Metabolic and Proteomic Analyses of Product Selectivity and Redox

Groeger et al. Microb Cell Fact (2017) 16:64 DOI 10.1186/s12934-017-0678-9 Microbial Cell Factories

RESEARCH Open Access Metabolic and proteomic analyses of product selectivity and redox regulation in Clostridium pasteurianum grown on glycerol under varied iron availability Christin Groeger†, Wei Wang†, Wael Sabra, Tyll Utesch and An‑Ping Zeng*

Abstract Background: Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes signifcantly with the composi‑ tion of the culture medium. Among others iron content in the medium was shown to strongly afect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses. Results: 1,3-PDO is most efectively produced under iron limited condition (Fe ), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe ). With increased iron availability the− BuOH/1,3-PDO ratio increased signifcantly from 0.27 mol/mol (at Fe ) to 1.4+ mol/mol (at Fe ). Additionally, hydrogen production was enhanced signifcantly under Fe condition. Proteomic− analysis revealed+ diferentiated expression of many proteins including several ones of the central+ carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydro‑ genases, and several electron transfer favoproteins was found to be strongly up-regulated under Fe condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently+ their infuences on the product selectivity in C. pasteurianum. Of particular signifcance is the fnding that H 2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which signif‑ cantly afects the redox balance and thus the product selectivity. Conclusions: The metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under diferent iron conditions. Keywords: n-Butanol, 1,3-Propanediol, C. pasteurianum, Proteomics, Product selectivity, Metabolic analysis

Background of a wide range of substrates [1–3], production of a wide Clostridium pasteurianum is an emerging and promis- spectrum of products [4, 5] and robust growth in sim- ing microbial cell factory for the production of chemicals ple media even under unsterile conditions [6]. Recently, and fuels because of some unique features, e.g. utilization C. pasteurianum was shown to accept electrons from the cathode by direct electron transfer [7], which make it an attractive candidate for new bioelectrical systems. *Correspondence: [email protected] †Christin Groeger and Wei Wang are frst authors and contributed equally Terefore, C. pasteurianum has received considerable to this work interests for the production of chemicals and fuels such Institute of Bioprocess and Biosystems Engineering, Hamburg University as 1,3-propanediol (1,3-PDO) and n-butanol (BuOH), of Technology, Denickestr.15, 21073 Hamburg, Germany

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Groeger et al. Microb Cell Fact (2017) 16:64 Page 2 of 16

which represents attractive bioprocesses for the use of bioreactor fermentation. Te bioreactor medium con- renewable resources, like biodiesel-derived glycerol or tained the following ingredients in 1 L of distilled water glucose from biomass hydrolysates [8–14]. In such bio- (modifed from [4]): glycerol, 80 g; yeast extract, 1 g; processes, several other fermentation products like gases K2HPO4, 0.5 g; KH 2PO4, 0.5 g; MgSO 4·7H20, 0.2 g; (carbon dioxide and hydrogen), ethanol as well as acetic, (NH4)2SO4, 5 g; CaCl 2 2H2O, 0.02 g; cysteine-HCl, 0.5 g; butyric and lactic acid are produced [2, 4, 15], in addi- resarzurin, 0.005 g; trace element solution SL-7 (DSMZ), tion to 1,3-PDO and BuOH. Even though the formation 2 mL. Iron concentrations were varied in the bioreactor of organic acids is inevitable for the maintenance of the medium as follows: Iron excess (Fe+) condition means 2+ intracellular redox balance, it represents a loss of car- the addition of 10 mg/L FeSO 4·7H2O (2 mg Fe /L) into bons at the expense of the target products. Moreover, the the medium and iron limitation (Fe−) condition means resulting product distribution, especially the selectivity no iron addition. Iron originally present in the pre- of either 1,3-PDO or BuOH, is mainly infuenced by the culture (0.07 mg Fe2+/L) and those present in the yeast cultivation conditions and/or media supplements. For extract (up to 0.05 mg Fe2+/L) were the sole iron sources instances, several studies analyzed the efect of pH [16], in the Fe− cultivations. Cultivations were run at 35 °C, inoculum conditions [17], supplementations of yeast pH 6 and 500 rpm agitation in a stirred tank bioreactor extracts and ammonia [18], or acetic and butyric acid [2, (Bioengineering) with a working volume of 1.2 L. During 19], or phosphate and iron [18, 20]. Among others, iron the fermentation pH was maintained at 6 with 5 M KOH. seems to have extensive efects, since its absence lead to To achieve anaerobic condition prior to the inoculation, a strongly reduced BuOH formation [16, 20]. In real fer- the autoclaved medium was sparged with sterile O 2-free mentation processes, especially under conditions more N2. Te experiments were performed in duplicates. Total relevant to industrial applications, with raw substrates volume of the efuent fermentation gas was determined and high concentrations of products, the product selec- with a Milli-Gascounter (Dr.-Ing. Ritter Apparatebau tivity and yield often strongly fuctuate and are hardly GmbH & Co. KG), and its composition was measured reproducible. Te underlying mechanism(s) of selectiv- with the mass spectrometer OmniStar 300 (Balzer Instru- ity and regulation of intracellular metabolic pathways ments/Pfeifer Vacuum GmbH). Te MS took samples in are still unclear, even though a combined efect of many an interval of 0.5 mL/min for the concentration analysis iron-related enzymes has been assumed [4]. Indeed, sev- of H2, CO2, O2, N2 and Ar. eral iron containing enzymes are involved in clostridia metabolism, e.g. nitrogenases, ferredoxin coupled Analytical methods and calculations enzymes, and alcohol dehydrogenases. Tese enzymes Te optical density of cell suspension was measured tur- play key roles in the maintenance of intracellular redox bidometrically at 600 nm and correlated with cell dry balance and a limited functionality of them, e.g. due to weight: biomass BM (g/L) = OD600 × 0.336. Te spe- iron limitation, will be refected by a metabolic shift and cifc growth rate µ (h−1) was determined from biomass thus change of product selectivity. data (smoothed using the software Origin 8.5.1 G SR1, In this work, the variations of product selectivity and OriginLab Corporation, Northampton, USA) accord- the underlying mechanisms of pathway regulation in C. ing to Eq. 1, where x1 and x2 are the concentrations of pasteurianum DSMZ 525 grown on glycerol under var- biomass (g/L) at the times t1 and t2, respectively. Te ied iron availability are studied with an integrated sys- substrate and product titers in the supernatant were ana- tems biology approach, particularly with stoichiometric, lyzed via HPLC equipped with a refractive index detec- kinetic and proteomic analyses. tor and an ultraviolet detector. HPLC was performed on an Aminex HPX-87H column (300 7.8 mm) at 60 °C, Methods × with 0.005 M H 2SO4 as mobile phase at a fow rate of Microorganism, medium and cultivation 0.6 mL/min. Clostridium pasteurianum DSMZ 525 was routinely For the measurement of 3-HPA, the method described maintained as cryoculture at −80 °C in Reinforced by Oehmke and Zeng [21] was used, in which 3-HPA is Clostridial Medium (RCM, Oxoid Deutschland GmbH) converted into acrolein and the concentration of acr- containing 20% (v/v) glycerin. Te cryoculture was used olein is determined spectrometrically by external calibra- for the pre-culture carried out in anaerobic bottles with tion. Briefy, 100 µL of cell free culture supernatant were RCM medium at 35 °C and pH 7 without shaking. Te mixed with 200 µL of HCl (37%) and 50 µL of tryptophan RCM contained 1 mg/L resazurin (7-hydroxy-10-oxi- solution in a cooled 96 well plate. Te tryptophan solu- dophenoxazin-10-ium-3-one) as a redox indicator for tion consisted of 10 mM DL-tryptophan, 0.05 M HCl anaerobiosis and 2 g/L CaCO3 as pH-bufering agent. and 24 mM toluene. After 40 min incubation at 37 °C, After 24 h this pre-culture was used as inoculum for the absorbance of the mixtures were determined with a Groeger et al. Microb Cell Fact (2017) 16:64 Page 3 of 16

