E1 E2 E3 E4 cultivation mode chemostat batch chemostat chemostat (D = 0.04 h-1) (D = 0.05 h-1) (D = 0.05 h-1) e-donor 5 mM acetate 1 mM acetate 5 mM acetate 10 mM acetate &10mLH2* e-acceptor 55 mM Fe(III)citrate 55 mM Fe(III)citrate 28 mM fumarate 20 mM fumarate

*: H2 was provided in the headspace of the culture (initial partial pressure ~ 0.60 atm).

13 [U- C6]Fe(III)citrate) and 10 mM non-labeled acetate Comparison of Amino Acid Labeling Patterns 13 13 with 27.5 mM fumarate (30% [2,3- C2]fumarate), C-labeling patterns of metabolic products including respectively. The carbon mass isotopomer distributions proteinogenic amino acids are not direct measures of the (MIDAA) of alanine, glycine, valine, leucine, isoleucine, in vivo flux distributions, but information on the flux dis- phenylalanine, serine, aspartate, and glutamate were tributions is recorded in the labeling patterns. Therefore, compared to those obtained from an identical culture gross metabolic discrepancies or similarities induced by containing only non-labeled compounds. the different electron donor/acceptor conditions can be Linear regression of the two sets of MIDAA measured visualized by directly comparing the MIDAA values in the acetate/Fe(III)citrate culture gave a slope of 1.003 obtained from these conditions. As shown in Figure 1, the 2 with R of 1.000 and an intercept of 0.000. In addition, comparison of MIDAA values suggested that electron the root mean square (RMS) [38] of the difference acceptor variation (Figure 1-B, C, D, E: Fe(III) vs. fuma- 13 between the two MIDAA sets was 0.0006, which was sig- rate) resulted in a larger discrepancy in C labeling pat- nificantly less than the average MIDAA determined mea- terns than electron donor variation (Figure 1-A: acetate vs. surement error (ca. 0.003) [27]. This result indicates hydrogen). In terms of the Euclidian distance, the measure that the citrate carbons were not utilized by G. sulfurre- ofthedifferenceoftwosetsofdata;theMIDAA values ducens cells, Fe(III)citrate served exclusively as an elec- from E3 to E1 (Figure 1-B) or E4 to E1 (Figure 1-C) was tron acceptor, and that acetate was the sole carbon larger than that from E2 to E1 (Figure 1-A). The distance source in experiments E1 and E2. from E2 to E3 or E4 (Figure 1-D, E) was smaller than that In contrast, the acetate/fumarate culture gave a dis- from E1 to E3 or E4, however larger than the distance tinct difference from the natural abundance (slope = between E1 and E2. The most similar amino acid 13C 1.151, intercept = 0.030, R2 = 0.9326) with an RMS of labeling patterns were found between E3 and E4, in which 0.011. For instance, MIDAA of aspartate gave the largest only donor and acceptor concentrations were varied discrepancy from the natural abundance (RMS = 0.08), (Figure 1-F). Nevertheless, detectable differences of followed by glycine (RMS = 0.054), glutamate (RMS = MIDAA between E3 and E4 indicate that the physiological 0.049), serine (RMS = 0.044), phenylalanine (RMS = state of G. sulfurreducens is altered in response to varying 0.032), isoleucine (RMS = 0.012), alanine, leucine, and donor and acceptor concentrations. valine (RMS = 0.004). Our results demonstrate that fumarate carbons can be metabolized into different Overview of in vivo Fluxes amino acid biosynthesis precursors in the central meta- To assess in vivo flux distributions from the four cul- bolic pathways. Cellular fumarate utilization was investi- tures of G. sulfurreducens, 500 stochastic numerical gated more quantitatively in terms of 13CMFA. optimization runs were performed by generating ran- dom numbers of measurements (from the normal distri- Assessment of Biological Reproducibility butions estimated using experimentally determined The consistency between biological replicates was evalu- means and standard deviations) and starting points ated by comparing the MIDAA of replicates. The (from a continuous uniform distribution between the 2 squared correlation coefficients (R ) between the MIDAA upper and lower bound of each parameter). The details values of the biological replicates (n =3)werehigher enzyme reactions comprised in the metabolic network than 0.9990 in all the chemostat experiments (E1, E3, are listed in Table 2, and the 95% confidence intervals E4), suggesting a high consistency between the repli- for the resulting flux estimates are depicted in Figure 2. cates. Thus, the experimental data were considered to The fluxes were normalized to acetate uptake and can be unbiased by biological variations, and the intracellu- be understood as molar yield coefficients on acetate. For lar metabolic fluxes were computed using the mean each experiment, the experimental measurements and values of replicate MIDAA and measured effluxes. the corresponding model-predicted values gave a high Yang et al. Microbial Cell Factories 2010, 9:90 Page 4 of 15 http://www.microbialcellfactories.com/content/9/1/90

1 1 1 ABC 0.8 0.8 0.8

0.6 0.6 0.6 + Fe(III) 2 0.4 0.4 0.4 E2: H E2: 0.2 R: 0.931 0.2 R: 0.881 0.2 R: 0.860 D: 0.760 E3: acetate + fumarate D: 1.314 E4: acetate + fumarate D: 1.381 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 E1: acetate + Fe(III) E1: acetate + Fe(III) E1: acetate + Fe(III)

1 1 1 DEF 0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 R: 0.955 0.2 R: 0.937 0.2 R: 0.987 E3: acetate + fumarate + acetate E3: D: 0.868 fumarate + acetate E4: D: 0.971 fumarate + acetate E4: D: 0.410 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 E2: H + Fe(III) E2: H + Fe(III) E3: acetate + fumarate 2 2 Figure 1 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. degree of consistency; their correlation coefficients (R) reactions of the TCA cycle (CS/ACONT) was slightly were higher than 0.9970 at the optimum with the slope higher than 1, suggesting that acetate was not the sole and y-axis intercept close to 1 and 0, respectively. source of carbon for the TCA cycle. In addition to acet- Under the examined four conditions, the majority of ate, fumarate served as carbon source in these cultures consumed acetate was oxidized through the TCA cycle and likely introduced the additional carbons into the (Figure 2). When comparing fluxes in E1 and E2, the TCA cycle. The in vivo flux distribution suggests that flux through the initial reaction of the TCA cycle (CS/ when fumarate was provided as the electron acceptor, ACONT) in the H2/Fe(III) culture with 1 mM acetate fumarate was not only reduced to succinate but also (E2) was significantly less than that obtained from the converted to malate by fumarase (FUM) and further to acetate/Fe(III) culture (E1). The addition of hydrogen oxaloacetate via malate dehydrogenase (MDH). During increased the flux from acetyl-CoA to pyruvate (POR: cell growth with fumarate as the electron acceptor, pyruvate synthase) as well as other gluconeogenetic synthesis of oxaloacetate from fumarate contributed sig- fluxes and resulted in a 2-fold higher biomass yield on nificantly to gluconeogenesis via its conversion to phos- acetate (cf.YXS values in Figure 2). The coupling of Fe phoenolpyruvate by phosphoenolpyruvate carboxykinase (III) reduction to H2 oxidation, an energy generating (PEPCK/PEPCKG in Figure 2). In addition, there was a process resulted in decreased demand for energy genera- significant net conversion of phosphoenolpyruvate into tion by the oxidation of acetyl-CoA via the TCA cycle acetyl-CoA (via the combined actions of PDH and/or and therefore increased the fluxes of acetate carbons for POR) in the fumarate cultures. This flux accounted for gluconeogenesis, biomass synthesis, and other anabolic the elevation in the TCA cycle relative to the acetate activities. uptake flux. The fumarate uptake was slightly higher Surprisingly, when fumarate was provided as the elec- than the succinate secretion, indicating the consumption tron acceptor in both E3 and E4, flux through the initial of fumarate. Our 13CFMA results suggest that fumarate Yang et al. Microbial Cell Factories 2010, 9:90 Page 5 of 15 http://www.microbialcellfactories.com/content/9/1/90

