Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

international journal of hydrogen energy 34 (2009) 7417–7427

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Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains Seohyoung Kima, Eunhee Seola, You-Kwan Ohb, G.Y. Wangc, Sunghoon Parka,* a

Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735, Republic of Korea Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-543, Republic of Korea c Department of Oceanography, University of Hawaii at Manoa Honolulu, HI 96822, USA b

article info

abstract

Article history:

Escherichia coli can produce H2 from glucose via formate hydrogen lyase (FHL). In order to

Received 3 August 2008

improve the H2 production rate and yield, metabolically engineered E. coli strains, which

Received in revised form

included pathway alterations in their H2 production and central carbon metabolism, were

13 May 2009

developed and characterized by batch experiments and metabolic flux analysis. Deletion of

Accepted 14 May 2009

hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of

Available online 12 June 2009

two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H2 production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase

Keywords:

(ldhA) and fumarate reductase ( frdAB) further improved the H2 yield; 1.80 mol/mol glucose

H2 production yield

under high H2 pressure or 2.11 mol/mol glucose under reduced H2 pressure. Several batch

Glucose fermentation

experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/

Metabolic engineering of Escherichia

L) were conducted for the strain containing all these genetic alternations, and their carbon

coli

and energy balances were analyzed. The metabolic flux analysis revealed that deletion of

Carbon and energy balance

ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading

Metabolic flux analysis

to H2 production by FHL, not to the pentose phosphate pathway. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Hydrogen is a highly relevant energy carrier and its conversion to heat or power is simple and clean. When it is combusted with oxygen, only water is formed and no pollutants are generated or emitted [1]. In addition, H2 can be produced from a variety of different feedstocks including fossil resources, biomass and water [1,2]. When fossil fuel and water are used as a feedstock, energy-intensive thermochemical and electrochemical methods are required for H2 production. In comparison, biological methods that use biomass as a feedstock are less energy-intensive and considered to be neutral for CO2 emissions [3].

Among the available biological methods, microbial fermentation of organic carbon sources has been extensively studied. Glucose is a monomer of cellulose, the most abundant biomass in the world and a good source for H2 production by many microorganisms. However, low yield has been a major obstacle for the hydrogen production through microbial fermentation of glucose. For example, Clostridium species can use 1 mol of glucose to produce at the most 4 mol of H2 while Escherichia coli can only produce at the most 2 mol of H2 from the same amount of glucose [4–9]. As of now, the low production yields, ranging from 1 to 2 mol H2/mol glucose, have commonly been reported with actual glucose fermentations.

* Corresponding author. Tel.: þ82 51 510 2395; fax: þ82 51 510 2716. E-mail address: [email protected] (S. Park). 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.05.053

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international journal of hydrogen energy 34 (2009) 7417–7427

Recently, efforts have been made to improve H2 production yield and rate using metabolic engineering approaches [10,11,22]. Most of these efforts have been focused on modification of hydrogen (e.g., uptake hydrogenases) and central carbon (lactate dehydrogenases) metabolism pathway. Yoshida et al. reported that ldh- and frd-inactivated E. coli strain produced 1.8 mol H2/mol glucose, which corresponds to 90% of the theoretical maximum value for E. coli [10]. Redwood et al. found that the deletion of the two uptake hydrogenases of E. coli strains FTD67 and FTD89 improved hydrogen production by 37% over the parent strain [22]. Still, Maeda et al. reported that, by altering the regulation of FHL expression, the H2 production rate increased by 141-fold and, by disrupting uptake hydrogenases, the yield from glucose increased by 50%. Clearly, there are discrepancies in the production yield of these genetically engineered E. coli strains, potentially resulting from fermentation medium and the poor performance of the parental strains used in these experiments. The improvement in H2 production rate and or yield cannot be fully appreciated if the control strain and medium are not properly selected [10,11]. The effect and advantages of metabolic-pathway engineering can be best evaluated by analyzing the carbon and energy balances and/or metabolic fluxes. Recently, we have reported that the amount of major metabolites produced from glucose fermentation by the chemoheterotrophic bacterium Citrobacter amalonaticus Y19 (H2, ethanol, acetate, lactate, CO2 and cell mass) varied with the initial glucose levels [12,13]. A brief metabolic-pathway fluxes model for the strain Y19 was also constructed using 81 biochemical reactions, and its applicability was verified by comparing simulation results with those of batch experiments conducted at several different initial glucose concentrations [12]. The enhanced production of NAD(P)H from the pentose phosphate pathway and its funneling to H2 production through a non-native NAD(P)H-dependent hydrogenase were suggested as an optimal metabolic pathway for maximizing H2 production from glucose fermentation. The purpose of this study is to develop E. coli strains for improved H2 production yield and quantitatively analyze the performance of these strains. Specifically, we developed a series of E. coli strains by deleting uptake hydrogenases, FHL transcriptional inhibitor, and several genes involved in the glycolytic pathway. Then, the growth, H2 production and carbon metabolites of these strains were determined. Finally, analysis and comparison of the changes in carbon and energy balances and the carbon metabolic fluxes of both the wild type and mutant E. coli strains were performed to understand the effect of metabolic-pathway engineering on H2 production. This report represents one of the most thorough analyses of metabolically engineered E. coli strains for hydrogen production.