Multiskan® Spectrum plate reader (Termo Fisher Scien- condition, respectively). One of the main diferences tifc) at 560 nm. observed was the relatively shorter lag phase under Fe− Te yield coefcient (Y) for either product or substrate condition, which was accompanied by an early growth (i) was calculated according to Eq. 2. Based on the stoi- cessation (Fig. 1a). With excess iron in the medium, a chiometric equations for glycerol utilization in C. pasteu- higher biomass production with a maximum concentra- −1 rianum [11], carbon and redox recovery were calculated tion of 5.1 ± 0.09 g/L and µmax of 0.31 ± 0.01 h were according to Eqs. 3 and 4, respectively. Here C [−] is the reached, whereas under iron limitation condition only number of carbon atoms in the products and substrate, a biomass concentration of 3.2 ± 0.01 g/L and a µmax of −1 c is the concentration of products in (mmol/L) and bio- 0.23 ± 0.01 h could be achieved (Table 1). Te cessa- mass (BM) in (g/L). tion of growth under iron limitation was obviously not due to butanol toxicity, as the highest titer of BuOH ln x2 − ln x1 µ = (1) reached did not exceed 3.7 g/L (Table 1), which was t − t 2 1 lower than the toxic concentration level of BuOH for C. pasteurianum (>5 g/L [11]). Depletion of the intracellu- i2 − i1 Yi/X = (2) lar iron pool and/or the accumulation of 3-hydroxypro- x2 − x1 pionaldehyde (3-HPA), a very toxic intermediate in the formation of 1,3-PDO [22, 23], may cause the relatively Cproducts Crecovery [%]= (3) earlier growth cessation under Fe condition. As shown Csubstrate − in Fig. 1b, under Fe+ condition the 3-HPA concentration c1,3−PDO did not exceed 8 mg/L, whereas under Fe− condition up NADHrecovery [%]= 2cacetate + 2cbutyrate + clactate + 13.2cBM to 30 mg/L 3-HPA were produced. In this time range of (4) relatively high concentrations of 3-HPA a growth cessation was observed. Indeed, it has been reported that the Comparative proteomic analysis growth of vegetative cells of C. tyrobutyricum was com- Samples for proteomics were taken in the exponential pletely inhibited at 38 mg/L externally added 3-HPA [24]. growth phase and stationary phase during parallel fermentations of C. pasteurianum DSMZ 525 under iron excess and iron limited conditions. Te detailed methodi- cal procedure for comparative proteomic analysis was previously described by Sabra et al. [11].

Results and discussion Efects of iron availability on the growth and product formation of C. pasteurianum Diferent concentrations of iron have been reported for the optimization of butanol or 1,3-PDO formation using C. pasteurianum [2, 18, 20]. Using a fractional facto- rial experimental design, Moon et al. used 60 mg/L FeSO 4·7H2O for optimum butanol formation in C. pasteurianum in serum anaerobic bottle experiments, while no iron sulphate was supplemented for a better 1,3-PDO production [18]. In controlled bioreactor, we have found that 10 mg/L FeSO 4·7H2O is enough to support a similar butanol productivities (0.9 g/L×h) and an almost doubled butanol concentration (21 g/L butanol) by the same strain [2]. Terefore, in the current investigation, 0 and 10 mg/L FeSO 4·7H2O were chosen, respectively, to describe the growth and product formation under iron limited and iron excess conditions in our glycerol fermentation. Te same pre-culture was used to inoculate two bioreactors Fig. 1 a Cell growth behavior and b 3-HPA formation and glycerol containing the growth medium supplemented either with consumption in cultivation of C. pasteurianum DSMZ 525 under iron excess (Fe ) and iron limitation (Fe ) conditions. Arrows indicate or without 10 mg/L FeSO 4 7H2O (hereinafter termed + − · time points of sampling for proteomic analysis as iron excess (Fe+) condition or iron limitation (Fe−) Groeger et al. Microb Cell Fact (2017) 16:64 Page 4 of 16 − 1.1 ± 0.11 0.04 ± 0.02 0.25 ± 0.05 Fe + 0.5 ± 0.07 0.03 ± 0.01 0.08 ± 0.02 Fe Formate − 7.5 ± 2.58 0.28 ± 0.02 1.64 ± 0.37 Fe + 0.1 ± 0.00 0.02 ± 0.00 0.007 ± 0.003 Fe Lactate − 2.8 ± 0.13 0.13 ± 0.01 0.80 ± 0.17 Fe + 1.7 ± 0.45 0.14 ± 0.02 0.39 ± 0.08 Fe Butyrate − 1.5 ± 0.02 0.07 ± 0.03 0.39 ± 0.09 Fe + 0.8 ± 0.01 0.04 ± 0.004 0.10 ± 0.01 Fe Acetate − 0.9 ± 0.60 0.03 ± 0.003 0.17 ± 0.13 Fe + 1.0 ± 0.12 0.06 ± 0.02 0.15 ± 0.06 Fe EtOH − 4.4 ± 0.70 0.16 ± 0.04 0.97 ± 0.13 Fe ) conditions. Fermentations were were + ) and iron Fermentations DSMZ 525 under ironC. pasteurianum − ) conditions. (Fe (Fe limitation excess + 0.53 ± 0.11 12.3 ± 0.06 1.88 ± 0.25 Fe BuOH − 0.74 ± 0.02 16.6 ± 2.26 4.40 ± 0.52 Fe + Y P/X 0.66 ± 0.17 9.4 ± 1.44 1.16 ± 0.09 Fe 1,3-PDO − 3.2 ± 0.01 0.16 ± 0.03 13.9 ± 1.2 Fe + Y S/X 0.39 ± 0.00 5.1 ± 0.09 9.6 ± 0.3 Fe Biomass Product formation during theProduct formation of growth - 1 (g/g BM) tion rate tion rate (g/L*h) at exponent ial growth phase i/X Y ‑ Produc Final titer (g/L) titer Final performed in duplicates Table Groeger et al. Microb Cell Fact (2017) 16:64 Page 5 of 16

