Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase
© Kim et al; licensee BioMed Central Ltd. 2012
Received: 22 December 2011
Accepted: 4 January 2012
Published: 4 January 2012
Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions.
Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%.
Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.
Keywordsbiohydrogen Escherichia coli proteorhodopsin light-driven proton pump light-powered cell factory
Since the Industrial Revolution, energy consumption has increased exponentially and most energy has been derived from fossil fuels. Currently, we still depend on fossil fuels for more than 80 percent of our demands for electricity, transportation, and industries, although concerns about the exhaustion of fossil fuels and global warming have led to increased attention to renewable energy . Among various renewable energy sources, solar energy is the most abundant and ultimate source. The total amount of solar energy absorbed by the Earth's surface is 1.74 × 105 terawatts (TW) , which is a tremendous amount compared to the world's energy consumption (~13 TW) . Thus, the conversion of solar energy to fuels may constitue the most sustainable way to solve the energy crisis.
In the field of biotechnology, the photosynthetic process in algae and cyanobacteria has been actively investigated for the conversion of solar energy to useful biofuels [3–5]. Photosynthesis requires a highly complex photosystem composed of numerous proteins and photosynthetic enzymes, such as Rubisco . In addition, many challenges still remain for engineering photosynthetic microorganisms [1, 6]. Recently, a new type of rhodopsin, called proteorhodopsin, was discovered in the metagenome of uncultured marine γ-proteobacteria . Proteorhodopsin can be heterologously expressed in Escherichia coli to possess proton-pumping activity , which is different from bacteriorhodopsin found in halobacteria [1, 8]. This property of proteorhodopsin enables the investigation of its impact on cellular energy and phototrophy . There have also been reports of the enhancement of cell viability or growth via light-driven proton pumping by proteorhodopsin under nutrient-limited conditions [9–11]. However, there have been no substantial applications in biofuel production using proteorhodopsin, although this potential has been mentioned recently .
Hydrogen (H2) has been recognized as one promising alternative energy source to fossil fuels. It does not emit carbon dioxide during combustion and can be easily converted to electricity using fuel cells. In addition, it has a higher energy density than other energy sources. Although the current production of H2 mainly depends on thermochemical methods using fossil fuels , biological approaches have been actively investigated to generate H2 in a more sustainable manner [13–17]. Among them, photobiological H2 production has attracted great attention due to its eco-friendly properties, such as its usage of solar energy and carbon assimilation. Nevertheless, there are still many obstacles to overcome, including slow cell growth, the low conversion efficiency of light to H2, the inhibitory effect of oxygen on hydrogenase activity, and others [16, 17].
E. coli has been used widely as a cell factory for many types of bio-products (including biofuels), but it cannot utilize light energy. Therefore, constructing E. coli capable of absorbing light energy and converting it to other biofuels through the introduction of proteorhodopsin might increase biofuel production efficiency. It has been shown that protons generated by rhodopsin can migrate along the membrane surface  and thus, they can act as substrates of hydrogenase for H2 evolution. Thus, in the present work, for the first time (to our knowledge), we introduced proteorhodopsin into E. coli, generating bacteria capable of utilizing light and investigated its effect on H2 production yield using the previously constructed recombinant E. coli expressing Hydrogenovibrio marinus-originated [NiFe]-hydrogenase .
