Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)
© The Author(s) 2017
Received: 17 November 2016
Accepted: 22 March 2017
Published: 29 March 2017
Biologically, hydrogen (H2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H2 production yield is unsatisfactorily low as <4 mol H2/mol glucose. To address this challenge, simultaneous production of H2 and ethanol has been suggested. Co-production of ethanol and H2 requires enhanced formation of NAD(P)H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden–Meyerhof–Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition.
Here, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli Δpgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgi mutant could produce 1.69 mol H2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol−1 glucose, indicating that some carbon is metabolized through the Entner–Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol−1 glucose along with 1.74 mol H2 mol−1 glucose, which are close to the theoretically maximal yields, 1.67 mol mol−1 each for ethanol and H2. However, the attempt to delete the ED pathway in the Δpgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful.
By deletion of pgi and overexpression of heterologous zwf and gnd in E. coli ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB, two important biofuels, ethanol and H2, could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions.
KeywordsBiohydrogen Co-production of hydrogen and ethanol Phosphoglucose isomerase deletion Pentose-phosphate pathway Escherichia coli
Hydrogen (H2) is considered as a promising alternative to fossil fuel as it is an efficient energy carrier and produces zero carbon emission. Currently, H2 is produced by steam reforming process using natural gas, a non-renewable source. Therefore, as an alternative, biological H2 production by photolysis, photofermentation, or dark fermentation has been studied for decades due to its dependence on renewable energy source. Dark fermentation, owing to its rapidity and simplicity, usually is the preferred approach [1–3], though its theoretical H2 yield is low: typically <2 mol mol−1 glucose for mesophilic bacteria such as Escherichia coli  and <4 mol mol−1 glucose for strict anaerobes such as Clostridia, Thermoanaerobacter tengcongensis, Pyrococcus furiosus, and others [5–7]. To improve H2 production yield in E. coli, introduction of heterologous pathways such as ferredoxin- or NAD(P)H-dependent H2 production pathways has been attempted. The heterologous pathways, though functional in E. coli, have been shown to be highly inefficient and, as such, non-conducive to practical improvements in H2 yield . In the case of strict anaerobes, higher yield, close to 4 mol mol−1 glucose, have been reported , albeit still not high enough to be commercially interesting. Due to the lack of a genetic tool box and/or the difficulty for gene manipulation, serious pathway engineering in strict anaerobes is yet to be attempted. As alternative means of addressing dark fermentation’s low H2 production yield, hybrid processes such as dark plus photofermentation, hythane process (production of H2 in the first stage and methane in the second), etc., have been studied [10–12]. Albeit efficient and feasible on the laboratory scale, these hybrid processes’ industrial application is highly challenging due to complicated reactor configurations and/or operation. We have suggested, as an alternative, a simple process by which H2 and ethanol are co-produced in a single bioreactor . Ethanol in fact is a good liquid biofuel, and can easily be separated from gaseous H2. Co-production of ethanol with H2, moreover, can significantly increase the energy recovery of dark fermentation and, thereby, make H2 production from glucose more attractive .
In the present study, we determined that Δpgi mutants can grow anaerobically when Zwf and Gnd are overexpressed. Consequently, we investigated complete blockage of the carbon flux through the EMP pathway and the concomitantly improved co-production of H2 and ethanol. Also, to address the two major disadvantages of Zwf and Gnd of E. coli—their dependence on NADP+ as the cofactor and serious inhibition of their activities by NADPH at the enzyme level—expressions of heterologous Zwf and Gnd from other microorganisms have been studied. We also attempted deletion of the ED pathway for operation of the PP pathway as the sole glycolytic route. Our results demonstrate that Δpgi mutants can grow under anaerobic conditions and co-produce H2 and ethanol at near-theoretical yields. Additionally, the data obtained show that the developed strains can be used as an interesting platform when generation of considerable reducing power is needed in anaerobic glucose metabolism [17, 18].