Fig. 2 1,3-PDO and BuOH formation (a) and acid formation (b) in C. pasteurianum DSMZ 525 during glycerol fermentation under iron excess (Fe ) and iron limitation (Fe ) conditions + − Fig. 3 a Measured cumulative hydrogen and carbon dioxide produc‑ tion and b calculated (calc) vs. measured (meas) hydrogen produc‑ tion under iron excess (Fe ) and iron limitation (Fe ) conditions in C. + − Figure 2 shows the formation of fermentation prod- pasteurianum DSMZ 525 cultures ucts in C. pasteurianum DSMZ 525 under iron excess and iron limitation conditions. With higher iron concentration 12 g/L of BuOH and 11 g/L of 1,3-PDO were Redox regulation and H 2 production in C. pasteurianum produced. In comparison, at iron limitation signifcantly DSMZ 525 less BuOH, i.e. 3.7 g/L, was formed, accompanied with For the growth and metabolism of C. pasteurianum, the formation of 14.5 g/L of 1,3-PDO. In fact, the molar particularly when growing on a more reduced substrate ratio of BuOH to 1,3-PDO decreased from 1.34 mol/mol like glycerol, the maintenance of intracellular redox bal- under Fe+ condition to 0.27 mol/mol under Fe− condi- ance is crucial. Te shift of metabolism under conditions tion. Te yield of 1,3-PDO per biomass increased nearly of iron excess and limitation shown above is postulated 4 times from 1.16 ± 0.09 g/g at Fe+ to 4.4 ± 0.52 g/g at to be strongly related to the redox regulation which is Fe−, whereas the specifc yield of BuOH was halved from addressed below frst from a stoichiometric point of view. 1.88 ± 0.25 g/g at Fe+ to 0.97 ± 0.13 g/g at Fe−. Next To check the stoichiometry and consistency of the fer- to this, the acid formation changed remarkably, especially mentation data, fermentation balance analysis was frst the lactate production, which was shown to increase sig- done. A very good consistency in carbon recovery was nifcantly in the Fe− culture (Table 1). Te specifc lac- observed for the fermentations. Te carbon recovered as tate yield increased signifcantly from 0.02 ± 0.0 g/g in fermentation products represented approximately 98% the Fe+ culture to 1.6 ± 0.4 g/g in the Fe− culture. Also of the carbon source consumed. On the other hand, the acetate and butyrate yield increased under Fe− condi- calculated recovery of the reducing equivalents accord- tion, but to less extent than that of lactate (Table 1). Te ing to Eq. 4 reached 91% at Fe+ and 94% at Fe−, indicat- reason(s) for these dramatic changes of metabolism are ing a lower consistency in reducing equivalent recovery not clear yet, but of fundamental importance for the according to the assumed pathways of redox regulation. development of C. pasteurianum as an emerging micro- C. pasteurianum contains ferredoxin-dependent hydro- bial cell factory for the production of chemicals and fuels. genases, which catalyze the re-oxidation of reduced Terefore in the following redox regulation and compara- ferredoxin with the formation of H2. Reduced ferredox- tive proteomic analysis were performed. ins are generally formed in the enzymatic step of forming Groeger et al. Microb Cell Fact (2017) 16:64 Page 6 of 16

acetyl-CoA from pyruvate catalyzed by pyruvate: ferre- butyryl-CoA formation route one mole NADH 2 is addi- doxin oxidoreductase (PFOR). Hence, under the assump- tionally required for the formation of one mol butanol tion that the formation of one mole acetyl-CoA from or one mol butyrate, accompanied with the formation pyruvate is accompanied with the formation of one mole of one mole more H 2, in addition to the H 2 formation H2, the theoretical H 2 production would be calculated counted in Eq. 5. Consequently, the calculation of reduc- according to Eq. 5, where q is the formation rate of each ing equivalent recovery should be modifed as follows compound (mmol/g×h): (Eq. 9), by also taking into account the diference of calculated and measured H values (c ), representing the qH2 = qethanol + qacetate + 2qbutyrate + 2qbutanol (5) 2 ∆H2 additionally consumed NADH2:

In repeated fermentations to those shown in Fig. 1 c1,3−PDO + c�H2 under similar conditions we measured the evolution NADHrecovery (%) = 2cacetat + 2cbutyrat + clactate + 13.2cBTM of CO2 and H 2 in efuent gas. Te results are given in (9) Fig. 3a. Under Fe+ conditions a cumulative amount of 452 mmol/L H2 and 399 mmol/L CO 2 were produced, Using Eq. 9, a more satisfying reducing equivalent compared to 245 mmol/L H 2 and 177 mmol/L CO 2 pro- recovery of 105% under Fe+ condition and 100% under duced under Fe− condition. Referred to the biomass Fe− condition was obtained, giving a strong support for formed, H2 and CO2 production increased signifcantly the involvement of the BCdH-ETF complex. Particularly, from 63 (±3.3) and 49 (±3.1) mmol/g biomass at Fe− the results from Fe+ condition are in agreement with the condition to 91 (±1.9) and 82 (±3.9) mmol/g biomass at corrected Eq. 9, where more BuOH and hydrogen were Fe+ condition, respectively. Interestingly, the theoreti- produced, and the deviation between the calculated and cally calculated H2 production values were lower than the measured H2 was higher. However, this is in contrary measured ones (Fig. 3b), particularly under Fe+ condi- to what was reported for C. acetobutylicum. For a more tions. Obviously, the re-oxidation of reduced ferredoxin efective butanol production, a lower hydrogenase activ- generated in the enzymatic step catalyzed by PFOR ity and hydrogen production was favored in C. aceto- was not the only source of hydrogen formation. Similar butylicum [29]. To shed more light on the mechanisms behavior was also noticed previously in cultures of C. underlying the efect of iron on the regulation of glycerol butyricum or Klebsiella pneumoniae [25, 26]. metabolism in C. pasteurianum, comparative proteomic It is known that butyryl-CoA is generally formed from studies were performed as described below. crotonyl-CoA by the NADH dependent trans-2-enoyl- CoA reductase (Ter) enzyme (Eq. 6) [27]. Comparative proteomic analysis of the iron efect For proteomic analysis of the efects of iron concentra- Ter Crotonyl-CoA + NADH → NAD + Butyryl-CoA (6) tion on the metabolism of C. pasteurianum, samples were taken from the two bioreactors in the exponential growth But recently, Buckel and Tauer [28] proposed a new phase (termed as Fe+ early and Fe− early, respectively) indirect route of H 2 formation in C. pasteurianum from and the stationary growth phase (Fe+ late and Fe− late, NADH and ferredoxin in two steps, catalyzed succes- respectively) (Fig. 1). Each sample was analyzed in tripli- sively by the ferredoxin-dependent butyryl-CoA dehy- cates. After 2-DE separation of the intracellular proteins, drogenase/electron transferring favoprotein complex protein spots showing statistically signifcant changes (BCdH-ETF) (Eq. 7) and a hydrogenase (Eq. 8). between Fe+ early and Fe− early, Fe+ late and Fe− late, Fe early and Fe late, as well as Fe early and Fe late Fd + 2NADH + Crotonyl-CoA + + − − ox were further identifed by LC–MS/MS. Proteins which BCdH−ETF (7) −→ Fdred + 2NAD + Butyryl-CoA were identifed as single protein present in a spot on the 2-D gels are summarized in Table 2 according to their functional categories and accession numbers, together + H2−ase Fdred + 2H −→ Fdox + H2 (8) with the information of their expression changes. Te existence of more than one values of fold change for a Since the measured H2 production values were signif- single protein indicates that this protein appeared as cantly higher than the theoretically calculated ones based multiple spots on the 2-D gels. on Eqs. 7 and 8 (see Fig. 3b), it is reasonable to assume that in C. pasteurianum DSMZ 525, BCdH-ETF together The pyruvate acetyl‑CoA node: a focal point in the with Ter is actively involved in the step of converting metabolism of C. pasteurianum crotonyl-CoA to butyryl-CoA, giving rise to an additional Te conversion of pyruvate to acetyl-CoA linking gly- source of H2 formation. Tus, with this new suggested colysis to TCA cycle is a fundamental metabolic step Groeger et al. Microb Cell Fact (2017) 16:64 Page 7 of 16 2.0 1.1 1.6 Late − higher at 2.1 2.3 1.6 2.0 0.9 1.5 Early Fe 1.8 2.5 2.6 2.0 6.1 0.8 2.3 1.6 1.9 2.1 Late + higher at 1.7 2.4 2.2 2.7 1.3 1.9 2.2 2.1 2.2 1.9 Early Fold change growth-related Fold Fe + 2.0 1.5 1.7 1.6 2.9 1.5 Fe − 1.8 0.6 4.0 2.1 1.9 1.7 Fe Late phase Late higher at + 1.7 1.6 1.7 2.4 3.7 2.5 1.6 1.8 Fe − 2.0 4.8 4.1 1.5 7.9 0.4 2.7 1.9 1.6 1.6 1.8 1.9 Fe Fold change iron-related Fold Early phase higher at 158 491 277 409 179 696 503 520 695 549 233 440 519 460 329 594 220 594 312 190 187 449 89 298 Spot no. DAK1 TpiA GapA DivIVA Eno CotF LysM FixB IscU PdxS GlgA FldA PduE ThrC GlnA3 CysK CarB Lap4 Conserved Conserved protein domain COG2376 COG0149 COG1543 COG0057 COG3599 COG0148 COG5577 COG1388 COG2025 COG0822 COG0214 COG0297 COG0716 No COG COG4910 COG0498 COG3968 COG0031 COG0458 COG1362 Cluster of ortholCluster - ogous groups (COG) subunit Dihydroxyacetone kinase Dihydroxyacetone Triosephosphate isomerase Triosephosphate Glycoside hydrolase Glycoside phosphate dehydrogenase 3 - phosphate Glyceraldehyde Cell division protein Cell Enolase Spore coat protein Frelated protein Frelated coat protein Spore - binding protein Peptidoglycan Electron transfer favoprotein subunit alpha favoprotein transfer Electron related domain containing protein NifU - related Pyridoxal biosynthesis lyase Pyridoxal Glycogen synthase Glycogen Subunit favodoxin Glycerol dehydratase reactivation factor large factor reactivation large dehydratase Glycerol Propanediol dehydratase small dehydratase Propanediol Threonine synthase Threonine Glutamine synthetase type III Cysteine synthase a Cysteine Carbamoyl phosphate synthase large subunit synthase large phosphate Carbamoyl Amino peptidase 1 Protein name Protein ) conditions, as well+ ) and iron− ) conditions, (Fe between iron as between (Fe compared limitation excess levels, changed expression signifcant showing Proteins 2 F502_12758 F502_06077 F502_07098 F502_06087 F502_08238 F502_06067 F502_01965 F502_00655 F502_06282 F502_05017 F502_07578 F502_03937 F502_07638 F502_03417 F502_03412 F502_18676 F502_17572 F502_07028 F502_05412 F502_05097 Cell cycle division Cell control/cell Cell wall/membrane/envelope biogenesis wall/membrane/envelope Cell Energy production and conversion production Energy Coenzyme transport and metabolism Carbohydrate transportCarbohydrate and metabolism Amino acid transport and metabolism Table and the stationary phase (early) phase (late) growth the exponential no. Accession Groeger et al. Microb Cell Fact (2017) 16:64 Page 8 of 16 1.8 1.6 1.9 1.6 Late − higher at 1.5 2.0 2.0 Early Fe 4.7 5.3 1.8 3.7 1.9 1.6 2.1 2.1 1.6 2.0 2.3 2.1 2.0 2.0 1.7 1.7 Late + higher at 1.5 0.6 1.6 2.5 2.3 3.6 1.9 Early Fold change growth-related Fold Fe + 3.0 2.9 2.4 2.4 1.7 2.1 2.7 2.3 2.8 2.8 2.3 2.3 4.5 1.8 1.6 1.9 2.4 2.5 2.7 2.8 2.8 Fe − 2.9 1.5 1.9 2.4 8.0 Fe Late phase Late higher at + 2.4 1.5 2.0 2.2 2.2 2.1 4.5 5.1 1.5 2.4 1.5 2.2 2.1 Fe − 2.3 1.6 1.5 2.7 2.1 2.2 1.7 1.6 14.3 Fe Fold change iron-related Fold Early phase higher at 537 70 75 487 717 119 715 76 Spot no. 77 87 90 40 57 303 558 303 315 736 223 224 58 156 71 611 645 646 575 59 730 131 383 NfnB PorA FixA AdhE Conserved Conserved protein domain PorA YotD Nar1 PurB GlcD AdhE NuoG AcnA PycA SorL AtpD FldA NorV Buk COG0778 COG0674 COG2086 COG1012 Cluster of ortholCluster - ogous groups (COG) COG0674 COG0015 COG1592 COG4624 COG0277 COG1012 COG1034 COG1048 COG1038 COG2033 COG0055 COG0716 COG0426 COG3426 phosphate - 3 phosphate ‑ dehy CoA/alcohol - - 1 dependent glyceraldehyde - tase. homodimeric tase. beta - like protein drogenase dehydrogenase Nitroreductase ‑ oxidoreduc (favodoxin) ferredoxin Pyruvate: Electron transfer favoprotein subunit alpha/ favoprotein transfer Electron Bifunctional acetaldehyde Protein name Protein Pyruvate: ferredoxin oxidoreductase ferredoxin Pyruvate: Adenylosuccinate lyase Adenylosuccinate Rubrerythrin [Fe] hydrogenase [Fe] Glycolate oxidase Glycolate NADP Hydrogenase Hydratase (aconitase A) Pyruvate carboxylasePyruvate Desulfo ferrodoxin Desulfo F0F1 ATP synthase subunit beta F0F1 ATP Flavodoxin Rubredoxin/favodoxin/oxidoreductase Butyrate kinase continued F502_07493 F502_07643 F502_06287 F502_06447 F502_07648 F502_04707 F502_15080 F502_14390 F502_16610 F502_18651 F502_18287 F502_09488 F502_11976 F502_12878 F502_12091 F502_13493 F502_09238 F502_11871 2 Table no. Accession Groeger et al. Microb Cell Fact (2017) 16:64 Page 9 of 16 6.9 Late − higher at 1.9 7.4 1.6 3.4 2.0 Early Fe 4.4 2.8 1.5 2.4 3.0 2.0 2.6 2.1 2.4 2.1 1.5 1.8 2.2 Late + higher at 1.7 1.7 1.7 2.2 1.5 1.7 3.0 2.9 Early Fold change growth-related Fold Fe + 2.4 1.5 1.5 1.6 3.8 2.2 2.1 Fe − 2.5 1.6 2.1 1.9 2.0 1.6 5.7 6.5 2.8 Fe Late phase Late higher at + 2.1 3.4 7.1 2.6 Fe − 2.5 1.8 1.6 2.2 1.8 1.6 1.8 2.5 2.3 3.6 2.3 2.0 3.7 4.5 8.0 2.5 1.7 1.6 Fe Fold change iron-related Fold Early phase higher at 716 138 206 196 613 600 553 258 165 168 281 302 472 637 318 107 106 635 400 362 481 181 174 175 178 597 217 Spot no. ClpA HtpG GroES IbpA ThiJ GroEL ClpA PurH AccC CaiD YbjQ Cym1 GrpE PfD Dank Conserved Conserved protein domain COG0542 COG0326 COG0234 COG0071 COG0693 COG0459 COG0542 COG0138 COG0439 COG1024 COG0393 COG2607 COG1026 No COG No COG COG1453 COG0576 COG1882 No COG COG0443 Cluster of ortholCluster - ogous groups (COG) - binding CoA dehydratase CoA dependent Clp protease ATP dependent Clp protease - chaperonin - subunit zolecarboxamide formyltransferase/IMP formyltransferase/IMP zolecarboxamide cyclohydrolase Clpb protein Heat shock protein 90 Heat shock protein Co Heat shock protein (molecular chaperone IbpA) (molecular chaperone Heat shock protein Thij/PfpI family protein Thij/PfpI Chaperonin ATP Bifunctional phosphoribosylaminoimida‑ Biotin carboxylase 3 - Hydroxybutyryl - Hypothetical protein Hypothetical protein peptidase Hypothetical protein Hypothetical protein (GGGtGRTHypothetical protein) protein Aldo/keto reductase Heat shock protein (molecular chaperone GrpE) (molecular chaperone Heat shock protein Formate acetyltransferase Formate Hypothetical protein Molecular chaperone DnaK Molecular chaperone Protein name Protein continued F502_18446 F502_15425 F502_06247 F502_10228 F502_07608 F502_06242 F502_05557 F502_17300 F502_10483 F502_06297 F502_16320 F502_06682 F502_03987 F502_05962 F502_05012 F502_02435 F502_03242 F502_19556 F502_15420 F502_03247 Nucleotide transport and metabolism Lipid transport and metabolism Function unknown/general function only prediction Function Posttranslational modifcation/Protein turnover/Chaperones modifcation/Protein Posttranslational 2 Table no. Accession Groeger et al. Microb Cell Fact (2017) 16:64 Page 10 of 16 2.1 2.0 1.6 1.9 Late − higher at 1.5 1.4 2.7 1.8 1.6 2.1 Early Fe 2.4 1.7 3.9 2.4 2.6 3.6 2.1 3.4 2.0 2.4 1.7 2.5 Late + higher at 4.7 1.5 2.7 2.3 1.8 2.0 1.7 2.4 Early Fold change growth-related Fold Fe + 1.5 2.1 1.5 1.9 Fe − 1.8 3.9 4.5 2.9 1.8 1.9 2.6 1.5 2.1 1.7 4.8 2.2 2.2 Fe Late phase Late higher at + 1.7 2.1 1.7 2.4 2.1 2.4 2.4 1.7 Fe − 2.4 2.3 6.1 3.5 5.3 3.1 3.8 6.2 3.7 4.7 2.1 2.5 4.5 3.9 3.6 Fe Fold change iron-related Fold Early phase higher at 327 497 160 565 753 752 681 548 544 500 504 567 638 47 201 200 197 193 147 149 148 606 Spot no. 572 155 TufB RpsB Tex RaiA RplF RplE RplA CotJC AbrB RplY AhpC GlnK TypA PrkA ClpA ClpA CBS Conserved Conserved protein domain Tpx CheA COG0050 COG0052 COG2183 COG1544 COG0097 COG0094 COG0081 COG3546 COG2002 COG1825 COG0450 COG0347 COG1217 COG2766 COG0542 COG0517 Cluster of ortholCluster - ogous groups (COG) COG2077 COG0643 - - 1) containing protein associated protein Y (PSrp protein - associated - like domain) adaptor - binding protein L25/TL5 binding domain protein CheW - II Elongation factor Tu 30S ribosomal protein S2 30S ribosomal protein Transcription accessory protein Transcription Ribosome 50S ribosomal protein L6 50S ribosomal protein 50S ribosomal protein L5 50S ribosomal protein 50S ribosomal protein L1 50S ribosomal protein Spore coat protein Spore Stage V sporulation protein T V sporulation protein Stage loop binding protein Ctc/ Ribosomal 5S rRNA E - loop binding protein Nitrogen regulatory protein P Alkyl hydroperoxide reductase Alkyl hydroperoxide GTP Serine protein kinaseSerine protein ATPase with chaperone activity with chaperone clpC. two ATP ATPase CBS domain - Protein name Protein Lipid hydroperoxide peroxidase Lipid hydroperoxide Chemotaxis histidine kinase. CheA (contains continued ) collection database ( http://www.biocyc.org/organism-summary?object = CPAS1262449 BioCyc DSMZ 525 by C. pasteurianum for the annotation to according COG: ) ( http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml Classifcation NCBI Conserved Domains the defnition by and Protein to according Domain Family: Conserved Protein F502_18808 F502_04537 F502_12326 F502_12196 F502_18963 F502_18948 F502_18833 F502_17637 F502_18092 F502_06817 F502_16565 F502_17612 F502_04082 F502_14770 F502_18743 F502_13258 F502_10768 F502_07703 1 2 Translation/Ribosomal structure and biogenesis structure Translation/Ribosomal Transcription/Defense mechanisms Transcription/Defense Signal transduction/stress response/defense mechanism response/defense transduction/stress Signal * 2 Table no. Accession * Groeger et al. Microb Cell Fact (2017) 16:64 Page 11 of 16