2.

Materials and methods

2.1.

Materials used

Restriction and DNA-modifying enzymes were obtained from New England Bio-labs (Beverly, MA, USA). Taq polymerase

enzyme was acquired from Bioneer (Daejeon, Republic of Korea). A miniprep kit and DNA gel purification kit were purchased from Qiagen (Mannheim, Germany). Primers were synthesized from Bioneer. Yeast extract (Cat. 212750) was obtained from Difco (Becton Dickinson and Company). All other chemicals, unless indicated, were obtained from Sigma– Aldrich (St. Louis, MO, USA).

2.2.

Microorganisms and culture conditions

Bacterial strains, plasmids and primers used in this study are presented in Table 1. The bacterial strain E. coli BW25113 was used as the parental strain for developing H2 producing mutants. Luria Bertani (LB) medium was used for regular genetic engineering and culture maintenance work. Ampicillin at a concentration of 100 mg/mL or kanamycin at 30 mg/mL or both were added to the culture media when necessary. For the production of H2, E. coli strains were cultured in M9 medium containing glucose. The culture medium was maintained at pH 7.0  0.2 and contained the following components (per liter): K2HPO4, 19.3 g; KH2PO4, 9.4 g; MgSO4$7H2O, 0.25 g; NaCl, 1.0 g; NH4Cl, 1.6 g; yeast extract, 3.0 g; CaCl2, 1.0 g; and 2 mL of Pfennig and Lippert’s trace element solution. The trace element solution contained the following compounds (per liter): FeSO4$7H2O, 0.21 g; ZnSO4$7H2O, 50 mg; MnCl2$4H2O, 50 mg; H3BO3, 100 mg; CuSO4$5H2O, 6.7 mg; 15 mg; CoCl2$6H2O, NiCl2$6H2O, 10 mg; Na2SeO3 5.0 mg; and Na2MoO4$2H2O, 15 mg. Glucose was sterilized separately and added in the range of 2.5–10 g/L. All experiments were conducted in a serum bottle (165 mL; working volume, 50 mL) at 37  C. After inoculating the seed culture, the bottles were flushed with argon (Ar) gas (99.9%) for 10 min to ensure that the bottles were completely deprived of O2. The bottles were then sealed with butyl rubber septum and aluminum caps. The serum bottles were incubated at 37  C in an orbital incubator shaker (VS-8480 SF, Vision Scientific, Co. Ltd., Republic of Korea) at 200 rpm for 12–24 h. Samples were periodically withdrawn to determine the cell growth, residual substrate, and metabolite content. In low H2 partial pressure (LHP) experiments, a needle was struck through the rubber septum of the culture bottle top, through which the gases generated were continuously purged out. The gases were directed to 2 N NaOH solutions via silicone tubing to remove CO2 and H2 gas was collected by the water substitution method [11].

2.3.

Construction of E. coli strains

Genetic manipulations were done as previously described by Datsenko and Wanner [15] and Baba et al. [16]. In brief, hybrid primers of hycA, hyaAB, hybBC, ldhA, and frdAB that are complementary to E. coli K-12 chromosomal genes and to the antibiotic cassette (FRT-kan-FRT) in pKD4 were designed. The FRT-kan-FRT cassette was amplified by PCR using the primers listed in Table 1 and pKD4 as the template and digested with DpnI and purified. After purification, plasmid DNA was electroporated (Bio-Rad) into E. coli containing pKD46 at 2.5 kV with 25 mF and 200 U, immediately followed by the addition of 1 mL of LB medium. After incubation for

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international journal of hydrogen energy 34 (2009) 7417–7427

Table 1 – Primers, plasmids and strains used this study. Genotype Strain BW25113 SH1 SH2 SH3 SH4 SH5

lacIq rrnBT14 DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78 BW25113 DhycA SH1 DhyaAB SH2 DhybBC SH3 DldhA SH4 DfrdAB

Plasmid pKD4 pKD46 pCP20

PCR template for Km cassette and FRT, Apr Red recombinase system, Apr FLP recombinase expression plasmid, Apr