Table 2 Abbreviations and EC numbers of the enzymes suggested that fumarate was not utilized as a carbon considered in the central metabolic network of G. source or an electron donor. sulfurreducens for 13C metabolic flux analysis In contrast to the gluconeogenesis during growth with abbreviation EC number enzyme fumarate, no significant gluconeogenetic flux through ACKr 2.7.2.1 acetate kinase PEPCK/PEPCKG was observed during growth with Fe PTAr 2.3.1.8 phosphotransacetylase (III) citrate, where the conversion of acetyl-CoA into PFK 2.7.1.11 phosphofructokinase pyruvate by PDH/POR was the main gluconeogenetic FBP 3.1.3.11 fructose-bisphosphatase flux (Figure 2). In all cases, a small flux through PC was FBA 4.1.2.13 fructose-bisphosphate aldolase observed that seemed to be necessary for anaplerosis TPI 5.3.1.1 triose-phosphate isomerase towards oxaloacetate to initiate the TCA cycle. As stated GAPD 1.2.1.12 glyceraldehyde-3-phosphate above, PEPCK/PEPCKG also showed anaplerotic func- dehydrogenase (NAD) tion including ATP production and carboxylation in PGK 2.7.2.3 phosphoglycerate kinase both Fe(III) cultures (E1 and E2), although its contribu- PGM 5.4.2.1 phosphoglycerate mutase tion to the anaplerosis in the culture with hydrogen (E2) ENO 4.2.1.11 enolase was insignificant. PYK 2.7.1.40 pyruvate kinase The flux distributions in acetate/fumarate chemostats PPDK 2.7.9.1 pyruvate phosphate dikinase E3 and E4 revealed that growing G. sulfurreducens PPS 2.7.9.2 phosphoenolpyruvate synthase under donor-limiting conditions (E3) versus acceptor- PDH 1.2.1.51 pyruvate dehydrogenase limiting conditions (E4) resulted in differences in fuma- POR 1.2.7.1 pyruvate synthase (pyruvate ferredoxin rate uptake and succinate secretion effluxes, as well as oxidoreductase) in the utilization of the fumarate carbon source. In both TKT1 2.2.1.1 transketolase cases, the net fumarate uptake flux was higher than the TAL 2.2.1.2 transaldolase sum of succinate secretion, further suggesting that TKT2 2.2.1.1 transketolase fumarate was utilized as an additional carbon source. CS 4.1.3.7 citrate synthase The reductive flux of SUCOAS/ATO/FRD5 was higher ACONT 4.2.1.3 aconitase in the donor-limited chemostat than that in the accep- ICDHy 1.1.1.42 isocitrate dehydrogenase (NADP) tor-limited conditions. Fumarate uptake and succinate AKGD 1.2.4.2 2-oxoglutarate dehydrogenase secretion effluxes were also larger in E3 than in E4. In OOR 1.2.7.3 2-oxoglutarate synthase addition, the oxidative TCA fluxes (CS/ACONT, SUCOAS 6.2.1.5 succinyl-CoA synthetase (ADP-forming) AKGD/OOR, FUM, MDH) in E3 were slightly higher ATO 2.8.3.8 acetate CoA-transferase than those in E4. Our results suggest that excessive FRD5 1.3.5.1 succinate dehydrogenase (menaquinone 7) fumarate in E3 resulted in elevation of the reductive FUM 4.2.1.2 fumarase flux of succinate dehydrogenase in conjunction with MDH 1.1.1.37 malate dehydrogenase increased oxidative TCA fluxes. Furthermore, excessive PC 6.4.1.1 pyruvate carboxylase fumarate present in E3 elevated the initial flux of gluco- ME1 1.1.1.38 malic enzyme (NAD) neogenesis carried out by PEPCK/PEPCKG. A more ME2 1.1.1.40 malic enzyme (NADP) detailed investigation of the fumarate cultures is pro- PEPCK 4.1.1.49 phosphoenolpyruvate carboxykinase vided in a later section entitled ‘Fumarate Utilization’. PEPCKG 4.1.1.32 phosphoenolpyruvate carboxykinase (GTP) ASPTA1 2.6.1.1 aspartate transaminase Branch Point Flux Distributions THRS 4.2.3.1 threonine synthase Basedonaone-wayANOVAconductedatasignifi- THRD 4.3.1.19 threonine deaminase cance level of 0.05, none of the relative flux estimates LEUB 1.1.1.85 3-isopropylmalate dehydrogenase were equal in all four experiments, with the exception THRLAD 4.1.2.5 threonine aldolase of the anaplerotic flux of PC. Likewise, comparison of ADA 1.2.1.10 acetaldehyde dehydrogenase relative fluxes for all possible pair-wise distributions GHMT 2.1.2.1 glycine hydroxymethyltransferase revealed only a few instances in which fluxes were iden- PSP 3.1.3.3 phosphoserine phosphatase tical. Although many of the flux discrepancies between SERD 4.3.1.17 serine deaminase 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 phospho- serves not only as an electron acceptor, but also as a glycerate to glucose 6-phosphate in gluconeogenesis and carbon source and to a lesser extent, as an electron pentose phosphate pathway fluxes should be propor- donor. This finding contrasts with those of previous cul- tional to the anabolic demand under all four conditions. tivation and genetic studies [13,33,35,37] which To gain detailed understanding of the metabolic Yang et al. Microbial Cell Factories 2010, 9:90 Page 6 of 15 http://www.microbialcellfactories.com/content/9/1/90

aspartate THRS [.034, .037] acetaldehyde [.004, .005] [.005, .006] [.009, .012] PFK, FBP, FBA, TPI threonine THRLAD glycine THRD [.033, .037] ADA E1: [.004, .004] [.002, .002] E2: [.008, .008] [.000, .000] [.033, .037] [.002, .003] E3: [.021, .021] [.001, .001] [.002, .002] [.007, .009] E4: [.020, .021] [.000, .000] [.002, .003] fructose-6P/-BP GAPD, PGK [.000, .000] [.007, .009] (+) [.008, .004] 2-oxobutanoate GHMT ASPTA1 [.019, .020] ycerate gl [.036, .040] [.049, .051] [-.035, -.032] glyceraldehyde-3P ospho (+) LEUB [.009, .009] [.048, .050] ph serine [.001, .002] PSP [.005, .007] [.018, .019] (+) [.000, .000] [.137, .156] SERD [.000, .002] [.000, .000] [.022, .024] PGM, ENO [.133, .154] [.168, .190] [.003, .003] [.146, .165] [.328, .354] [.129, .150] [.003, .003]