of living organisms in general. In anaerobes, pyruvate to iron availability. Furthermore, whether PFOR2 trans- can be metabolized through a variety of pathways but it fers the electrons generated during the decarboxylation is often oxidized to CO2 and acetyl-CoA with the con- reaction to a ferredoxin or favodoxin remains elusive. comitant reduction of a low-potential redox protein, Beside the ferredoxin (favodoxin)-dependent PFORs, like ferredoxin or favodoxin. Te enzyme responsible acetyl-CoA can be synthesized from pyruvate through for this oxidative decarboxylation of pyruvate in many the pyruvate formate-lyase (PFL) with the formation of anaerobic bacteria and archaea is pyruvate: ferredoxin formate. Tere are three genes (F502_19556, F502_15690, oxidoreductase (PFOR). PFORs contain thiamin pyroph- F502_15710) in the genome of C. pasteurianum DSMZ osphate (TPP) for the cleavage of carbon–carbon bonds 525 being annotated to encoding enzymes functioning next to a carbonyl group, as well as iron-sulfur clusters as pyruvate formate lyase (PFL). Only one of these PFLs for electron transfer (see [30, 31] and references therein). (F502_19556, also named formate acetyltransferase) was For example, PFOR from C. pasteurianum W5 (ATCC unambiguously identifed in four protein spots (Fig. 4). 6013) was characterized to be an air-sensitive homodi- Interestingly, the molecular weight of the two acidic iso- mer with each subunit containing eight iron atoms in two forms (spots 178 and 181) appeared lower than that of [4Fe–4S] clusters, for which pyruvate is the best substrate the two basic isoforms (spots 174 and 175). Compared found among several α-ketoacids [30]. In the genome of to the iron excess condition, where the expression of PFL C. pasteurianum DSMZ 525, three homologue enzymes was nearly not detectable, all these four PFL isoforms of PFOR are present, namely two pyruvate ferredoxin were signifcantly but diferently up-regulated under the oxidoreductases (F502_01955 and F502_07648), and iron limitation condition. While the two acidic isoforms one pyruvate:ferredoxin (favodoxin) oxidoreductase showed 2.5 and 3.7 folds increased abundances only in (F502_07643) [32]. In this study, F502_07648 (termed as the Fe− early sample, the expression of the two basic iso- PFOR1) and F502_07643 (termed as PFOR2) were iden- forms were up to eightfold strongly up-regulated in the tifed among the proteins showing signifcant expression Fe− early sample and about sixfold in Fe− late sample. changes. On the 2-D gels both PFORs appeared as a chain Tus, under iron limitation, it was apparently favorable of spots (Fig. 4). Protein identifcation showed that the for C. pasteurianum to use the PFL-catalyzed reaction spots 70, 75, 76, 77, 87, 90 are pI isoforms of the homodi- for the conversion of pyruvate to acetyl-CoA. Corre- meric protein pyruvate: ferredoxin (favodoxin) oxidore- spondingly, under this condition the formate yield was ductase (F502_07643), whereas the spots 40, 57, 58 and clearly higher than that under the Fe+ condition, namely 59 are pI isoforms of pyruvate:ferredoxin oxidoreductase 0.25 ± 0.05 g/g biomass in contrast to 0.08 ± 0.02 g/g (F502_07648). F502_07643 and F502_07648 are homolo- biomass. Nevertheless, the expression levels of the two gous proteins with a sequence identity match of 66% and PFORs, especially the pyruvate:ferredoxin (favodoxin) positive match of 81%. Tey have nearly identical molec- oxidoreductase (PFOR2), were visibly higher than that ular weights but the pI value of F502_07648 is more basic of PFL. Since protein synthesis is an energy-demanding than that of F502_07643, which was also obvious on the process, cells usually do not produce useless enzymes 2-D gels. Although previous studies showed that under in noticeable amounts. Te presence of the two PFORs conditions of iron limitation many anaerobes synthesize under Fe− condition may point to a fact that, in the favodoxins as substitution of ferredoxins for many enzy- absence of iron, the two PFORs, especially the pyru- matic reactions [28], all the isoforms of the two PFORs vate: ferredoxin (favodoxin) oxidoreductase (PFOR2), showed, in general, higher expression at iron excess than probably use favodoxin instead of ferredoxin as the at iron limitation (Fig. 4). Te expression patterns of the electron acceptor. Indeed, the expression of a favo- isoforms of PFOR1 were similar to each other, with the doxin (F502_13493) was strongly up-regulated under highest expression at Fe+ late. In contrast, the expres- iron limitation for 14.3 folds in the exponential growth sion patterns of the isoforms of pyruvate:ferredoxin phase and remained high even after entering the station- (favodoxin) oxidoreductase (PFOR2) were diferent ary phase (8.0 fold higher in Fe− late than in Fe+ late). to each other. While the expression level of the spot 87 However, whether or not the up-regulated expression of in the middle of the spot chain did not change between this favodoxin was coupled to the functionality of the Fe+ early and Fe+ late, the more basic isoforms (spots 70 PFORs remains to be verifed. In case it is, it did not help and 90) showed higher expression at Fe+ early, and the much in sustaining the production of butanol under the more acidic isoforms (spots 75, 76 and 77) were up-regu- Fe− condition. lated at Fe+ late which was similar to the expression pattern of PFOR1. Terefore, based on the proteomic results Regulation of the ferredoxin pool alone, it is not clear whether these two PFORs function in For the proper function of PFORs, ferredoxin (red), which synchronization or are diferently regulated in response is generated in the PFOR-catalyzed pyruvate oxidative Groeger et al. Microb Cell Fact (2017) 16:64 Page 12 of 16