Primer hycAp2 hycAp1 hyaAp1 hyaBp2 hybCp2 hybBp1 ldhAp2 ldhAp1 frdBp2 frdAp1

50 -AACGCCTGCAAAACGGGCAAAGCCTCAGCTCATGCTGCCGGGCTTTGTCCCATATGAATATCCTCCTTAG-30 50 -CAAAAAATGCTTAAAGCTGGCATCTCTGTTAAACGGGTAACCTGACAATGGTGTAGGCTGGAGCTGCTTC-30 50 -AAGCGCCCGGTGTCCTGCCGGTGTCGCAAGGAGGAGAGACGTGCGATATGGTGTAGGCTGGAGCTGCTTC-30 50 -TTCTGTTGCATGATGATTCTCCTTCGCTGTTAACGCACCTGCACGGAGATCATATGAATATCCTCCTTAG-30 50 -ATTGCCGACCCCTAAGACTAAAATACGCATTACAGAACCTTCACTGAAACCATATGAATATCCTCCTTAG-30 50 -GGTTCGTCGCAACACCAAAAACGACCATCACGACGGAGGAGACGATCATGGTGTAGGCTGGAGCTGCTTC-30 50 -CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGCACATATGAATATCCTCCTTAG-30 50 -TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGGTGTAGGCTGGAGCTGCTTC-30 50 -GTTTACGTTTAGTCGTCATGTTGCACTCCTTAGCGTGGTTTCAGGGTCGCCATATGAATATCCTCCTTAG-30 50 -ACCCTGAAGTACGTGGCTGTGGGATAAAAACAATCTGGAGGAATGTCGTGGTGTAGGCTGGAGCTGCTTC-30

Description CGSCa This study This study This study This study This study CGSCa CGSCa CGSCa

This This This This This This This This This This

study study study study study study study study study study

a Coli Genetic Stock Center, USA.

2 h at 37  C, one-fifth of the inoculation was spread onto LB agar plate. The resulting kanamycin-resistant recombinant E. coli contained FRT-kan-FRT in the deleted region. After verification of gene deletion with locus-specific primers, the kmr gene was cured from the chromosome with FLP recombinase by using a temperature-conditional helper plasmid (pCP20).

2.4.

Enzyme assay

H2 uptake activity was assayed at 30  C in 4-mL cuvettes under anoxic conditions. Potassium phosphate buffer (pH 7.0, 50 mM) was used in the cell suspension. For H2 uptake activity, the oxidized form of benzyl viologen (BV) was used as the electron acceptors and the reduction of BV was measured calorimetrically at 580 nm. The reaction mixture and an electron acceptor at 2 mM were equilibrated with 100% H2. For formate hydrogen lyase activity, the reaction mixture, which contained bis–tris buffer (pH 6.8, 50 mM), 1 mM sodium dithionite, and 20 mM formate, as electron donors, was equilibrated with 100% argon. The formation of H2 was analyzed from the gas phase using gas chromatograph.

2.5.

were analyzed using a Gel-Doc instrument (Bio-Rad, 2000). Glucose, ethanol and other organic acids were determined by an HPLC equipped with an auto sampler, RI and PDA detectors (HPLC, Agilent 1100 series, USA). The culture samples at the appropriate intervals were collected and centrifuged at 10,000  g for 5 min and the supernatant was filtered through 0.2 mm Tuffryn-membranes (Acrodisc, Pall Life Sciences). Finally the samples were eluted through the column, Aminex HPX-87H; 300  7.8 mm (Bio-Rad) at 50  C using 5.41 mM H2SO4. The flow rate was set at 0.5 mL/min. The flow cell temperature was set as 35  C. The analog output range was set at 0.1 V with positive polarity for RID. H2 and CO2 concentrations were measured using a gas chromatograph (DS 6200; Donam Inst. Inc, Republic of Korea) equipped with a TCD and a stainless steel column (1.8 m  1/800 packed with Molecular Sieve 5A; Alltech Deerfield, IL, USA). The temperatures of the injector, oven and detector were set at 90, 80 and 120  C, respectively. Argon gas was used as the carrier gas at a flow rate of 30 mL/min.

2.6. In silico model construction and metabolic flux analysis (MFA)

Analytical methods

Bacterial growth was measured at 600 nm using a double beam spectrophotometer (Lamda 20, Perkin Elmer, USA). The biomass was determined by measuring cell dry weight using a pre-determined correlation between OD (1 OD600 ¼ 0.3 g/L DCW of washed cells) and cell dry weight. Agarose gel electrophoresis was carried out using a Bio-Rad electrophoretic apparatus. The band intensities on gels

Glucose metabolic network for E. coli was developed from physiological experiments and published data [17,18]. The model contains 66 reactions: glycolyis, 15; pentose phosphate pathway, 8; tricarboxylic acid cycle, 11; pyruvate metabolism, 8; energy metabolism, 12; transport reaction, 11; and growth flux, 1. The metabolic demands of precursors and cofactors required for the biomass formation were adopted from another report [18]. A list of reactions and enzymes is provided in Appendix A.