Strains, plasmids, and materials
The Escherichia coli BW25113 mutant strain (SH5) from our previous study  was used as a base strain in this work. Restriction and DNA-modifying enzymes were obtained from New England Bio-Labs (Beverly, MA, USA). The Miniprep and DNA gel extraction kits were purchased from Qiagen (Mannheim, Germany). The primers were synthesized by Macrogen Inc. (Seoul, Korea). The yeast extract (Cat. 212750) and Bacto™ tryptone (Cat. 211705) were acquired from Difco (Becton–Dickinson; Franklin Lakes, NJ, USA). Unless indicated otherwise, all of the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Construction of recombinant E. coli strains
Strains and plasmids used in this study
BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB
Kim et al. 
SH5Δpgi harboring pEcZ
SH5Δpgi harboring pEcZG
SH5Δpgi harboring pEcZGU
SH5Δpgi_Z L G E
SH5Δpgi harboring pLmZ-EcG
SH5Δpgi_Z Z G E
SH5Δpgi harboring pZmZ-EcG
SH5Δpgi_Z E G C
SH5Δpgi harboring pEcZ-CgG
SH5Δpgi_Z L G C
SH5Δpgi harboring pLmZ-CgG
SH5Δpgi_Z Z G C
SH5Δpgi harboring pZmZ-CgG
SH5Δpgi_Z E G G
SH5Δpgi harboring pEcZ-GoG
SH5Δpgi_Z L G G
SH5Δpgi harboring pLmZ-GoG
SH5Δpgi_Z Z G G
SH5Δpgi harboring pZmZ-GoG
SH5ΔpgiΔedd harboring pEcG
SH5ΔpgiΔedd harboring pEcZG
SH5ΔpgiΔedd_Z L G G
SH5ΔpgiΔedd harboring pLmZ-GoG
Kleiner et al. 
pDK7 carrying zwf of E. coli BW25113
Sundara Sekar et al. 
pDK7 carrying zwf, gnd of E. coli BW25113
pDK7 carrying zwf, gnd, udhA of E. coli BW25113
pDK7 carrying zwf of L. mesenteroides and gnd of E. coli BW25113
pDK7 carrying zwf of Z. mobilis and gnd of E. coli BW25113
pDK7 carrying zwf of E. coli BW25113 and gnd of C. glutamicum
pDK7 carrying zwf of L. mesenteroides and gnd of C. glutamicum
pDK7 carrying zwf of Z. mobilis and gnd of C. glutamicum
pDK7 carrying zwf of E. coli BW25113 and gnd of G. oxydans
pDK7 carrying zwf of L. mesenteroides and gnd of G. oxydans
pDK7 carrying zwf of Z. mobilis and gnd of G. oxydans
Luria-Bertani broth was used to culture the cells for genetic engineering and culture maintenance work. Production studies were performed in modified M9 medium containing 5.0 g L−1 glucose or gluconate, 1.0 g L−1 yeast extract, 3.0 g L−1 Na2HPO4, 1.5 g L−1 KH2PO4, 0.5 g L−1 NH4Cl, 0.25 g L−1 NaCl, 0.25 g L−1 MgSO4, and 0.01 g L−1 CaCl2. Kanamycin (50 µg mL−1) and chloramphenicol (25 µg mL−1) were added to the medium for culturing of the recombinant strains. The medium was also supplemented with 0.2 mg L−1 NiSO4, 1.4 mg L−1 FeSO4, 0.2 mg L−1 Na2SeO3, 0.2 mg L−1 Na2MoO4, and 8.8 mg L−1 cysteine HCl for supporting the synthesis of co-production-related enzymes. The cells were cultured anaerobically with 50 mL of M9 medium in 165 mL serum bottles. The serum bottles with the media were flushed with argon for 15 min to create the anoxic condition for fermentation. The cells were cultured at 37 °C in an orbital shaker rotating at 200 rpm. The expressions of Zwf and Gnd were induced by the addition of 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), unless stated otherwise.