Fig. 4 Expression patterns of enzymes catalyzing the conversion of pyruvate to acetyl-CoA under Fe and Fe conditions at exponential growth + − phase (early) and stationary phase (late). PFOR 1 (F502_07648, pyruvate: ferredoxin oxidoreductase) and PFOR 2 [F502_07643, pyruvate:ferredoxin (favodoxin) oxidoreductase] are up-regulated at Fe ; PFL (pyruvate formate lyase) is up-regulated at Fe + − decarboxylation reaction, must be oxidized to regen- either as substrate or as product. In general, nitroge- erate ferredoxin(ox). C. pasteurianum DSMZ 525 pos- nases and hydrogenases are the two enzyme classes sesses a big number of ferredoxins and the regeneration capable of hydrogen production in Clostridia [33]. But of ferredoxin(ox) can be achieved using diferent electron Hallenbeck and Benemann [34] reported that hydro- acceptors. Te fact that the redox potentials of ferre- genases are much more efcient, with more than 1000 doxins (−400 mV) are in the range of H2 electrodes times higher turnover than nitrogenases. Hydrogenases (−414 mV, at pH 7) reveals that in most energy metab- are divided into two main groups in Clostridia based on olisms where ferredoxins are active, H 2 is also involved, their metaollocenter composition, namely [NiFe] and Groeger et al. Microb Cell Fact (2017) 16:64 Page 13 of 16