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Glucose 1 4

Phosphoenolpyruvate

Oxaloacetate 5

2 3

x

Lactate

Malate

Pyruvate

6 8

Fumarate

Acetyl-CoA

x

Formate

9

Succinate

11 Acetalehyde

7

Hyb

13

Acetylphosphate

2e

Quinone Pool 10

2H

12

Ethanol

CO2

-

H2

+

H2

Acetate

Hya

Cytoplasm H2

2e

-

2H

+

H2

Periplasm

Fig. 1 – Fermentative pathway of glucose and hydrogen metabolism. ldhA, frdAB, hycA and uptake hydrogenase 1, 2 were deleted to enhance hydrogen production yield in E. coli. Enzymes: 1, EMP enzymes; 2, pyruvate kinase; 3, lactate dehydrogenase; 4, phosphoenolpyruvate carboxlyase; 5, malate dehydrogenase; 6, fumarase; 7, fumarate reductase; 8, pyruvate-formate lyase; 9, acetaldehyde dehydrogenase; 10, alcohol hydrogenase; 11, phospho-transacetylase; 12, acetate kinase; 13, formate hydrogen lyase.

Metabolic abbreviations are described in our earlier report [12]. The underdetermined system was solved by linear optimization using the program package MetaFluxNet [19].

3.

Results and discussion

3.1.

Construction and characterization of E. coli strains

Fig. 1 is a schematic of the strategy used to develop the strains in this study. Five strains were constructed

sequentially. Growth rate, FHL activity, H2 uptake activity, and H2 production yield of these five strains are summarized in Table 2. The deletion of hycA improved whole-cell H2 production from formate by 90% compared to the parental strain (BW25113). However, the deletion did change H2 production yield from glucose (1.20 mol/mol glucose). The strain SH3 (DhycADhyaABDhybBC ) which resulted from disrupting two uptake hydrogenases; hydrogenase 1 (hya) and hydrogenase 2 (hyb) lost almost all H2 uptake activity and gave substantially increased hydrogen production yield, i.e., 1.48 mol/mol glucose. Furthermore, the strain SH5

Table 2 – Specific cell growth rate, formate hydrogen lyase (FHL) activity, hydrogen uptake activity and hydrogen production yield of E. coli strains constructed in this study.a

Specific growth rate (h1) FHL activity (mmol H2 min1 mg1 DCW) H2 uptake activity (mmol BVred min1 mg1 DCW) H2 yield (mol H2/mol glucose)

BW

SH1

SH2

SH3

SH4

SH5

BW-LHPb

SH5-LHPb

0.69 0.51 0.16 1.20

0.66 0.97 ndb 1.17

0.58 0.77 ndb 1.37

0.55 0.82 <0.01 1.48

0.52 0.76 <0.01 1.61

0.48 0.87 <0.01 1.80

0.68 ndc ndc 1.62

0.64 ndc ndc 2.11

a Cells were grown on M9 minimal medium containing 10 g/L glucose and 3 g/L yeast extract. b Low H2 partial pressure. c Not determined.

international journal of hydrogen energy 34 (2009) 7417–7427

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Table 3 – Carbon mole balance of E. coli strains constructed in this study.

Glucose Pyruvate Succinate Lactate Formate Acetate Ethanol Cell mass H2 CO2 Recovery (%)

BW

SH1

SH2

SH3

SH4

SH5

BW-LHPa

SH5-LHPa

13.59 0.00 1.56 2.10 0.29 3.65 3.81 1.28 2.73 2.30 110

13.59 0.00 1.57 1.92 0.13 3.72 4.08 1.35 2.73 2.30 111

13.59 0.00 1.69 1.77 0.12 3.81 4.13 1.35 3.20 2.70 115

13.59 0.00 1.48 1.74 0.12 4.08 3.86 1.35 3.46 2.80 114

13.59 0.42 1.80 0.01 0.32 4.22 4.18 1.37 3.75 2.90 112

13.59 0.30 0.15 0.13 0.39 4.56 5.63 1.14 4.13 3.40 116

13.90 0.00 1.48 1.41 0.00 3.87 4.14 3.20 3.77 3.14b 124

13.90 0.17 0.18 0.11 0.14 4.88 5.20 3.02 5.01 4.17b 128

a Low H2 partial pressure. b CO2 production was estimated by assuming that the ratio of CO2 production to H2 production was 1.20, the average value obtained from all mutant strains under the condition of high H2 partial pressure.

Table 4 – Carbon material balance, reduction degree balance and product molar yield of anaerobic glucose metabolism by E. coli strain SH5 at varying initial glucose concentrations. Data refer to the consumption of 1 mol of glucose. Glucose concentration (g/L)

Carbon material balance Reactant (C-mol) Glucose Product (C-mol) Pyruvate Succinate Lactate Formate Acetate Ethanol Biomass CO2a Total products (%) Error (%) Reduction degree balance Reactant Glucose Product Pyruvate Succinate Lactate Formate Acetate Ethanol Biomass H2 O2 Total Errors (%) Molar yield YATP/G Yx/G YATP/x

2.5

5

10

6

6

6

0.23 0.27 0.04 0.19 1.7 1.66 1.08 0.69 97.8 2.1

24

0.44 0.16 0.03 0.21 1.75 1.46 0.9 0.9 97.3 2.7

24

0.56 0.11 0.04 0.15 1.66 1.49 0.77 1.03 96.6 3.2

24

0.56 0.98 0.08 0.38 6.8 9.96 4.23 2.74 0.52 25.21 5.0

1.2 0.56 0.08 0.42 7.04 8.76 3.26 2.94 0.4 23.86 0.6

1.52 0.42 0.08 0.3 6.64 8.88 3.09 3.18 0.44 23.67 1.36

2.42 1.04 2.33

2.57 0.8 3.23

2.58 0.76 3.33

a CO2 in the gas phase was ignored.