TAL (+) [.153, .174] [.167, .193] [.315, .341] [.000, .000] [.379, .405] [.159, .185] [.000, .000] [.217, .243] acetateex [.001, .001] (+) PYK, PPDK, PPS pyruvate [.001, .001] acetate ruvate [.160, .179] PDH, POR, lpy [.161, .182] transport -eno TKT2 erythrose-4P o [.159, .183] [.022, .025] [.051, .053] [.001, .001] phosph [.017, .031] ACKr, PTAr [-.127, -.108] acetyl-CoA 1.00 [.002, .002] PEPCK, PEPCKG (+) PC acetate

(+) [-.058, -.044] [.005, .005] CS, ACONT [-.014, -.014] [.014, .016] (+) [.005, .005] (+) [.012, .014] [.999, 1.00] [-.006, -.003] [.006, .043] [.927, .930] [.221, .238] [.020, .045] [1.05, 1.07] sedoheptulose-7P [.201, .226] citrate/ [1.00, 1.01] isocitrate oxaloacetate ME1, ME2 TKT1 MDH [-.014, -.011] ICDHy

[.000, .000] (+) [1.01, 1.02] pentose-5P [.003, .005] (+) [.999, 1.00] pool [.000, .000] [.000, .032] [.918, .922] [.927, .930] [.001, .001] [-.045, -.022] [1.26, 1.31] (+) [1.05, 1.07] [.001, .001] [1.19, 1.22] [1.00, 1.01] (+) malate FUM α-ketoglutarate biomass [.997, 1.00]

(+) AKGD, OOR Y [g/mmol] [.922, .925] XS [1.28, 1.32] [.997, 1.00] E1: [.003, .003] [1.16, 1.19] fumarate succinate/succinyl- [.922, .925] E2: [.006, .006] CoA [1.04, 1.06] [.984, .997] E3: [.015, .016] dicarboxylic acid transporter E4: [.015, .015] SUCOAS, ATO, FRD5 fumarateex succinateex N.A [.997, 1.00] N.A N.A [.922, .925] N.A [3.09, 3.10] [-1.81, -1.77] [2.83, 2.86] [2.67, 2.68] [-1.52, -1.49] [2.48, 2.50] (+) 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 arrows indicate bidirectional reactions, and the black arrowa with plus signs correspond to the net direction of a positive value for the bidirectional reactions. Unidirectional reactions were indicated by gray single-headed arrows. The subscript “ex“ denotes extracellular metabolites. The abbreviations (pathway enzymes) are listed in Table 2, and precursor demand for biomass synthesis (precursors in yellow color) in Table 4. response of G. sulfurreducens under varying growth con- phosphoenolpyruvate biosynthesis for gluconeogenesis. In ditions, relative flux distributions at key branch points in the acceptor-limited culture (E4), phosphoenolpyruvate gluconeogenesis, anaplerosis, the TCA cycle, and amino for gluconeogenesis was synthesized almost exclusively acid metabolism were examined in detail (Figure 3). from oxaloacetate (ca. 93%), whereas in the donor-limited The synthesis of phosphoenolpyruvate is one key branch culture ca. 60% of phosphoenolpyruvate for gluconeogen- point of gluconeogenesis. As shown in Figure 3-A, the esis was generated from oxaloacetate by PEPCK/PEPCKG major pathway for phosphoenolpyruvate synthesis for glu- by consuming ATP/GTP. coneogenesis varied with electron acceptors. When Fe(III) Conversion of pyruvate to oxaloacetate by pyruvate was the electron acceptor (E1 & E2), phosphoenolpyruvate carboxylase (PC) plays a key role in anaplerosis. As for gluconeogenesis was generated almost exclusively (89 - shown in Figure 3-B this branch point of anaplerosis was 99%) from pyruvate by PYK/PPDK/PPS, and phosphoenol- less sensitive towards electron acceptor variation than pyruvate synthesis via the decarboxylation of oxaloacetate gluconeogenesis. In the both Fe(III) cultures, ca. 9% of (PEPCK/PEPCKG) did not contribute significantly to glu- pyruvate was converted into oxaloacetate for anaplerosis, coneogenesis. In contrast, when fumarate was provided as whereas the amount of pyruvate that was diverted to ana- the electron acceptor (E3 & E4), the decarboxylation of plerosis varied from 4 to 15% during growth with fuma- oxaloacetate (PEPCK/PEPCKG) was the main route for rate. This variation appeared to reflect the degree to Yang et al. Microbial Cell Factories 2010, 9:90 Page 7 of 15 http://www.microbialcellfactories.com/content/9/1/90

A.gluconeogenesis B.anaplerosis

phosphoenolpyruvate phosphoenolpyruvate pyruvate pyruvate pyruvate pyruvate other other pathways pathways E1: [98, 99] % E1:[8,10]% E3: [3, 5] % E2: [98, 99] % E3: [57, 61] % E4: [90, 95] % E2:[8,10]% E4: [14, 17] % oxaloacetate oxaloacetate oxaloacetate oxaloacetate

Fe(III)citrate fumarate Fe(III)citrate fumarate

C. TCA cycle

(1)pyruvate pyruvate (2) phosphoenolpyruvate phosphoenolpyruvate

E1: [97 97] % E3: [94, 95] % E1: [96, 96] % E3: [81, 82] % E2: [92 93] % E4: [93, 94] % E2: [99, 99] % E4: [81, 82] %

acetyl-CoA acetyl-CoA oxaloacetate oxaloacetate citrate citrate citrate citrate

Fe(III)citrate fumarate Fe(III)citrate fumarate

D. amino acid metabolism

pyruvate + acetyl-CoA (1)2-oxobutanoate (2)glyceraldehyde 3-P (3) E1: [68, 73] % E1: [99, 99] % E1: [87, 89] % E2: [1, 2] % E2: [47, 51] % E2: [80, 82] % E3: [88, 90] % E3: [66, 72] % E3: [86, 86] % E4: [91, 93] % E4: [80, 84]% E4: [75, 76] %