Total RNA isolation and real-time PCR
The recombinant strains were induced with IPTG and harvested during the late exponential growth phase. RNAprotect reagent (Qiagen Inc., USA) was added to the cell pellets, which were stored at −80 °C to prevent RNA degradation. Total RNA was extracted using the Nucleospin® RNA isolation kit (Macherey-Nagel, Germany) and converted to cDNA using the SuperScript III first-strand synthesis system (Invitrogen, USA). The RT-PCR primers were designed using Primer Express® software. RT-PCR analysis was performed using the StepOne real-time PCR system (Applied Biosystems, USA). The experiment was conducted in duplicate using the SYBR Green method, and the relative mRNA was quantified by the ΔCT method . rpoD was utilized as the endogenous control.
Determination of enzyme activities
The enzyme activities of Gnd and Zwf were measured as described in Moritz et al. , with slight modifications. Briefly, the enzyme activities were performed in 50 mM Tris-HCl (pH 7.5) containing 0.2 mM NADP+, 1 mM MgCl2, and 0.5 mM glucose-6-phosphate or 6-phosphogluconate. The reduction of NADP+ was observed at 340 nm. The extinction coefficient (ε 340) of 6.22 mM−1 cm−1 was used for calculating the amount of NADPH formed in the assay. All measurements were performed at 30 °C.
Cell growth was monitored by UV spectrophotometry (Lambda 20, Perkin Elmer, USA) measurement of the optical density (OD600) at 600 nm. Gases such as H2 and CO2 were measured by gas chromatography (DS6200 Donam Systems Inc., Seoul, Korea) equipped with a TCD detector. The stainless steel column of gas chromatography was packed with either Hayesep Q (for CO2 analysis, Alltech Deerfield, IL, USA) or Molecular Sieve 5A (for H2 analysis, Alltech Deerfield, IL, USA). Argon was used as the carrier gas and its flow rate was set at 30 mL min−1. The temperature of injector, column oven, and TCD detector was maintained at 90, 80, and 120 °C, respectively, during analysis. Glucose, ethanol, and all of the other metabolites were measured by high-performance liquid chromatography (Agilent Technologies, HP, 1200 series) equipped with a refractive index (RID) and photodiode array (DAD) detectors. The post-fermentation medium was centrifuged and filtered, and samples were eluted through a 300 mm × 7.8 mm Aminex HPX-87H (Bio-Rad) column at 65 °C using 2.5 mM H2SO4. The protein concentrations of the samples used in the enzyme activities were determined by the Bradford method as described previously  using bovine serum albumin as the standard.
Results and discussion
Growth of E. coli mutant lacking pgi under aerobic and anaerobic conditions
To confirm our hypothesis, zwf was homologously overexpressed by a multi-copy plasmid under the IPTG-inducible tac promoter. The resultant recombinant, SH5Δpgi_Z could grow well on glucose under anaerobic conditions, though rather slowly compared with its parental strain SH5 (Fig. 2b). In order to determine if gnd expression can further improve cell growth, SH5Δpgi_ZG was developed and tested. No difference from SH5Δpgi_Z was observed. It was concluded that the incapacity of E. coli Δpgi for anaerobic growth is rooted in the inefficient conversion of G6P to 6PG.