(DhycADhyaABDhybBCDldhADfrdAB), which resulted from further deletion of lactate dehydrogenase (ldhA) and fumarate reductase ( frdAB) in strain SH3 was constructed to increase the carbon flux of glucose metabolism to pyruvateformate lyase (PFL) reaction. The H2 production yield was further improved and reached 1.8 mol/mol glucose, which corresponds to 90% of the theoretical maximum of E. coli. However, cell growth rate gradually decreased as the number of deletions accumulated. The specific growth rate of strain SH5 was reduced by 30% compared to the wild type strain. At lower H2 pressure, culture experiments were repeated using the parental strain BW and the final construct SH5 (Table 2, columns 7 and 8). The H2 production yields increased to 1.62 mol/mol glucose for the parental strain BW25113 and to 2.11 mol/mol glucose for SH5, respectively. This is consistent with the report of the negative effects of H2 pressure on H2 production [2]. It should be noted that the yield with SH5 is even higher than the theoretical maximum (2.0 mol/mol glucose), suggesting a potential contribution of the yeast extract to H2 production. We also observed that the lowered growth rate of the SH5 mutant strain was significantly recovered when the cells were grown under reduced H2 partial pressure. This suggests that the growth of mutated E. coli cells is more inhibited by H2 than that of the parental strain. Recently, several efforts have been made to increase hydrogen production of E. coli using metabolic engineering approaches. The deletion of the hycA gene resulted in an increase in FHL activity by more than 50% [20,21]. The deletion of the two uptake hydrogenases improved H2 production yield by 37% over the parent strain [22]. Still, inactivation of ldhA and frdBC increases the H2 yield from 1.08 mol/mol glucose to 1.82 mol/mol glucose [10]. The production rate and yield of the E. coli strains resulting from this study were higher than those of the similar strains reported before. However, due to different media and conditions used in these studies, results of this study are not comparable to those of previous studies. Nevertheless, our results indicate that E. coli strains with different genetic backgrounds have different growth rate and H2 production yield.

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Table 5 – Experimental-measured metabolic rates and constraints used in in silico MFA.a Glucose (g/L)

2.5

Experimental/in silico Specific uptake rate (mmol/g cell h) Specific production rate (mmol/g cell h)

5 b

Experimental Glucose Acetate Formate Succinate Lactate Ethanol H2

H2 yield (mol/mol glucose) Specific growth rate (h1)

18.30

in silico

c

14.90 7.57c 0.00 0.00 12.83 28.67 1.45 0.47

Experimental 23.01

c

10 b

in silico

Experimentalb

in silico

c

c

23.39

c

0.00 0.00 20.01 37.19 1.59 0.40

0.00 0.00 14.40 26.47

19.76 7.00c 0.00 0.00 17.64 31.28

0.00 0.00 19.25 34.21

20.55c 5.40c 0.00 0.00 19.35 29.30

1.46 0.45

1.48 0.45

1.49 0.46

1.60 0.44

a Specific growth rate was used as an objective function for MFA. b Measured during exponential growth phase of batch cultures. c Used as the constraints in the model.

3.2. Carbon balance of glucose fermentation using engineered E. coli strains To better understand the fluxes, the carbon mass balances of glucose fermentation for the E. coli strains (Table 3) were examined. The experiments were conducted in a batch mode with the medium containing 10 g/L glucose and 3 g/L yeast extract. The parental strain BW25113 exhibited a typical mixed acid fermentation with succinate, lactate, acetate and ethanol as major products. The effects of individual deletion were clearly reflected in the carbon mass balances. For instance, the strain SH1, which had an improved FHL activity, showed a reduced formate accumulation compared to the parental strain BW25113 (Table 3). The SH4 and SH5 strains, both of which had the deletion of ldhA, showed a drastic decrease in lactate accumulation. Furthermore, the SH5 strain, which had an incomplete succinate pathway, had less than 90% of succinate compared to the parental strain (Table 3). However, the deletion of ldhA and frdAB did not completely limit the production of lactate and succinate, indicating the existence of unknown pathways leading to lactate and succinate production. In addition, the carbon flow to acetate, CO2 and ethanol increased when the production pathway of lactate and/or succinate was blocked. Clearly, the increases are ascribed to the increases of the carbon flux to FHL (Table 2 and Fig. 1). Pyruvate was detected in the fermentation of strains SH4 and SH5, suggesting that the deletion of ldhA and/or frdAB caused some imbalance between the production and utilization of pyruvate. The total amount of recovered carbon for all the engineered strains was consistently w10% higher than the theoretical value. This can be attributed to the carbon content in the yeast extract (28–38%; [14]). When H2 pressure was lowered, biomass production of parental strain and SH5 increased by more than twofold. This finding is consistent with the previous reports that high H2 pressure inhibits the cell growth [23–25]. Under the low H2 pressure condition, the

decreased production of pyruvate and formate was observed for the strain SH5.