2-oxobutanoate threonine threonine glycine 3-phosphoglycerate serine

Figure 3 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). which oxaloacetate was diverted to gluconeogenesis. In utilization (Figure 3-C.2) revealed that nearly all of the the acceptor-limited culture (E4), where 90-95% of the oxaloacetate produced during growth via Fe(III) reduc- oxaloacetate synthesized from fumarate was used for glu- tion (96 - 99%) was utilized to initiate the TCA cycle, coneogenesis, about 15% of pyruvate was utilized for ana- whereas only 82% to 85% of oxaloacetate utilized to plerosis. In contrast, in the donor-limited culture (E3), initiate the TCA cycle in the two fumarate cases. This 57-61% of oxaloacetate was consumed for gluconeogen- difference can be attributed to the utilization of fuma- esis and 4% of pyruvate was diverted to anaplerosis. rate as an additional carbon source for gluconeogenesis The condensation of acetyl-CoA and oxaloacetate (CS/ and amino acid biosynthesis. ACONT) is the initiation step of the TCA cycle. As A recent study [3] indicated that G. sulfurreducens has shown in Figure 3-C.1, the relative amount of acetyl- two pathways for isoleucine biosynthesis: the precursor CoA (between 92% and 97%) entering the TCA cycle 2-oxobutanoate can be generated (i) by the deamination was comparable for all cultivation conditions. The rela- of threonine and (ii) from pyruvate and acetyl-CoA tive amount of acetyl-CoA entering the TCA cycle was through the citramalate pathway. Risso et al. (2008) [3] nearly identical for the two fumarate cultures (E3 and observed that the citramalate pathway accounted for the E4). In the Fe(III) cultures, the percentage of acetyl-CoA majority of isoleucine biosynthesis during growth with entering the TCA cycle in the culture with acetate fumarate. This was also the case in the present study as the sole electron donor (97%) was higher than that where 88 to 93% of 2-oxobutanoate was synthesized from in the presence of both hydrogen and acetate as the pyruvate and acetyl-CoA in the two fumarate cultures electron donors (92%). Examination of oxaloacetate (Figure 3-D.1). A smaller fraction of 2-oxobutanoate, ca. Yang et al. Microbial Cell Factories 2010, 9:90 Page 8 of 15 http://www.microbialcellfactories.com/content/9/1/90

70%, was produced via the citramalate pathway in the flux through serine deaminase that observed by 13CMFA acetate Fe(III) culture (E1). In contrast, the 2-oxobutano- is plausible. ate was synthesized almost exclusively from threonine It has been hypothesized that futile cycles in cellular (98 - 99%) in E2 culture containing hydrogen, Fe(III), and metabolism are involved in the regulation of biochem- 1 mM acetate. During growth in the presence of hydro- ical pathways. Quian and Beard (2006) [39] suggested gen, decreased demand for energy production by the that futile cycles actively shift the effective equilibrium TCA cycle may result in TCA cycle intermediates such by expending energy where the magnitude of changes in as oxaloacetate (main precursor for aspartate and threo- effective equilibria and sensitivities is a function of the nine), more available for other uses such as amino acid amount of energy used by a futile cycle. Also, it has biosynthesis. In fact, a complete TCA cycle is not been suggested that cellular regulation of the ATP/ADP required during growth on hydrogen [13]. ratio depends on both the specific growth rate and the G. sulfurreducens also has two pathways for the bio- environmental conditions [40]. Tang et al. (2007) [31] synthesis of glycine: through the conversion of threonine has found futile cycle activity in Geobacter metalliredu- to glycine and acetaldehyde by threonine aldolase cens, however metabolic function of futile cycles is yet (THRLAD) and from the conversion of serine to glycine fully understood. The observed futile cycle might be by glycine hydroxymethyltransferase (GHMT). 13CMFA related to an important metabolic function, e.g., in regu- indicated that the percentages of threonine converted lating substrate utilization for gluconeogenesis and the into glycine and acetaldehyde varied under different oxidative TCA cycle. conditions (Figure 3-D.2). In E1, 99% of threonine was converted to glycine and acetaldehyde. The acetaldehyde Specific Metabolic Fluxes was further converted to acetyl-CoA (generating In addition to the relative branch point flux distributions, NADH) and the acetyl-CoA was then recycled into the we compared the specific metabolic fluxes, which are central metabolic pathways. quantities scaled by cell growth (see Methods Eq. 4), to Another interesting observation which has not been understand the growth-associated metabolic activities. As previously described in the literature was that a large shown in Table 3, the specific fluxes from phosphoglyce- portion of 3-phosphoglycerate (75 - 89%) was converted rate to hexose 6-phosphate of the lower gluconeogenesis to serine (Figure 3-D.3). At this metabolic branch point, and pentose phosphate pathways were invariant under the flux into glyceraldehyde 3-phosphate for gluconeo- different culture conditions. These results suggest that genesis was kept at a relatively low level corresponding these pathways operated purely to satisfy anabolic to anabolic demands. Serine was subsequently converted demand (Table 4) and that the differences in the relative to pyruvate by serine deaminase. As shown in Figure 2, fluxes through these pathways shown in Figure 2 were there were significant fluxes through serine. During due to different biomass yields on acetate under different growth with Fe(III) as the electron acceptor (E1, E2), conditions. The specific fluxes of the upper gluconeogen- there was a ATP-consuming futile cycle consisting of esis pathway from pyruvate to phosphoenolpyruvate were phosphoenolpyruvate, phosphoglycerate, serine, and pyr- similar to those from phosphoenolpyruvate to phospho- uvate, where phosphoenolpyruvate synthase (PPS) and/ glycerate during growth with Fe(III) (E1 & E2). In com- or pyruvate phosphate dikinase (PPDK) consume ATP parison, these specific fluxes were considerably lower when converting pyruvate to phosphoenolpyruvate. than those from phosphoenolpyruvate to phosphoglyce- In spite of its small contribution to 2-oxobutanoate rate when fumarate was the electron acceptor. This biosynthesis, a high level of threonine deaminase activity appeared to be due to the substantial contribution of the (25-higher than that of citramalate synthase) has been phosphoenolpyruvate carboxykinase to gluconeogenesis previously detected in soluble extracts of G. sulfurredu- by converting oxaloacetate to phosphoenolpyruvate. cens growing on fumarate [3]. The threonine deaminase As indicated in Figure 2, the initial step in gluconeo- of G. sulfurreducens is also capable of deaminating ser- genesis was the generation of pyruvate from acetyl- ine to pyruvate [3], and is likely the enzyme deaminating CoA (POR) in the Fe(III) cultures and phosphoenol- serine in G. sulfurreducens, as this species does not con- pyruvate formation from oxaloacetate by PEPCK/ tain the Fe(II)-dependent and oxygen sensitive serine PEPCKG in the fumarate cultures. As shown in Table deaminases gene found in Clostridia and plants. More- 3, the specific fluxes of POR were identical in the two over, knocking out the threonine deaminase eliminated Fe(III) cultures, and the specific fluxes of PEPCK/ all detectable serine deaminase activity in soluble PEPCKG in the fumarate cultures were similar. Thus, extracts prepared under aerobic conditions [3]. Given the initial gluconeogenetic flux per unit biomass synth- the presence of relatively high levels of serine deaminase esis remained constant when the same electron accep- activity in G. sulfurreducens extracts, the relatively high tor was utilized. Yang et al. Microbial Cell Factories 2010, 9:90 Page 9 of 15 http://www.microbialcellfactories.com/content/9/1/90

Table 3 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. pathway enzymes specific metabolic fluxes pathway description E1 E2 E3 E4 ACKr, PTAr 369 166 65.2 66.3 acetate uptake PFK, FBP, FBA, TPI 1.38 1.38 1.38 1.38 lower gluconeogenesis GAPD, PGK 3.25 3.25 3.25 3.25 PGM, ENO 57.5 27.1 25.6 15.3 upper gluconeogenesis PYK, PPDK, PPS 62.6 28.4 11.1 1.60 PDH, POR 8.68 8.68 -7.63 -3.37 pyruvate ↔ acetyl-CoA PEPCK, PEPCKG -4.62 -0.77 15.0 14.2 gluconeogenesis initiation TKT1 0.05 0.05 0.05 0.05 pentose phosphate pathway TAL 0.05 0.05 0.05 0.05 TKT2 0.31 0.31 0.31 0.31 CS, ACONT 370 154 69.3 66.5 TCA cycle ICDHy 370 154 69.3 66.5 AKGD, OOR 370 153 68.5 65.7 FRD5 370 153 -116 -99.5 FUM 370 153 84.8 77.8 MDH 374 153 83.8 80.0 decarboxylic acid transporter / / 202 177 fumarate uptake / / 185 165 succinate secretion Enzyme abbreviations are listed in Table 2.

