Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery
© The Author(s) 2017
Received: 30 December 2016
Accepted: 22 March 2017
Published: 28 March 2017
Citrobacter amalonaticus Y19 is a good biocatalyst for production of hydrogen (H2) from oxidation of carbon monoxide (CO) via the so-called water–gas-shift reaction (WGSR). It has a high H2-production activity (23.83 mmol H2 g−1 cell h−1) from CO, and can grow well to a high density on various sugars. However, its H2-production activity is expressed only when CO is present as an inducer and in the absence of glucose.
In order to avoid dependency on CO and glucose, in the present study, the native CO-inducible promoters of WGSR operons (CO dehydrogenase, CODH, and CODH-dependent hydrogenase, CO-hyd) in Y19 were carefully analyzed and replaced with strong and constitutive promoters screened from Y19. One engineered strain (Y19-PR1), selected from three positive ones after screening ~10,000 colonies, showed a similar CO-dependent H2-production activity to that of wild-type Y19, without being affected by glucose and/or CO. Compared with wild-type Y19, transcription of the CODH operon in Y19-PR1 increased 1.5-fold, although that of the CO-hyd operon remained at a similar level. To enhance the activity of CO-Hyd in Y19-PR1, further modifications, including an increase in gene copy number and engineering of the 5′ untranslated region, were attempted, but without success.
Convenient recombinant Y19-PR1 that expresses CO-dependent H2-production activity without being limited by CO and glucose was obtained.
KeywordsCitrobacter amalonaticus Water–gas-shift reaction CODH CO-Hyd narG gapA
Although several microorganisms perform the WGSR, their use is restricted due to various challenges encountered in cell growth and biocatalytic activity. Most of these microorganisms require high temperatures and lavish nutrients (e.g., hyperthermophilic bacteria such as Thermococcus onnurineus and Morella thermoacetica) or sunlight (e.g., photosynthetic bacteria such as Rhodospirillum rubrum, Rubrivivax gelatinosa, and Rhodopseudomonas palustris) for growth and expression of WGSR enzymes [2–4]. Moreover, they usually grow only slowly and to a low cell density. Our laboratory strain, a chemotrophic Enterobacter, Citrobacter amalonaticus Y19, not only can perform WGSR but also grows rapidly to a high cell density on diverse and cheap carbon sources .
The purpose of this study was to engineer C. amalonaticus Y19 strains for their best WGSR use. Specifically, the use of glucose as the carbon source and the removal of CO dependency for CODH and CO-Hyd expression were targeted. To this end, the regulatory region of the operons for the two-enzyme complexes was carefully analyzed and replaced by several constitutive promoters screened from Y19. Furthermore, to improve CO-linked H2-production activity, overexpression of non-membranous CO-Hyd genes at the plasmid level and chromosomal 5′UTR modification of membraneous CO-hyd genes were also attempted. The newly developed recombinant C. amalonaticus Y19 could grow on glucose and express CO-dependent H2-production activity constitutively, based on which results it was considered to be a highly convenient biocatalyst for the WGSR.
Construction of recombinant strains
Bacterial strains and plasmids used in this study
Strains and plasmids
Genotype and description
Citrobacter amalonaticus Y19; Ampr
Y19 with chromosomal replacement of native coo promoters with Pgap and Pnar*
Y19-PR1 with UTR1(threefold) cooM
Y19-PR1 with UTR1(6.5-fold) cooM
Y19-PR1 harboring pHyd-CO plasmid
Y19-PR2 harboring pHyd-CO plasmid
Y19-PR3 harboring pHyd-CO plasmid
E. coli DH5 alpha
pUCPK/Pgap or Pnar*
ColE1 ori, green florescent protein under the control of Pgap or Pnar*
P15A ori; Cmr
pDK7 p15A Pnar* cooKLXUH
To verify the strengths of the selected native promoters (Pgap and Pnar*) in C. amalonaticus Y19, about 300 bp of intergenic regions were PCR-amplified and cloned under green fluorescent protein (GFP) as a fluorescent marker.
Citrobacter amalonaticus Y19 was cultivated in modified M9 medium fortified with potassium phosphate buffer (100 mM; pH 7.0). The medium contained the following constituents: 1.0 g/L MgSO4·7H2O, 1.0 g/L NaCl, 1.0 g/L NH4Cl, and 3.0 g/L yeast extract. Maltose and glucose were used as the sole carbon sources at 5 g/L, respectively, wherever indicated. l-Cysteine (1.0 mM), sodium selenate (2 μM), sodium molybdate (2 μM), NiCl2 (10 μM), and FeSO4 (25 μM) were added to the culture medium as essential micronutrients supportive of cell growth. All the experiments were performed in 165 mL serum bottles (working volume, 50 mL) at 30 °C. The bottles were sealed with a butyl rubber septum and aluminum cap before inoculating the seed culture. The bottles were flushed with argon (Ar) gas (99.9%) for 10 min to ensure O2 deprivation. Unless stated otherwise, the culture head space contained an Ar/CO (80:20, v/v) mixture.