3.3. Carbon and energy balances of glucose fermentation for E. coli strain SH5 The strain SH5, exhibiting a high H2 production yield, was studied in detail at the glucose concentrations ranging from 2.5 to 10 g/L (Table 4). Since the yeast extract caused an unexpected value of carbon recovery (over 100%) and overestimation of the glucose to H2 yield [26–28], its concentration was lowered to 0.3 g/L (Table 3). In this study, the use of a low concentration of yeast extract yielded a satisfactory carbon recovery at 97.2  0.6%, regardless of the initial glucose concentration. The carbon mass balance between the substrate and the various metabolites produced is summarized in Table 4. The fractional distribution of glucose carbon to various metabolites varied slightly, depending on the initial glucose concentration. As the glucose concentration increased, the production of pyruvate and CO2 increased and yield of the biomass and succinate decreased. The increase of pyruvate excretion at higher glucose concentrations confirmed the effect of multiple deletion in strain SH5 (Table 3). The deletion of ldhA and frdAB directed more carbon flux to the pyruvate node by blocking the production of lactate and succinate without accelerating the downstream pathways of pyruvate except for FHL. The imbalance between pyruvate formation and consumption seems to become more obvious as glucose uptake rate increased through increasing medium glucose concentration. Since pyruvate excretion decreased carbon for H2 production, it should be limited as much as possible. Limiting pyruvate excretion can be achieved by increasing the rate of downstream metabolic reactions of pyruvate using pathway engineering approaches. The degree of reduction (3) for the substrate and all products is shown in Table 4. It represents the number of available

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international journal of hydrogen energy 34 (2009) 7417–7427

0.00 0.00 0.00

D6PGC 0.00 0.00 0.00

RL5P

0.33 0.33 0.29

R5P

G6P 18.00 22.90 23.31

-0.33 -0.33 -0.29

-0.07 -0.07 -0.06

S7P

D6PGL

0.00 0.00 0.00

F6P 17.63 22.52 22.98

FDP

X5P

17.63 22.52 22.98

-0.25 -0.25 -0.22

F6P

-0.07 -0.07 -0.06

18.1 23.0 23.4

DHAP

GAP

T3P1

34.96 44.74 45.69

E4P

GLC

13DPG 34.96 44.74 45.69

3PG

34.28 44.05 45.09

0.00 0.00 0.00

2PG 24.38 44.05 45.09

PEP

0.00 0.00 0.00

14.64 19.50 20.32

PYR

0.00 0.00 0.00

MAL

0.00 0.00 0.00

0.00 0.00 0.00

CIT

GLX

FUM 0.00 0.00 0.00

SUC

FOR

0.49 0.50 0.44

OA

ICIT 0.00 0.00 0.00

COA 31.47 41.21 42.59

1.31 1.32 1.16

LAC

23.90 34.21 37.19

H2

ACCOA 14.90 19.76 20.55 0.49 0.50 0.44

ACTP 14.40 19.25 20.10

0.49 0.50 0.44

AKG

ETH

14.90 19.76 20.55

AC

Fig. 2 – Distribution of optimized metabolic flux (mmol/g cell h) in SH5 when maximization of specific growth rate was used as an objective function at varying glucose concentrations (2.5/5.0/10.0 g glucose/L). A negative sign indicates that the reaction proceeds in the opposite direction of the arrow. The constraints used for MFA are shown in Table 5. The detailed reactions are shown in Appendix A.

electrons per unit carbon atom and is calculated from the following equation [29,30]: 3 ¼ 4C þ H  2O  3N where, C, H, O and N denote the atomic coefficients of carbon, hydrogen, oxygen and nitrogen. This expression

includes the 3N factor, which compensates for the electrons donated to the biomass from ammonia and thereby omits ammonium consumption in the calculation [30]. Table 4 indicates that the reduction degrees of the reactant glucose and sum of all products listed are well balanced with relative errors that are less than 5%. This indicates

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that, along with the satisfactory carbon recovery, the metabolites in Table 4 were well identified and the quantification of each metabolite was accurate. Based on the carbon mass balance, various molar yields were also determined. The metabolic equations and calculations of molar yields have been reported in detail in our previous work [12]. As the initial glucose concentration increased from 2.5 g/L to 10 g/L, the H2 production yield increased from 1.45 to 1.60 mol/mol glucose and the ATP-tocell yield (YATP/x) increased from 2.33 to 3.33; while the cellto-glucose yield (Yx/G) decreased from 1.04 to 0.76. The increase in H2 production yield at increasing glucose concentrations appeared to be affected by pH variance. Final pH values were 6.4, 6.2 and 5.8 at the glucose concentrations of 2.5, 5.0 and 10 g/L, respectively. Therefore, our findings also agree with the reports that H2 productivity increases with the decrease of pH [10,31]. However, biomass is low at low pH because more maintenance is required [32–34]. In this study, while ATP formation yield was constant over the entire range of glucose concentrations tested, the values of Yx/G and Yx/ATP decreased as the glucose concentration increased (Table 4). In other words, from an energetic point of view, the progressive accumulation of acidic products made cell growth more expensive while H2 productivity was enhanced due to low pH.