[FeFe] hydrogenases [33]. In this study, the expression of a main route of Fd ox regeneration in C. pasteurianum hydrogenase-1 (F502_18287), which belongs to the [FeFe] DSMZ 525. Based on this assumption, we compared group, was highly up-regulated under iron excess, show- the hydrogen yield from glycerol between the Fe− and ing up to fvefolds higher expression level in the exponen- the Fe+ conditions. As shown in Fig. 5a, hydrogen yield tial growth phase under the Fe+ condition compared to decreased signifcantly from 0.75 mol/mol glycerol under the Fe− condition. After entering the stationary phase Fe+ condition to 0.21 under Fe− condition, which was in the expression of hydrogenase-1 (H 2-ase) was down- agreement with the higher expression of hydrogenase-1 regulated for two to threefolds under the Fe+ condition, under the Fe+ condition. which could be possibly in response to a depletion of the In addition, as described in the above redox balance intracellular iron pool in the Fe+ late sample required for analysis, a possible involvement of a ferredoxin-depend- this [FeFe]-hydrogenase. However, it was still nearly two- ent butyryl-CoA dehydrogenase/electron transferring folds higher than its expression level under the Fe− con- favoprotein complex (BCdH-ETF) in H2 production was dition. An additional [FeFe] hydrogenase (F502_14390) proposed. In the BCdH-ETF catalyzed reaction electron was also identifed which showed expression regula- transfer favoproteins (ETFs) are involved in the reduc- tions similar to that of the hydrogenase-1 (F502_18287). tion of crotonyl-CoA to butyryl-CoA, coupled with Nevertheless this hydrogenase did not appear as a spot ferredoxin(ox) reduction by bifurcating electrons from containing only a single protein on the 2-D gels and NADH (Fig. 5) [28, 37]. In this proteomic study, two therefore could not be quantifed for comparison. Te ETFs, namely ETFs subunit alpha (F502_06282) and sub- higher expression of hydrogenase-1 (F502_18287) coin- unit alpha/beta-like protein (F502_06287), were identi- cided with the higher H2 production in the fermentation fed among the most abundant proteins regardless of the culture under iron excess and should have signifcantly iron availability; however, their abundances were slightly contributed to the regeneration of ferredoxin(red) to higher (1.5–1.7 folds) in the late phase of the Fe+ culture ferredoxin(ox). compared to the Fe− late condition. Tis might indi- However, it is to notice that the regulation of hydro- cate a relative increase in the oxidized ferredoxin pool genase-1 (F502_18287) expression is rather in agree- necessary to carry out the BCdH-ETF reaction and also ment with that of the basic iso-forms of the ferredoxin contributed to the stronger H 2 production in the late fer- (favodoxin)-dependent PFOR2 than with the expression mentation phase under Fe+ condition. patterns of the ferredoxin-dependent PFOR1. Terefore, Nevertheless, not only the hydrogenase-1 but also the it is tempting to suggest that under the given experi- two PFORs were down-regulated under Fe− condition mental conditions the hydrogenase-1 catalyzed reaction compared to that under Fe+ condition. Terefore, the rel- should not be the only route of ferredoxin(ox) regenera- ative changes of the expression levels of the two enzymes tion. Te PFORs-catalyzed pyruvate oxidation to acetyl- might be indicative of the overall Fdox regeneration state CoA might be coupled with other but yet unknown (Fig. 5). Te expression levels shown as the protein spot ferredoxin(ox) regenerating reaction(s) catalyzed either by intensities of both enzymes under Fe− and Fe+ conditions other unidentifed hydrogenases (at least 5 genes in the at the two time points were thus compared. As shown genome of C. pasteurianum DSMZ 525 encode hydro- in Fig. 5b, the relative expression of PFOR to hydroge- genases) or ferredoxin reductases. In addition, it has also nase-1 (H2-ase) showed a positive correlation with the been reported that PFOR can transfer the electrons gen- 1,3-PDO production rate. Te decrease in the Fdox frac- erated in the decarboxylation reaction directly to protons tion under Fe− condition due to reduced H 2-ase presence to generate molecular hydrogen [35]. will decrease the Fdox coupled synthesis of butyryl-CoA Within the cells of anaerobes including Clostridia, 90% catalyzed by the BCdH-ETF complex, a crucial step in of ferredoxins were reported to be present in reduced butyrate and especially butanol biosynthesis. Moreover, form, allowing them to serve as electron donors in difer- the intermediate acetyl-CoA will be favorably channeled ent reactions [28]. In general, this is achieved in C. pas- into the Fd ox-independent acetate formation route than teurianum by the following three ferredoxin-dependent the Fdox-dependent butyrate formation route, as shown redox reactions: the oxidation of pyruvate to acetyl-CoA by the increase in the butyrate/acetate ratio from 1.3 at and CO2 (−500 mV), the oxidation of formate to CO 2 Fe− to 1.5 at Fe+ (Fig. 5c). Consequently, it seems that (−430 mV, [36]) and the favoprotein based electron under Fe− condition, Fd ox dependent conversion steps bifurcation involved in the reduction of crotonyl-CoA are reduced and the resulting free reducing power, usu- to butyryl-CoA (Eq. 7). On the other hand, the oxida- ally needed for butanol formation, could be redirected for tion of Fd red by NAD is excluded due to the absence of the sake of redox balance to the production of 1,3-PDO the ferredoxin: NAD oxidoreductase activity [28]. Tere- and lactate. Indeed, the overall yield of 1,3-PDO and lac- fore, hydrogen production via hydrogenase should be tate were much higher under iron limitation than under Groeger et al. Microb Cell Fact (2017) 16:64 Page 14 of 16