Also shown in Table 3, the specific flux through the generation via hydrogen oxidation did not appear to TCA cycle during growth with Fe(III) was approximately completely replace energy generation via acetate oxida- 2.4-fold larger in E1 (5 mM acetate) than E2 (H2 +1 tion by the TCA cycle. During growth in the presence mM acetate). This is due to the direct energy produc- of hydrogen and 1 mM acetate, flux through the TCA tion via hydrogen oxidation which decreased the energy cycle was higher than what was required for the ana- production demand for the TCA cycle. However, energy bolic demand for a-ketoglutarate and oxaloacetate, and a significant amount of acetate was oxidized via the Table 4 Precursor demand of G. sulfurreducens from the TCA cycle. One reason of the energy production via intermediary metabolites involved in the central acetate oxidation in the presence of hydrogen might be ’ metabolic pathways in Figure 2 the relatively poor solubility of H2 in water. The Henry s -4 -1 -1 Precursor demand of G. sulfurreducens constant of H2 in water is only 7.8 × 10 molL atm -1 at 298°K and therefore the dissolved H2 concentration precursor demand [mmolgbiomass ] at 0.60 atm H2 is approximately 0.5 mM. This can also glucose 6-phosphate 0.964 be due to other metabolic factors such as hydrogenase fructose 6-phosphate 0.056 activity [41,42] or cell preference for generating redu- ribose 5-phosphate 0.405 cing power through the oxidative TCA cycle. erythrose 4-phosphate 0.259 In the fumarate cultures, the specific acetate uptake glyceraldehyde 3-phosphate 0.192 and TCA cycle activity was dramatically lower than that 3-phosphoglycerate 0.062 in the Fe(III) cultures (Table 3). The specific acetate phosphoenolpyruvate 0.519 uptake in the two fumarate cultures was 65 to 66 mmol pyruvate 1.842 acetyl-CoA per gram biomass synthesis as compared to acetyl-CoA 3.556 166 to 369 mmol of acetyl-CoA per gram biomass synth- a-ketoglutarate 0.755 esis for the Fe(III) cultures (Table 3). The standard aspartate 0.814 potential of the half reaction Fe3+ + e- ® Fe2+ (E°=771 serine 0.490 mV) is higher than that of fumarate2- +2e- +2H+ ® suc- glycine 0.576 cinate2- (E° = 31 mV). Thus, it is expected that a higher threonine 0.173 electromotive force (ΔE°) would be generated by the acet- 2-oxobutanoate 0.198 ate/Fe(III) pair than the acetate/fumarate pair. However, Yang et al. Microbial Cell Factories 2010, 9:90 Page 10 of 15 http://www.microbialcellfactories.com/content/9/1/90

the fumarate cultures consumed less acetate per unit bio- acetateex mass formation than the Fe(III) cultures, suggesting that the cells in the fumarate cultures utilize acetate more effi- gluconeogenesis acetyl-CoA ciently than in the Fe(III) cultures. Higher biomass yields on acetate in the fumarate cultures relative to Fe(III) have been previously reported [2,32,36] and is likely due E3: [.41, .43] E3: [.34, .34] to the fact that Fe(III) is reduced on the outside of the Q8 Q6 E4: [.43, .44] E3: [.60, .65] E4: [.37, .37] Q2 cells which results in an additional energetic cost for E4: [.85, .94] exporting electrons. E3: [.57, .59] Qnet E4: [.56, .57] E3: [.92, .92] Q7 fumarate succinate E4: [.93, .94] Fumarate Utilization 13 G. sulfurreducens fumarateex succinateex The CMFA results suggest that uti- Q1 1.00 E3: [.02, .07] Q3 lizes fumarate not only as the electron acceptor for acet- E4: [.29, .37] ate oxidation but also as an additional carbon source. Figure 4 95% confidence intervals given for key TCA fluxes (νi) Based on efflux measurements by HPLC, fumarate in the fumarate cultures (E3, E4), normalized by fumarate ν ν ν uptake and succinate secretion were found to be very uptake. net = 2 - 3. close in both fumarate cultures (cf. ‘Steady-State Effluxes in Chemostat Cultures’ section). However, the optimal solutions found by the stochastic 13CMFA runs resulted between fumarate and succinate were smaller in the in larger fumarate uptake than succinate secretion donor-limited culture (E3) than in the acceptor-limited (Figure 2) and the efflux estimates were within the sta- culture (E4). In particular, the flux through the oxidative tistical range given by the means and standard devia- direction was only 2 to 7% per unit fumarate uptake in tions determined experimentally (95% confidence E3 with the corresponding reductive flux of 60 to 65%, interval). As a result, the small percentage (< 10%) of as compared to the oxidative flux of 29 to 37% with the fumarate consumed as a carbon source in the fumarate reductive flux of 85 to 94% under the donor-limiting cultures, which could not be determined by HPLC efflux condition. measurements was discovered by 13CMFA with more accurate GC/MS labeling measurements. Redox Balance As shown in Table 3, the uptake of fumarate increased We further calculated the net production of redox the specific oxidative TCA fluxes of fumarase (FUM) equivalents (NAD(P)H and menaquinol) and carbon and malate dehydrogenase (MDH) compared to that of dioxide from the in vivo flux estimates. Because G. sul- a-ketoglutarate dehydrogenase (AKGD/OOR). Conse- furreducens can utilize both NADH and NADPH for Fe quently, phosphoenolpyruvate carboxykinase (PEPCK/ (III) and fumarate reduction [43,44]; NADH and PEPCKG) was the enzyme responsible for initiating glu- NADPH are pooled for redox balance. NAD(P)H is pro- coneogenesis in the fumarate cultures, while pyruvate duced in the central metabolic network by the reactions synthase (POR) initiated gluconeogenesis in the Fe(III) ICDHy (isocitrate ®a-ketoglutarate), AKGD/OOR (a- cultures. In the fumarate cultures, acetyl-CoA was ketoglutarate ® succinyl-CoA), MDH (malate ® oxa- replenished from pyruvate (PDH/POR) which was pro- loacetate), ME1/ME2 (malate ® pyruvate), PDH/POR duced by phosphoenolpyruvate through 3-phosphoglyce- (pyruvate ® acetyl-CoA), GAPD (glyceraldehyde 3- rate and serine. Between the two fumarate cultures, cells phosphate ® 1,3-bisphosphoglycerate), and ADA (acet- in the donor-limited condition E3 had higher net fuma- aldehyde ® acetyl-CoA). In addition, cells consume 14.2 rate reduction, succinate secretion, oxidative TCA activ- mmol NADPH and produce 2.32 mmol NADH per ities, and gluconeogenesis activities than those in the gram biomass synthesis based on the genome-scale acceptor-limited condition E4 (Table 3). model [2]. 3-phosphoglycerate dehydrogenase involved To gain insight into fumarate utilization, we compared in serine biosynthesis produces NADH, yet the next relative fluxes around fumarate and succinate, which reaction carried out by 3-phosphoserine aminotransfer- were normalized by fumarate uptake (Figure 4). Overall, ase involves conversion of glutamate to 2-oxoglutrate relative fumarate utilization including net fumarate for transamination. Without 2-oxoglutamate secretion, reduction, fumarase flux, succinate secretion, etc., was glutamate has to be regenerated from 2-oxoglutarate similar in both cultures (Figure 4). A notable difference which consumes one redox equivalent. Hence, there is between the two fumarate cultures was found in the no net production of redox equivalent in this case. In G. reversibility of FRD5. FRD5 of G. sulfurreducens has a sulfurreducens, FRD5 reduces menaquinone to menaqui- dual function in fumarate reduction and succinate oxi- nol when succinate is oxidized to fumarate and vice dation [32]. Both reductive (ν2)andoxidative(ν3)fluxes versa for the fumarate reduction. The fluxes related to Yang et al. Microbial Cell Factories 2010, 9:90 Page 11 of 15 http://www.microbialcellfactories.com/content/9/1/90