Co-production of H2 and ethanol by E. coli Δpgi
Co-production by recombinant SH5Δpgi strains overexpressing Zwf and Gnd
Yields of metabolites (mol mol−1)
1.44 ± 0.07
0.79 ± 0.02
0.67 ± 0.04
1.81 ± 0.08
0.90 ± 0.02
0.86 ± 0.03
1.68 ± 0.06
1.44 ± 0.03
0.22 ± 0.02
1.72 ± 0.09
0.58 ± 0.01
1.31 ± 0.03
1.70 ± 0.05
0.68 ± 0.01
1.25 ± 0.04
1.69 ± 0.07
0.92 ± 0.02
0.85 ± 0.02
Relative transcription levels of key glycolytic enzymes in SH5Δpgi_Z and SH5Δpgi_ZG
SH5Δpgi_Z (0.1 mM)
SH5Δpgi_ZG (0.1 mM)
SH5Δpgi_ZG (0.2 mM)
2090.91 ± 146.36
2693.55 ± 212.52
8001.77 ± 400.09
6.03 ± 0.21
2155.47 ± 73.18
6039.95 ± 259.79
2.16 ± 0.11
3.59 ± 0.06
5.48 ± 0.13
2.59 ± 0.13
1.29 ± 0.03
1.19 ± 0.03
34.12 ± 1.54
44.01 ± 0.96
80.95 ± 3.24
1.41 ± 0.02
1.09 ± 0.01
0.87 ± 0.01
12.20 ± 0.24
5.72 ± 0.12
7.28 ± 0.16
42.99 ± 1.38
21.51 ± 0.65
37.04 ± 0.74
0.38 ± 0.01
0.52 ± 0.01
0.43 ± 0.01
0.45 ± 0.01
0.55 ± 0.01
0.28 ± 0.01
4.51 ± 0.05
7.22 ± 0.12
13.76 ± 0.47
Construction and characterization of SH5ΔpgiΔedd, the strain using PP pathway as sole glycolytic route
The improved activity of Zwf and Gnd could not completely eliminate acetate production in SH5Δpgi_ZG. Therefore, to make the PP pathway the sole glycolytic route, the ED pathway was blocked by disruption of the edd and eda genes from SH5Δpgi. The resultant SH5 ΔpgiΔedd strain could grow on glucose under aerobic conditions but not at all under anaerobic conditions, even after overexpression of Zwf and Gnd (see Additional file 1: Fig. S2). This result, albeit disappointing, confirmed that the ED pathway is functioning in the SH5Δpgi_ZG strain and metabolizing a portion of the glucose.
The inability of SH5ΔpgiΔedd_ZG to grow on glucose under anaerobic condition is attributed to redox imbalance. According to the carbon and energy balance, when one glucose is fully metabolized through the PP pathway, 0.33 NADPH is generated along with 1.67 H2 and 1.67 ethanol  (Fig. 1b). The supplementation of yeast extract in the medium could have worsen the redox imbalance because it contains complex amino acids and some carbohydrates which can contribute to the regeneration of NAD(P)H. If excess NADPH is accumulated, the PP pathway will be blocked along with termination of cell growth. To prove this hypothesis, two experiments were carried out. First, SH5ΔpgiΔedd _ZG was grown on gluconate as the gluconate is more oxidized than glucose, and so no excess NADPH is accumulated. As expected, SH5ΔpgiΔedd_ZG could grow on gluconate, though the rate is very slow (see Additional file 1: Fig. S2). In the course of that growth, ethanol and acetate were produced at yields of 0.79 and 0.84 mol mol−1, respectively, which makes the gluconate metabolism redox-balanced. In the second experiment, SH5ΔpgiΔedd_ZG was grown on glucose but in the presence of nitrate (Additional file 1: Fig. S3). Nitrate can be used as an external electron acceptor and regenerate NAD(P)+ under anaerobic conditions . The results showed that the addition of nitrate recovered the growth of SH5ΔpgiΔedd even without the expression of Zwf and Gnd (Additional file 1: Fig. S3). The overexpression of Zwf and Gnd in the presence of nitrate increased the glucose consumption. However, neither H2 nor ethanol was produced; instead, acetate was the sole metabolite. In the presence of nitrate, E. coli oxidizes NAD(P)H to reduce nitrate and produce ATP. In summary, these two experiments strongly suggest that the redox imbalance and/or excessive NADPH generated by the PP pathway prevented SH5ΔpgiΔedd_ZG growth on glucose under anaerobic conditions.
It is possible to determine the minimal glucose flux to the ED pathway allowing for redox-balanced glucose metabolism using the carbon balance equation of PP and ED pathway (Fig. 1b) (see Additional file 1: Fig. S4). When NAD(P)H used for cell growth is ignored, the estimated minimal flux ratio of the ED pathway (i.e., ED flux/sum of PP and ED fluxes) is 0.14. If the minimal flux ratio of the ED pathway is below 0.14, the production and consumption of NAD(P)H cannot be matched by the production of ethanol and acetate. On the other hand, at any flux ratios above 0.14, combined production of acetate and ethanol makes the glucose metabolism redox-balanced and allows cells to grow.