Functional expression of proteorhodopsin in E. coli
Co-expression effect of proteorhodopsin and hydrogenase on H2 production
Light intensity effect on H2 production by co-expression of proteorhodopsin and hydrogenase
We observed functional expression of proteorhodopsin in the recombinant E. coli BL21(DE3) and thus investigated the effect of co-expressing proteorhodopsin and H. marinus [NiFe]-hydrogenase on H2 production. Using a single plasmid system expressing both hydrogenase and proteorhodopsin without biosynthesis of endogenous retinal, we found that H2 production was improved by ~29% in the presence of added retinal under light. This indicates that proteorhodopsin function, gained with exogenous retinal, provided a synergistic effect on H2 production through the supplementation of protons. Similarly to the single plasmid system, we also observed a positive effect of proteorhodopsin using a dual plasmid system. H2 production levels of cells co-expressing hydrogenase, proteorhodopsin, and retinal synthesis proteins was ~1.3-fold higher at the final time point than that of the cells expressing only hydrogenase. It is noteworthy that H2 production from the strain co-expressing proteorhodopsin and hydrogenase quickly surpassed H2 production from the strain expressing only hydrogenase at a late phase. This retarded H2 production profile of the dual plasmid system might be attributed to the metabolic burden caused by the over-expression of multiple proteins required for the biosynthesis of retinal and proteorhodopsin.
Because the function of proteorhodopsin is driven by the absorption of light energy, light intensity can be a key factor for H2 production. We found that the H2 production from cells co-expressing hydrogenase and proteorhodopsin with endogenous retinal is strongly dependent on light intensity. Increasing the light intensity from 70 μmole photon/(m2·s) to 130 μmole photon/(m2·s) increased H2 production yield ~1.8-fold. However, cell growth was not different in the two cultures under different light conditions. This tendency was consistent with a previous report that proteorhodopsin contributes to cell growth only under nutrient-limited conditions . Thus, this indicates that H2 production was improved by the absorption of enhanced light energy through the proton-pumping function of proteorhodopsin. In the present study, it seems that the reduction from proton to H2 mediated by hydrogenase generates additional proton gradient, driving proteorhodopsin to pump protons, the substrates for hydrogenase. When we calculated the production rate per culture volume (21.9 mL H2/(L-culture·h)) and the conversion efficiency of light energy to H2 (~3.4%), the levels achieved in this study were comparable to the results of photobiological hydrogen production in previous studies: H2 production rate in green algae, 0.048-4.48 mL H2/(L-culture·h), cyanobacteria, 4.03-13 mL H2/(L-culture·h), photosynthetic bacteria, 7.6-131 mL H2/(L-culture·h), and light conversion efficiency in photoautotrophs, 3-10% with removal of O2 or 1-2%, and photoheterotrophs, 0.308-9.23%) [17, 19]. Although cell growth was mainly supported by exogenous nutrients in this system, these results are quite meaningful for the development of an E. coli system that can utilize light energy as a supplementary source.
Here, we demonstrated the substantial application of proteorhodopsin for light-driven biohydrogen production in a recombinant E. coli system. E. coli engineered to express H. marinus [NiFe]-hydrogenase and proteorhodopsin produced more H2 with the existence of retinal under light conditions. Engineered strains also produced more H2 as light intensity increased, although there was no difference in cell growth. These results suggest that our system works for converting light energy to H2 via the cooperation of proteorhodopsin and hydrogenase. In addition, engineering E. coli as light-powered cell factories could provide a solution for developing potential strategies for photobiological H2 production.
Bacterial strains and culture conditions
E. coli Top10 (Invitrogen, USA) was used for the manipulation and cloning of target genes. E. coli BL21(DE3) (Novagen, USA) was used for expression of recombinant proteins. We used LB medium including proper antibiotics (50 μg/mL ampicillin and 30 μg/mL chloramphenicol) for genetic manipulation. For protein expression, 1 mM (as a final concentration) of isopropyl-β-D-thiogalactopyranoside (IPTG; BioBasic, Canada) was added to each culture medium. For in vivo H2 production, cells were grown in M9 minimal medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, and 1 mg/L thiamine supplemented with 240.73 mg/L MgSO4, 11.09 mg/L CaCl2) including 5 g/L casamino acids and 5 g/L glucose. 0.1 M of FeSO4 and 1 M of NiSO4 solutions were added to M9 medium at a concentration of 30 μM for the functional expression of H. marinus [NiFe]-hydrogenase. All cultures were maintained under normal aerobic or micro-aerobic conditions . Cells were grown in serum bottles (125 or 500 mL; Wheaton, USA) sealed with rubber stoppers and aluminum capping at 37°C in an air-shaking incubator (Jeiotech, Korea) at a gyration rate of 200-230 rpm. For the assessment of the functional activity of proteorhodopsin, cells were irradiated by 20 W or 30 W fluorescent lamps. The light intensity (400-700 nm) at a given location in the culture was measured using a light meter (Apogee, USA) in units of μmol photon/(m2·s).