decarboxylation/carboxylation are ICDHy, AKGD/OOR, distributions of key metabolic branch points. Expanding PC, ME1/ME2, PEPCK/PECKG, PDH/POR, and other on this, we compared redox balances drawn from flux biosynthetic pathways. In addition, cells produce about estimates of the different cultures. From 13C labeling 1.36 mmol CO2 per gram biomass synthesis. Details of measurements and subsequent flux analysis, the greater anabolic NADPH net consumption, NADH net produc- metabolic discrepancies in the central metabolism, such tion, and net decarboxylation are listed in additional as different initial step in gluconeogenesis, were file 1. observed to be induced by the electron acceptor varia- In the acetate/Fe(III) culture (E1), the molar yields of tion of Fe(III) versus fumarate. As shown by the specific redox equivalents and CO2 net production on acetate fluxes, metabolic activities in the TCA cycle were per- were 3.97 ± 0.08 and 1.97 ± 0.05, respectively. These turbed not only by electron acceptor variation (Fe(III) corresponded to a molar yield of Fe(III) reduction on vs. fumarate) but also by electron donor variation (acet- acetateof7.94±0.16andaratioofFe(II)toCO2 pro- ate vs. hydrogen). Furthermore, we were able to obtain duction of 4.03 ± 0.13. Comparing the stoichiometry of detailed understanding of cellular fumarate utilization complete acetate oxidation with Fe(III), i.e., CH3COOH and demonstrated that fumarate acts simultaneously as 3+ 2+ + +8Fe + 10H2O ® 2CO2 +8Fe +8H3O , the Fe(III) the electron acceptor and an additional carbon source, reduction and CO2 production drawn from the in vivo as previously hypothesized elsewhere [46]. This seems to flux estimates for E1 are consistent with the theoretical be the reason why cells in the fumarate cultures stoichiometry. initiated gluconeogenesis via phosphoenolpyruvate dec- The direct oxidation of hydrogen coupled with Fe(III) arboxykinase and resulting in the higher biomass yield 3+ 2+ + reduction is H2 +2Fe +2H2O ® 2Fe +2H3O on acetate than in the Fe(III) cultures. [33,45]. Despite hydrogen utilization, cells in the hydro- In the hydrogen/Fe(III) culture, we found that cells gen/Fe(III) culture (E2) still produced 3.56 ± 0.05 moles still maintained the oxidative TCA cycle, with reduced of redox equivalents with 1.80 ± 0.04 moles of CO2 per activities as compared to the acetate/Fe(III) culture. mole of acetate taken up. These results corresponded to From redox balance calculations, we observed that cells a molar yield of Fe(III) reduction on acetate of 7.12 ± gained redox equivalents from both hydrogen and acet- 0.10 and a ratio of Fe(II) to CO2 production of 3.96 ± ate; i.e., acetate supplemented in the hydrogen culture 0.10. Thus, Fe(III) reduction together with CO2 produc- was not only utilized for anabolic demand but also oxi- tion coincides with the theoretical stoichiometry of Fe dized along with hydrogen by Fe(III). (III) reduction on acetate. In contrast to E1, cells oxi- The two fumarate cultures with different ratios of dized less acetate via the TCA cycle (smaller yield of Fe acetate and fumarate gave discrepancies in metabolic (III) reduction and reduced TCA fluxes) and conse- fluxes per unit acetate uptake or per unit biomass synth- quently doubled biomass yield on acetate (Figure 2 & esis including fumarate uptake and succinate secretion. Table 3). Nevertheless, the two cultures appeared to have some The complete acetate oxidation with fumarate follows physiological similarities. In particular, examination of CH3COOH + 4C4H4O4 +2H2O ® 2CO2 +4C4H6O4. TCA fluxes scaled by fumarate uptake elucidated meta- The net conversion of fumarate to succinate per acetate bolic similarities between the fumarate cultures in terms was 2.9 ± 0.1 in the donor-limited culture (E3) and of fumarate utilization. The notable discrepancy between 2.5 ± 0.2 in the acceptor-limited (E4). The molar yield the two cultures was reversibility of fumarate reductase. of CO2 production on acetate was comparable with the G. sulfurreducens has great potential for use in applica- theoretical stoichiometry: it was 2.0 ± 0.5 and 1.8 ± 0.3 tions such as harvesting bioelectricity and environmental in E3 and E4, respectively. These results may be due to remediation. In these applications, understanding the additional decarboxylation of fumarate carbons, e.g., glu- metabolism of G. sulfurreducens with different electron coneogenesis initiated from oxaloacetate by PEPCK/ donors and acceptors will allow for improved strategies PEPCKG and acetyl-CoA replenishment from pyruvate or designs. Along with in silico studies using material and by PDH. In addition, two fumarate carbons can poten- energy balances [2], the in vivo information gained from tially be released through these reactions. the 13C metabolic flux analysis in the present work can aid improvements of bioprocesses using this interesting Conclusions microorganism. For example, the flux distributions in In the present work, we characterized the intracellular donor limiting or acceptor limiting conditions coupled metabolic response of G. sulfurreducens towards differ- with microbial community models may help improve ent electron donor/acceptor conditions in vivo using bioremediation strategies. The flux results of acetate and 13 C-based metabolic flux analysis. The metabolic charac- H2 as donors can provide insights into certain co-cultures teristics were examined from different aspects of acetate or microbial communities containing G. sulfurreducens and fumarate utilization, biomass formation, and flux that produce bioelectricity in microbial fuel cells, and can Yang et al. Microbial Cell Factories 2010, 9:90 Page 12 of 15 http://www.microbialcellfactories.com/content/9/1/90