Limitations on PP pathway operation under anaerobic condition and expression of transhydrogenase
According to the redox-balance analysis results plotted in Additional file 1: Fig. S4, the current SH5Δpgi_ZG had a higher ED flux (~0.32) than the ideal case (0.14). We speculate that despite high expression by the multi-copy plasmid, the enzymatic activities of Zwf and Gnd were low under the physiological conditions, and that this might be the reason why the flux ratio to the PP pathway did not increase above the 0.1 mM IPTG shown in Fig. 3. It is known that Zwf and Gnd of E. coli are almost exclusively NADP+ dependent and that their enzymatic activities are highly inhibited by NADPH [24, 30] (see Additional file 1: Table S2). Because the roles of Zwf and Gnd are so important, we cloned and characterized these enzymes from our own host E. coli BW25113 (Additional file 1: Fig. S5, Additional file 1: Table S2). The enzymes were expressed with a C-terminal His-tag and characterized after purification by Ni–NTA chromatography. Both of them were shown to be strictly dependent on NADP+, and no activity was observed with NAD+ as the cofactor. The specific activities and K m values of the purified Zwf and Gnd were similar to those that have been reported  (Additional file 1: Table S2). Additionally, we found that the two enzymes were inhibited by NADPH at similar levels (K i = ~ 40 μM) but not at all by NADH.
The problem associated with high intracellular NADPH concentration and consequent inhibition on Zwf and Gnd can be solved in two ways: by (1) reducing the NADPH concentration and/or (2) employing less NADPH-sensitive enzymes. To explore the first approach, we overexpressed UdhA, the soluble transhydrogenase for the conversion of NADPH to NADH. E. coli strains have two transhydrogenases, one soluble (udhA) and the other membrane bound (pntAB) . Although both enzymes work reversibly, the former mainly catalyzes the reaction for the conversion of NADPH to NADH, and the latter, the reverse reaction . To our disappointment, even after the overexpression of UdhA under a strong tac promoter, no improvement in ethanol production was observed in SH5Δpgi_ZG (Additional file 1: Fig. S6). Further, deletion of both udhA and pntA from SH5Δpgi_ZG did not affect cell growth or metabolite formation: SH5ΔpgiΔudhAΔpntA_ZG grew similar to SH5Δpgi_ZG and produced similar amounts of H2, ethanol, and acetate (Additional file 1: Fig. S6). These results are puzzling, because they indicate that the roles of the two transhydrogenases are negligible in glucose metabolism, and also that ethanol production in SH5Δpgi_ZG and other derived strains might be NADPH dependent. In any case, it is clear that overexpression of transhydrogenases cannot be the solution to the problem that necessitates reduction of intracellular NADPH levels and/or enhancement of carbon flux through the PP pathway.
Use of heterologous zwf and gnd
In another attempt to improve the carbon flux to the PP pathway, we overexpressed heterologous Zwf and Gnd which are less inhibited by NADPH. If such enzymes can use NAD+ as the cofactor, the reduction of the intracellular NADPH level would be also expected. For the Zwf and Gnd reported in the literature and enzyme databases, cofactor specificity, activity, and NADPH-dependent inhibition were analyzed and compared (Additional file 1: Table S2). The Zwf from Zymomonas mobilis (ZZ) had an approximately sevenfold higher activity than that of E. coli (ZE; note that the subscript ‘E’ was added to avoid confusion) . Furthermore, it could use both NAD+ (K m, 210 µM) and NADP+ (K m, 40 µM), though preferring the latter more. The Zwf from Leuconostoc mesenteroides (ZL) also showed a sevenfold higher activity than that of ZE, and could use NAD+ as a cofactor, having a higher affinity (K m, 106 µM) than ZZ [33, 34]. Interestingly, the Gnd from Gluconobacter oxydans (GG) showed a higher affinity to NAD+ (K m, 64 µM) than to NADP+ (K m, 440 µM), whereas Gnd from Corynebacterium glutamicum (GC) showed a fivefold higher activity than that of E. coli (GE), though its use of NAD+ as a cofactor is not known [24, 35].