Primer sequences for amplification of genes related to β-carotene synthesis, retinal synthesis, and proteorhodopsin
Sequence (5'→3') (___: ribosome binding site,:___restriction site)
CCGAATTCAAGGAGATATACCA ATGAATAATCCGTCGTTACT CAATCATGC
CCGTCGACAAGGAGATATACAA ATGAAACCAACTACGGTAAT TG
CCAAGCTTGAAGGAGATATACCA ATGCAACCGCACTATGATC TGATTCTC
GGAGATCTAAGGAGATATACAT ATGGAGATAATATTTGGCCA GAATAAG
CCGGTACCAAGGAGATATACAA ATGGGTAAATTATTACTGAT ATTAGGTAG
CCGCGGCCGCAAGGAGATATACAA ATGGGTAAATTATTACTGAT ATTAGGTAG
Measurement of In vivo H2production
H2 gas produced in cell culture was obtained from the headspace of a sealed serum bottle (125 or 500 mL). Usually, 20-100 μL of the gas sample was analyzed using a gas chromatograph (GC; Younglin Instrument, Korea) equipped with a carboxen-1010 PLOT column (0.53 mm × 30 m, Supelco, USA) and pulsed discharge detector (Valco Instrument, USA). Elution was performed using helium as a carrier gas at a flow rate of 10 mL/min, and the temperatures of the injector, detector, and oven were set to 130, 250, and 100°C, respectively. The H2 concentration in the gas sample was calculated using a standard curve. The H2 amount was determined based on the H2 concentration and gas volume of headspace (including expanded volume).
Measurement of absorption spectra of proteorhodopsin
The absorption spectrum of cells expressing proteorhodopsin was measured using spectrophotometry . To prepare crude membrane fractions, recombinant E. coli were harvested by centrifugation at 4°C and 4,000 rpm for 15 min. The cell pellet was resuspended in 50 mM Tris-Cl (pH 6.8) and disrupted with a sonic dismembrator (Fisher Scientific, USA) for 10 min at 50% power (5 sec pulse on and 2 sec pulse off). The disrupted cell suspension (total cell lysate) was centrifuged at 4°C and 10,000 g for 20 min. The resultant supernatant (crude extract) was centrifuged at 4°C and 120,000 g for 120 min. The pellet was resuspended in 50 mM Tris-Cl (pH 8.0) and 5 mM MgCl2, which is regarded as the crude membrane. The absorption spectrum was measured at spectrum mode using a spectrophotometer (Shimadzu, Japan) after the addition of 20 μM of all-trans-retinal (Sigma).
Conversion efficiency of light energy to H2
The conversion efficiency of light energy to H2 was calculated using the following equation: conversion efficiency (%) = (H2 production amount × H2 energy content)/absorbed light energy × 100, where H2 energy content = 10.8 kJ/L (lower heating value of H2) . Absorbed light energy was calculated using the equation absorbed light energy (kJ/s) = 0.2176 IoSA, where Io = incident light intensity (μmol/(m2·s) measured by light meter, and SA = illuminated surface area (m2) = π × d × h (d = 0.075 m, h = 0.1 m for the culture in a 500 mL serum bottle) .
This work was supported by the Manpower Development Program for Marine Energy funded by the Ministry of Land, Transport and Maritime Affairs, Korea and the National Research Foundation Grant (NRF-2009-0093214) and the Brain Korea 21 Program funded by the Ministry of Education, Science and Technology, Korea. We thank Dr. Johannes von Lintig (Case Western Reserve Univ.) and Dr. Edward DeLong (MIT) for their gifts of plasmids.
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