be used to improve designs for higher electricity produc- monitored by high-performance liquid chromatography tion. Also, other fascinating metabolic capabilities of Geo- (HPLC) as previously described [46]. bacter species such as degrading variety of organic The amino acids obtained by cell protein hydrolysis materials, including aromatic compounds [1,47] and were silylated using N-methyl-N-tert-butyldimethylsilyl reducing toxic and radioactive metals [45,48,49], can be trifluoroacetamide as previously described [3,27]. Gas investigated using 13C metabolic flux analysis in the chromatography/mass spectrometry (GC/MS) analysis future. was carried out using a Hewlett-Packard HP G1723A GC-quadrupole mass selective detector (electron Methods impact), equipped with a DB-5 column (Agilent Tech- Experimental Design & Cultivation Conditions nologies). From the mass spectra of t-butyldimethylsilyl Geobacter sulfurreducens (ATCC51573) [33] was cul- (TBDMS)-amino acid derivatives (triplicate measure- tured at 30°C under strict anaerobic conditions with an ments), carbon mass isotopomer distributions (MID) N2:CO2 atmosphere (8:20) in a defined freshwater med- were computed using the method described earlier [27]. ium [50] either in pressure tubes or in chemostats as The metabolic fluxes were computed from the carbon previously described [36]. mass isotopomer distributions obtained from the mass To investigate the metabolic response of G. sulfurre- spectrometric labeling analysis of different fragment ions ducens to variations in electron donors and acceptors, of TBDMS-derivatives of proteinogenic amino acid three chemostat experiments (E1, E3, E4), in which acet- hydrolysates comprising alanine, glycine, leucine, isoleu- ate was provided as the electron donor and carbon cine, serine, phenylalanine, aspartate, and glutamate (see source and either Fe(III)citrate or fumarate served as the additional file 2). The GC/MS fragment ions for those electron acceptor, were conducted. Three biological TBDMS-amino acid derivatives are listed elsewhere [19]. replicates were performed for each of the three experi- ments and chemostats were run at a dilution rate of 13C Metabolic Flux Analysis either 0.04 h-1 (E1) or 0.05 h-1 (E3 and E4). Cells were From the carbon mass isotopomer distributions of pro- harvested at steady state, after five volume refills to teinogenic amino acid hydrolysates and measured achieve isotopic steady-state and to make the fraction of effluxes, in vivo metabolic fluxes were computed by sol- inoculum negligible. To confirm steady-state, substrate ving the following constrained nonlinear least-squares (s) and biomass (x) concentrations were measured to minimization problem (NLSP): verify no accumulation of substrate (d[s]/dt = 0) as well d x dt 1 T − as biomass ( [ ]/ = 0). min()− FF (ΘΣ )1 ()− ( Θ ) with Θ∈ [01 , ) (1) In addition, one batch experiment (E2) was performed Θ 2 in which both acetate and hydrogen were provided as electron donors and Fe(III)citrate served as the electron subject to ab≤≤ & =c()&Θ  =0 acceptor. This experiment consisted of four replicate ∑ carbon, i (2) cultures, which were harvested and pooled when the i concentration of Fe(II) approached 50 mM. All the In the objective function (Eq. 1), the covariance- batch cultures were transferred (1 vol/vol-%) into weighted (Σ) sum of squared of the difference between 13C-tracer containing minimal medium at least once the measurements h (only efflux and 13C-labeling mea- prior to the initiation of an experiment to extremely surements) and the corresponding model prediction minimize the fraction of non-labeled cells. The main F(Θ) are minimized with respect to [0,1)-scaled inde- cultures with the identical medium were inoculated with pendent flux variables Θ (12). As the covariance, experi- the mid-exponential phase 13C-precultures. mentally determined uncertainties associated with efflux All cultures contained a mixture of 70% non-labeled and labeling measurements were applied. The model and 30% uniformly labeled acetate (Sigma-Aldrich, St. F(Θ) describes the mathematical relationship between Louis, MO) as a 13C-tracer carbon source for 13CMFA. unknown fluxes (ν) and the measurements in terms of A summary of the cultivation conditions is provided in two connected equation systems, i.e., the stoichiometry Table 1. and the carbon isotopomer reactions in the metabolic network. The fluxes ν, which are the dependent vari- Analytical Methods ables of Θ in the stoichiometry system, consist of Cell growth was monitored by determining total protein immeasurable intracellular fluxes νu and measurable T by the bicinchoninic acid method with bovine serum effluxes νm, i.e., ν =(νu, νm) . Typically, realistic models albumin as a standard [51] and assuming a protein con- are underdetermined, that is, the rank of the stoichio- tent of 46% of cell dry weight [2]. Organic acids were metric matrix S is smaller than the number of entries in Yang et al. 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νu.Thisdifferencebetweenrank(S)andthenumberof In this metabolic network, the conversion from phos- entries of νu equals the number of fluxes that have to be phoenolpyruvate to pyruvate is catalyzed by pyruvate chosen as the design parameters, Θ. Those independent kinase, whereas the reverse reaction from pyruvate to fluxes are estimated from isotopic labeling measure- phosphoenolpyruvate by phosphoenolpyruvate synthase ments in 13CMFA. and/or pyruvate phosphate dikinase. Also, the conver- In the present work, in vivo flux estimation was done sion of pyruvate to acetyl-CoA is catalyzed by the irre- using the elementary metabolite unit (EMU) concept versible pyruvate dehydrogenase, and the bidirectional [15]. To solve the NLSP (Eq. 1) with the physical con- pyruvate synthase. Enzyme reactions catalyzing the iden- straints (Eq. 2), a gradient-based hybrid algorithm, tical substrate/product pairs in the same direction can- described in detail in Yang et al. (2008) [18], was not be discriminated by the 13C-based MFA. employed. The first inequality constraint in (Eq. 2) is The precursor demand of G. sulfurreducens applied to the feasible region in the flux space allowed by the stoi- the present study is listed in Table 4. The anabolic chiometry and experimental uncertainties given for the fluxes from the listed precursors into biomass synthesis effluxes νm. The region can be computed a priori by sol- can be calculated by multiplying the biomass yield coef- ving a quadratic programming problem for the stoichio- ficients with the precursor demand. metric system [2]. The second constraint is the elemental balance of carbon, i.e., the sum of carbons Flux Quantities incoming into the system equals that of outgoing the In the present work, we utilized two different flux quanti- system. Hereby, is not measured but can ties. The relative fluxes utilized in sections ‘Overview of in be derived from the stoichiometry, i.e., the net CO2 vivo Fluxes’ and ‘Fumarate Utilization’ are the rates nor- efflux equals the difference between the decarboxylation malized to acetate uptake and fumarate uptake, respec- (νdecarb) and the carboxylation (νcarb) fluxes. Hence, car- tively. They can be understood as molar yield coefficients bon balance can be computed from the effluxes (sub- on a particular substrate or the rates of enzyme reactions strate uptake, product and biomass formation) and the associated with the uptake of a unit substrate in unit time. net CO2 efflux, i.e., Another flux quantity introduced in section ‘Specific Metabolic Fluxes’ are the specific fluxes scaled by cell n m ⎛ p q ⎞ ⎜ ⎟ growth. We defined these specific fluxes as the relative cc−− − = 0. (3) -1 -1 ∑∑si,, si pj , pj ,⎜ ∑decard ,k ∑ carb ,l ⎟ flux vi (rate of a metabolite processed dci/dt [mmolL h ] i==11j ⎝ k = 1l = 1⎠ -1 -1 per acetate uptake rate [mmolL h ]) normalized by YXS. -1 th Thus, the specific flux qi [(mmol/h) (gbiomass/h) ]ofthei Here, νS and νP denote the effluxes of substrates and enzyme reaction equals products, respectively, and cS and cP the number of car- bons in the corresponding compounds. From precursor −  dc ⎛ dc ⎞ 1 relation, cells are expected to produce 1.36 mmol CO2 ==i iX qi ⎜ ⎟ , per gram dry cell weight. In practice, the constraint on Y dt ⎝ dt ⎠ XS (4) carbon balance can be defined as an inequality to con- −1 −1 dc ⎛ dc ⎞ dc ⎛ dc ⎞ sider measurement uncertainties, e.g., between ± 0.05. where  = iS⎜ ⎟ & Y = XS⎜ ⎟ . i dt ⎝ dt ⎠ XS dt ⎝ dt ⎠ Metabolic Network The specific flux can be understood as the rate of a 13 The framework of CMFA is to draw up an appropriate metabolic enzyme reaction associated with the synthesis metabolic network for the biological system of interest. of a unit biomass in unit time. Based on the genome-scale metabolic network of G. sul- furreducens reconstructed by Mahadevan et al. (2006) Software Implementation & Statistical Data Analysis G. sulfurreducens [2], the central metabolic network of All computations involved in carbon mass isotopomer 13 designed for CMFA comprises 26 intracellular fluxes analysis and 13CMFA were implemented using MATLAB containing 15 bidirectional flux pairs in the tricarboxylic (Version 7.8, The Mathworks Inc., Natick, MA). Numeri- acid (TCA) cycle, gluconeogenesis, pentose phosphate cal optimization was carried out using the Optimization (PP) pathway, amino acid metabolism, and anabolic Toolbox (Version 4.2) of MATLAB. demands for biomass synthesis. The details enzyme The in vivo metabolic fluxes were computed stochasti- reactions are listed in Table 2. In terms of the null cally by starting the numerical optimization runs from space investigation of the stoichiometric matrix [52], the arbitrary points and by using normal random numbers metabolic network was found to have 20 independent of measurement data. Hereby, 13C labeling patterns of flux variables that need to be determined from amino acids were computed directly if specified in the 13 C-labeling measurements. stoichiometric network model (Figure 2). Otherwise, Yang et al. Microbial Cell Factories 2010, 9:90 Page 14 of 15 http://www.microbialcellfactories.com/content/9/1/90