Co-production by recombinant SH5Δpgi and SH5ΔpgiΔedd strains overexpressing heterologous Zwf and Gnd
Relative growth rate
Yield of metabolites (mol mol−1)
1.44 ± 0.07
0.22 ± 0.03
1.48 ± 0.04
0.25 ± 0.02
1.49 ± 0.06
0.32 ± 0.01
1.35 ± 0.05
0.37 ± 0.01
1.46 ± 0.06
0.26 ± 0.01
1.32 ± 0.07
0.49 ± 0.01
1.52 ± 0.09
0.22 ± 0.02
1.62 ± 0.06
0.06 ± 0.01
1.46 ± 0.07
0.35 ± 0.01
ΔpgiΔedd_ZLG G a
1.01 ± 0.03
0.40 ± 0.01
The same plasmid expressing both Zwf of L. mesenteroides and Gnd of G. oxydans (pLmZ-GoG) was introduced to SH5ΔpgiΔedd to determine if these highly efficient Zwf and Gnd can enable its anaerobic growth. As expected, the resulting recombinant SH5ΔpgiΔedd_Z L G G could not grow anaerobically with glucose as the carbon source. This confirmed that redox imbalance does not permit use of the PP pathway as the sole glycolytic route of glucose metabolism under anaerobic conditions. The same SH5ΔpgiΔedd_Z L G G strain was also cultured on gluconate as the carbon source. In fact, it could grow much better than SH5ΔpgiΔedd_ZG (Additional file 1: Fig. S2), producing more ethanol (1.01 vs. 0.79 mol mol−1) and less acetate (0.40 vs. 0.85 mol mol−1). This result confirmed once again that the highly efficient Zwf (ZL) and Gnd (GG) can effectively activate the PP pathway.
The ΔpfkA strains could grow well after long adaptation to anaerobic growth  and produced good amounts of H2 and ethanol (~1.7 mol H2 mol−1 and ~1.40 mol ethanol mol−1) when Zwf and Gnd, the key enzymes of the PP pathway, were overexpressed. However, due to the active expression of pfkB which encodes for the isozyme of PfkA, up to 30% of glucose was metabolized through the EMP pathway in the ΔpfkA strains and substantial amount of acetate was produced (~0.15 mol mol−1). On the other hand, the Δpgi strains led us to understand the metabolic hurdles in achieving theoretical maximum yield. In addition, the usage of efficient Zwf and Gnd enzymes in the Δpgi strains led us to successfully achieve the theoretical maximum yield of H2 and ethanol (~1.6 mol mol−1 each).
The E. coliΔpgi mutant could grow on glucose under anaerobic conditions when Zwf was overexpressed, but when both Zwf and Gnd were overexpressed, diversion of the carbon flux through the PP pathway and efficient co-production of H2 and ethanol were possible. Operation of the PP pathway as the sole glycolytic route for glucose under anaerobic conditions, however, was not possible, due to the redox imbalance. When the EMP pathway was blocked by pgi deletion, there existed, for the ED pathway, a critical flux ratio (0.14) above which cell growth was possible. The flux distribution between the PP and ED pathways at the 6-phosphogluconate node, and the co-production yield of H2 and ethanol, were determined by the characteristics of Zwf and Gnd. When zwf from L. mesenteroides and gnd from G. oxydans, both of which use NAD+ and NADP+ as cofactors and are less inhibited by NADPH, were employed, the best co-production yield of H2 and ethanol (1.74 mol H2 mol−1 glucose; 1.62 mol ethanol mol−1 glucose), close to the theoretical maximum values (1.67 mol mol−1 glucose for each), resulted. Activation of the PP pathway, as presented in this work, will be found to be useful for developing efficient biocatalysts for other biofuels and biochemicals that require additional reducing power and need to be produced under anaerobic conditions.
- CO2 :
- H2 :
- OD600 :
BSS, ES, and SP designed the research. BSS and ES performed the experiments and wrote the manuscript. The manuscript was revised and critical comments were provided by SP. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This research was supported by C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016M3D3A1A01913248). This work was also supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT & Future Planning (ABC-2011-0031361). The authors are grateful also to the BK21 Plus program at Pusan National University.
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