Fig. 5 Revised metabolic pathway of glycerol bioconversion to 1,3-propanediol and n-butanol in C. pasteurianum. a Molar formation of H 2 over consumed glycerol b 1,3-PDO productivity over the ratio PFOR/H -ase c Molar ratio of butyrate to acetate under Fe and Fe conditions 2 + −

iron excess (Table 1). Nevertheless, lactate dehydroge- subunit of glycerol dehydratase reactivating factor (GDHt nase catalyzing the conversion of pyruvate to lactate or reactivase, GDHtR) was identifed in the spot containing 1,3-propanediol dehydrogenase catalyzing the formation this single protein (Fig. 6). GDHtR is a molecular chaper- of 1,3-propanediol from 3-HPA were not found among one participating in the reactivation of inactivated GDHt the proteins showing signifcant changes in expression in the presence of ATP and Mg2+ [38, 39]. Te expres- level. Both enzymes are not involved in energy production sion pattern of GDHtR indicates rather a correlation but constitute the cell’s back-up for stabilizing an internal of GDHtR expression to cell growth phase than to iron redox balance, and hence their constitutive production availability. Among the four samples compared by prot- may be a mechanism to withstand sudden perturbations eomics, the highest expression level of GDHtR was pre- in the NADH/NAD ratio. Nevertheless, it should also bear sent in the iron excess culture in the middle exponential in mind that higher or lower protein level does not always growth phase (Fe+ early) showing the highest specifc means higher or lower enzyme activity. growth rate (µ = 0.22). At this sampling time point, the culture under iron limitation (Fe− early) already entered Glycerol conversion to 1,3‑propanediol late exponential growth phase with reduced specifc In general the bioconversion of glycerol to 1,3-PDO takes growth rate (µ = 0.07), accompanied with lower GDHtR place in two steps, catalyzed successively by glycerol level. Te GDHtR abundance was further reduced to dehydratase (GDHt) and 1,3-propanediol dehydrogenase merely detectable levels in the stationary phase (Fe+ late (PDOR) (Fig. 5). It is known that glycerol dehydratase is and Fe− late), where the production of 1,3-PDO stag- the rate-limiting enzyme in this bioconversion. All the nated. 1,3-propanediol dehydrogenase (PDOR), the three subunit of GDHt encoded by pduC (F502_03402), responsible enzyme for the conversion of 3-HPA to 1,3- pduD (F502_03407) and pduE (F502_03412) were iden- PDO, was one of the highly abundant proteins on the tifed but unfortunately not as single protein spots and, 2-D gels and did not show signifcant expression changes therefore, could not be quantifed. Instead, the large under the diferent conditions (data not shown). Groeger et al. Microb Cell Fact (2017) 16:64 Page 15 of 16

Fig. 6 Expression pattern of the large subunit of glycerol dehydratase reactivating factor (GDHtR) under Fe and Fe conditions as well as early + − and late sampling point

Conclusion Authors’ contributions CG designed and performed the bioreactor experiments, interpreted the Te iron content in the fermentation medium was results and wrote the manuscript. WW carried out the proteomic studies, shown to infuence the product formation, especially the interpreted the results and wrote the manuscript. WS and TU helped in biore‑ actor experiments and results interpretation. APZ supervised the research. All 1,3-propanediol and butanol distribution, in Clostridium authors read and approved the fnal manuscript. pasteurianum DSMZ 525 grown on glycerol. Compared to the fermentation under iron limitation, it was shown Acknowledgements that the butanol to 1,3-propanediol ratio increased The authors also like to thank Jan Bomnüter and Anna Gorte for their excellent almost fvefold in fermentation under iron excess. To bet- assistance with the two dimensional electrophoresis of proteomic analysis. ter understand the efect of iron on the regulation of the Competing interests cell metabolism, physiological and proteomic analyses The authors declare that they have no competing interests. were performed. Several enzymes like pyruvate: ferredoxin oxidoreductase (PFOR), hydrogenase and bifunc- Availability of data and materials All data generated or analyzed during this study are included in this published tional acetaldehyde-CoA/alcohol dehydrogenase among article. others were found to be up-regulated under iron excess conditions. Te diferential expression of PFORs and a Funding Information This work was funded from the European Union Seventh Framework Program pyruvate formate lyase (PFL) in response to iron avail- (FP7/2007 2013) through the EuroBioRef project (grant agreement Nr. 241718). ability highlighted the impact of iron on the crucial step The funders had no role in study design, data collection and interpretation, of the central carbon metabolism in C. pasteurianum. or the decision to submit the work for publication. This publication was sup‑ ported by the German Research Foundation (DFG) and the Hamburg University Te importance of a hydrogenase in the regeneration of Technology (TUHH) in the funding programme, Open Access Publishing. of oxidized ferredoxin and therefore the maintaining of the redox balance was confrmed by its strong up- Publisher’s Note regulation under the iron excess condition. Beside the Springer Nature remains neutral with regard to jurisdictional claims in pub‑ release of molecular hydrogen in the pyruvate to acetyl- lished maps and institutional afliations.

CoA step catalyzed by PFORs, stoichiometric analysis Received: 7 December 2016 Accepted: 9 April 2017 showed a possible H 2 production coupled to the reaction catalyzed by the ferredoxin-dependent butyryl-CoA dehydrogenase/electron transfer favoprotein complex

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