3.4.

The pentose phosphate pathway (PP pathway) was barely activated at all of the glucose concentration used in this study, and most of the carbons were directed to the Embden–Meyerhof pathway. The PP pathway inactivation also took place in the parental BW strain for which flux analysis was carried out under the same conditions (data not shown). The main function of the PP pathway in E. coli cells is to supply precursors for supporting cell growth and, thus, the addition of the yeast extract and the availability of precursor molecules in the medium avoid the use of the PP pathway [37]. However, in our previous study with C. amalonaticus Y19, it was suggested that the PP pathway can have an important role in H2 production if NAD(P)Hdependent hydrogenases is functioning [36,14]. According to metabolic flux analysis, the H2 production yield could be increased more than 8.0 mol H2/mol glucose when glucose is metabolized through the PP pathway and the NAD(P)H produced by the PP pathway is utilized for producing H2. For the present model on E. coli, we could perform the same metabolic flux analysis with H2 production rate as an objective function. When non-native NAD(P)-linked hydrogenases (EC 1.12.1.2(3)) were incorporated in the model for the strain SH5, metabolism was mainly directed to the PP pathway (w80%), and a very high H2 production yield (w8.7 mol H2/mol glucose) was obtained (data not shown).

In silico model construction and MFA

In order to better understand the metabolic flow patterns that resulted from the genetic perturbations incorporated in this study, in silico metabolic flux analysis was conducted. The in silico model construction was carried out on the basis of published information and the results of the present study (see Section 2 for details). The objective function was cell growth rate. Results of the in silico model were compared to the data collected from batch experiments described in Section 3.3. In this model, the uptake rate of glucose and the production rate of acetate and formate were used as constraints and measured during the exponential growth phase of batch culture. Both the H2 production rate and the specific growth rate were crossexamined as criteria for the consistency between model and wet experiment. As shown in Table 5, the specific growth rate and H2 yield were in a very good agreement. However, as the initial glucose concentration increased, the gap in the H2 production rate between the experiments and the calculation increased. Fig. 2 is the graphical representation of the metabolic flux calculation of the strain SH5 at different initial glucose concentrations. All intermediate fluxes including the glucose uptake rate increased at higher glucose concentrations. The glucose utilization rate of the strain SH5 at the concentration of 2.5 g glucose/L was estimated as 18.3 mmol/g cell h. Verma and Palsson [38] have also reported almost the same rate (18.5 mmol/g cell h) with a wild type E. coli strain that was grown under anaerobic conditions at the concentration of 2 g glucose/L. However, compared to C. amalonaticus Y19 (12.6 mmol/g cell h), the strain SH5 had a 50% faster glucose uptake rate [12,14,35].

4.

Conclusion

A series of E. coli strains for enhanced H2 production were successfully developed using pathway engineering approaches. Deletion of hycA resulted in 2-fold increase of hydrogen production. The strain SH5, which resulted from combined deletion of hycA, uptake hydrogenase, ldhA and frdAB, produced H2 up to 2.11 mol/mol glucose under low H2 partial pressure condition. The concentration of the medium yeast extract affected anaerobic metabolism of glucose and the subsequent analyses of carbon and energy balances. When SH5 was cultured in a batch mode with a low yeast extract concentration (0.3 g/L), the glucose uptake rate and H2 production yield increased as initial glucose concentration increased from 2.5 to 10.0 g/L. Metabolic flux analyses indicated that the PP pathway was not functioning when the strain SH5 or the parental strain BW was cultured in the medium containing 0.3 or 3.0 g/L yeast extract.

Acknowledgement This work is a result of the fostering project of the Best Lab supported financially by the Ministry of Knowledge and Economy, Republic of Korea. Dr. SM. Raj is grateful to the Brain Korea 21 program, Pusan National University for financial assistance.

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Appendix A

List of enzymes and reactions used in metabolic flux model of E. coli Enzyme Glycolysis Glucose 6-phosphate isomerase 6-Phosphofructokinase Fructose 1,6-bisphosphatase Fructose-bisphosphate aldolase Triose-phosphate isomerase Glyceraldehyde 3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase Phosphoenolpyruvate synthase Pyruvate dehydrogenase

EC No.