13C labeling patterns were computed from the corre- Received: 6 August 2010 Accepted: 22 November 2010 sponding precursors. The procedure was repeated for Published: 22 November 2010

500 times from different starting points to get sample References distributions of the in vivo flux estimates resulting from 1. Lovley DR, Giovannoni SJ, White DC, Champine JE, Phillips EJ, Gorby YA, the numerical optimization. The symbolic operations Goodwin S: Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic required for metabolic flux modeling such as parametri- compounds to the reduction of iron and other metals. ArchMicrobiol zation and computing partial derivatives required for 1993, 159:336-344. EMU modeling were implemented using the Symbolic 2. Mahadevan R, Bond DR, Butler JE, Esteve-Nunez A, Coppi MV, Palsson BO, Schilling CH, Lovley DR: Characterization of metabolism in the Fe(III)- Math Toolbox (Version 3.2.3) of MATLAB. reducing organism Geobacter sulfurreducens by constraint-based Statistical data analysis such as random number gen- modeling. Appl Environ Microbiol 2006, 72:1558-1568. eration, confidence interval, analysis of variance 3. Risso C, Van Dien SJ, Orloff A, Lovley DR, Coppi MV: Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens. (ANOVA) and multiple comparison procedure were JBacteriol 2008, 190:2266-2274. conducted using the Statistics Toolbox (Version 7.1) of 4. Sun J, Sayyar B, Butler JE, Pharkya P, Fahland TR, Famili I, Schilling CH, MATLAB. One-way ANOVA was implemented to test Lovley DR, Mahadevan R: Genome-scale constraint-based modeling of Geobacter metallireducens. BMC Syst Biol 2009, 3:15. the hypothesis that the means of the flux estimates 5. Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR: given for the different culture conditions were identical, Extracellular electron transfer via microbial nanowires. Nature 2005, against the general alternative that they were not. In 435:1098-1101. 6. Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard TL, Lovley DR: Biofilm addition to this, multiple comparisons were performed and nanowire production leads to increased current in Geobacter to obtain information about which pairs of flux mean sulfurreducens fuel cells. Appl Environ Microbiol 2006, 72:7345-7348. estimates are significantly different or not. 7. Bond DR, Holmes DE, Tender LM, Lovley DR: Electrode-reducing microorganisms that harvest energy from marine sediments. Science 2002, 295:483-485. Chemicals 8. Holmes DE, Bond DR, O’Neil RA, Reimers CE, Tender LR, Lovley DR: 13 13 Microbial communities associated with electrodes harvesting electricity [U- C2] acetate was applied to CMFA in all the four from a variety of aquatic sediments. Microb Ecol 2004, 48:178-190. experiments (Table 1). To verify whether cells are cap- 9. Lovley DR: Bug juice: harvesting electricity with microorganisms. Nat Rev 13 able of using citrate as a carbon source, [U- C6]citrate Microbiol 2006, 4:497-508. was utilized. 10. Lovley DR: Microbial fuel cells: novel microbial physiologies and engineering approaches. Curr Opin Biotechnol 2006, 17:327-332. 11. Lovley DR: Cleaning up with genomics: applying molecular biology to Additional material bioremediation. NatRevMicrobiol 2003, 1:35-44. 12. Methe BA, Nelson KE, Eisen JA, Paulsen IT, Nelson W, Heidelberg JF, Wu D, Wu M, Ward N, Beanan MJ, et al: Genome of Geobacter sulfurreducens: Additional file 1: Anabolic precursor demand. Precursor stoichiometry metal reduction in subsurface environments. Science 2003, 302:1967-1969. given for the anabolic demand of G. sulfurreducens are listed. 13. Segura D, Mahadevan R, Juarez K, Lovley DR: Computational and Additional file 2: GC/MS mass isotopomer distributions. 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