Reaction

5.3.1.9 2.7.1.11 3.1.3.11 4.1.2.13 5.3.1.1 1.2.1.12

G6P 4 F6P F6P þ ATP / F16P þ ADP F16P / F6P þ PI F16P 4 T3P1 þ T3P2 T3P1 4 T3P2 T3P1 þ PI þ NAD 4 NADH þ a13P2DG

2.7.2.3 5.4.2.1 4.2.1.11 2.7.1.40 2.7.9.2

Phosphoenolpyruvate carboxykinase Phosphoenolpyruvate carboxylase Pyruvate-formate lyase

4.1.1.49 4.1.1.31 2.3.1.54

a13P2DG þ ADP 4 a3PDGL þ ATP a3PDGL 4 a2PDGL a2PDGL 4 PEP PEP þ ADP / PYR þ ATP PYR þ ATP / PEP þ AMP þ PI PYR þ COA þ NAD / NADH þ CO2 þ ACCOA OA þ ATP / PEP þ CO2 þ ADP PEP þ CO2 / OA þ PI COA þ PYR / ACCOA þ FOR

Pentose phosphate pathway Glucose 6-phosphate 1-dehydrogenase 6-Phosphogluconolactonase 6-Phosphogluconate dehydrogenase Ribose 5-phosphate isomerase Ribulose 5-phosphate-3-epimerase Transketolase Transaldolase Transketolase

1.1.1.49 3.1.1.31 1.1.1.44 5.3.1.6 5.1.3.1 2.2.1.1 2.2.1.2 2.2.1.1

G6P þ NADP 4 D6PGL þ NADPH D6PGL / D6PGC D6PGC þ NADP / NADPH þ CO2 þ RL5P RL5P 4 R5P RL5P 4 X5P R5P þ X5P 4 T3P1 þ S7P T3P1 þ S7P 4 E4P þ F6P X5P þ E4P 4 F6P þ T3P1

TCA cycle Citrate synthase Aconitase Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase

2.3.3.1 4.2.1.3 1.1.1.42

Succinyl-CoA synthetase Succinate dehydrogenase Fumarate reductase Fumarase Malate dehydrogenase Isocitrate lyase Malate synthase

6.2.1.5 1.3.99.1 1.3.99.1 4.2.1.2 1.1.1.37 4.1.3.1 2.3.3.9

ACCOA þ OA / COA þ CIT CIT 4 ICIT ICIT þ NADP 4 CO2 þ NADPH þ AKG AKG þ NAD þ COA / CO2 þ NADH þ SUCCOA SUCCOA þ ADP þ PI 4 ATP þ COA þ SUCC SUCC þ FAD / FADH þ FUM FUM þ FADH / SUCC þ FAD FUM 4 MAL MAL þ NAD 4 NADH þ OA ICIT / GLX þ SUCC GLX þ ACCOA / MAL þ COA

Pyruvate oxidation Alcohol dehydrogenase Phosphate acetyltransferase Acetate kinase Fomate hydrogen lyase Lactate dehydrogenase Malate dehydrogenase Fumarase Fumurate reductase Energy metabolism NADH dehydrogenase Cytochrome oxidase Transhydrogenase Succinate dehydrogenase complex Transhydrogenase F0F1-ATPase Aldenylate kinase ATP drain

2.3.1.8 2.7.2.1 1.1.1.28 1.1.1.37 4.2.1.2 1.3.99.1

1.6.5.3

3.9.3.14 2.7.4.3

ACCOA þ 2 NADH / ETH þ 2 NAD þ COA ACCOA þ PI 4 ACTP þ COA ACTP þ ADP / ATP þ AC FOR / CO2 þ H2 NADH þ PYR / LAC þ NAD NADH þ OA 4 MAL þ NAD MAL 4 FUM FUM þ NADH / NAD þ SUCC

NADH þ Q / NAD þ QH2 þ 2 HEXT 2 QH2 þ O2 / 2 Q þ 4 HEXT NADPH þ NAD / NADP þ NADH FADH þ Q / FAD þ QH2 NADP þ NADH þ 2 HEXT / NADPH þ NAD ADP þ PI þ 3 HEXT 4 ATP ATP þ AMP 4 2 ADP ATP / ADP þ PI (continued on next page)

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Appendix A (continued) Enzyme Formate dehydrogenase Uptake hydrogenase NAD kinase Lactate dehydrogenase

EC No.

2.7.1.23 1.1.2.3/4

Transport Glucose transport Succinate transport Acetate transport Ethanol transport Oxygen transport Carbon dioxide transport Phosphate transport Hydrogen transport Pyruvate transport Formate transport Lactate transport Growth Growth flux

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Reaction FOR þ Q 4 COA þ HEXT þ 2 QH2 H2 þ Q / QH2 þ 2 HEXT ATP þ NAD / ADP þ NADP LAC þ Q / PYR þ QH2 GLCxt þ PEP / G6P þ PYR SUCC 4 SUCCxt þ HEXT AC 4 ACxt þ HEXT ETH 4 ETHxt þ HEXT O2xt 4 O2 CO2 4 CO2xt PI 4 PIxt þ HEXT H2 4 H2ext PYR 4 PYRex þ HEXT FOR 4 FORex LAC 4 LACext þ HEXT

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