Measurement of whole-cell and crude-cell extract enzymatic activities
The enzymatic activities of the whole-cell or crude-cell lysates were examined as described previously . Briefly, the whole-cell activities were measured with cells harvested during the late exponential growth phase, washed twice with MOPS buffer (pH 7.0) and then resuspended in the same buffer. The cell suspensions were placed in a 9.5 mL serum bottle at 0.6–0.8 OD600 and charged with an Ar/CO (80:20, v/v) gas mixture to initiate CO-dependent H2 evolution. The activities of CODH and the hydrogenases (uptake and evolving hydrogenases) were measured using crude-cell lysates. The lysates were prepared by harvesting cells during the late exponential growth phase, washed twice with 100 mM cold phosphate buffer, and resuspended in 50 mM Tris–HCl buffer (pH 7.3, buffer A) containing 2 mM dithiothreitol and 1 mM sodium dithionate. The cells were disrupted using a bead beater (Fastprep FP120, Obiogene Inc., USA) following the standard protocols. The enzymes were assayed at 30 °C in either 9.5 mL serum bottles or 4 mL cuvettes under anoxic conditions. The CODH activity was determined by methyl viologen-dependent CO oxidation, as described previously . The reaction mixture, containing 15 mM of MV, 2 mM of SDT, and 1 mM of EDTA in 3[N-morpholino] propanesulfonic acid (100 mM MOPS; pH 7.0) buffer, was introduced into an anaerobic 10 mm quartz cuvette and bubbled with pure CO for 5 min, after which a sufficient amount of enzyme solution (0.6–0.8 mg mL−1) was added to initiate the reaction. The reduction of MV was monitored at 578 nm using a double-beam spectrophotometer (Lambda 20, Perkin Elmer, USA). For determination of the uptake hydrogenase activity, the oxidized form of MV was used as an electron acceptor, and the MV reduction was measured colorimetrically at 578 nm. The reaction mixture containing buffer A and the electron acceptor (MV) at 2 mM was equilibrated with 100% H2. The molar extinction coefficient ε578 for the reduced methyl viologen was 9.7 mM−1 cm−1. To assess the H2-formation activity, the reduced MV was used as an electron donor, and the MV-dependent H2 evolution was measured in the gas phase by gas chromatography.
For GFP-fluorescence measurements, 200 µL of culture was immediately chilled on ice and then measured (λ ex = 485 nm; λ em = 515 nm) on a Perkin Elmer/Wallac Victor 2 Multilabel Counter (1420-011). A non-GFP-producing C. amalonaticus Y19 culture was used as a blank for the fluorescence measurements.
Wild-type and recombinant Y19 were cultivated in a modified M9 medium under anaerobic conditions at 30 °C and agitated at 250 rpm in an orbital incubator shaker. The cells were harvested during the stationary growth phase. Approximately 2 × 108 cells were collected in vials containing two volumes of RNA protect reagent (Qiagen Inc., USA). The culture suspension was centrifuged at 10,000 rpm for 5 min. Pellets were applied for total RNA extraction using a total RNA isolation kit (Macherey–Nagel, Germany). Two micrograms of total RNA was used to synthesize the first-strand cDNA in a 20 µL reaction utilizing a SuperScript III first-strand synthesis system (Invitrogen, USA). Real-time PCR analysis was performed in a 20 µL reaction volume using the SYBR Green method with the StepOne Real-Time PCR system (Applied Biosystems, USA). Each 20 µL sample of the reaction mixture contained 300 ng of cDNA, 10 µL of 2× Power SYBR Green PCR Master Mix (Applied Biosystems, UK), 5 pmol of forward and reverse primers, and DEPC-treated water. The thermal cycling conditions were as follows: denaturation, 1 cycle of 95 °C for 30 s; amplification, 40 cycles of 95 °C for 15 s, 62 °C for 30 s, and 72 °C for 30 s. The PCR efficiencies of all the primers were determined experimentally and found to be suitable for reliable copy-number quantification. The relative quantification of the mRNA levels was calculated using the ΔΔCT method described previously [10, 11]. All the assays were performed in duplicate, and the reaction without a template was used as the negative control.
Bacterial growth was measured by spectrophotometry (Lamda 20, Perkin Elmer, USA) at 600 nm. The protein content was measured using the Bradford method with bovine serum albumin as the standard. The H2 and CO contents were quantified by gas chromatography (DS 6200; Doman Inst. Inc., Korea) equipped with a Thermal Conductivity Detector, utilizing stainless steel columns packed with Molecular Sieve 5A (for H2; Alltech Deerfield, IL, USA). The injector, column oven, and detector temperatures were 90, 80, and 120 °C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL min−1. Organic acid and alcohol analyses were carried out by HPLC (1100 series Agilent Technologies Foster, CA, USA).
Results and discussion
Effect of glucose on transcription of CO-dependent H2-production enzymes
According to a previous study , the WGSR activity of Y19, measured in both whole-cells and broken-cell extract, was negligible when the cells were grown on glucose. To confirm the mechanism, the transcription of a few selected genes in the two CO-responsible operons, CODH and CO-hyd, were analyzed in the wild-type C. amalonaticus Y19 (Fig. 1). The cells were cultured on either glucose or maltose, in the presence and absence of CO. Even with CO, wild-type Y19 grown on glucose showed a significantly lower transcription for the CODH (hypC, cooF, cooS) and CO-hyd (cooM, cooH) genes compared with the cells grown on maltose. Without CO, expression of all coo operon genes was low and, furthermore, not much difference between the two carbon sources was noticed. The highest transcription was exhibited when the cells were grown on maltose with CO. These results indicate that expression of the CODH and CO-hyd operons is under dual control at the transcriptional level by CO and the carbon source (CCR). Interestingly, the fold difference between the two carbon sources in the presence of CO was only 3–10, much smaller than that by CO (~30, grown on maltose) or that found in the well-known lac operon (~100) .
Transcription of the CODH genes gradually decreased at locations farther from the promoter site, due possibly to the polarity effect. By contrast, two genes of the CO-hyd operon, cooM, and cooH, though distantly located in the operon, did not show such difference. We also noticed that the CODH genes, hypC and cooF, were highly transcribed compared with cooM and cooH.
Analysis of CO-inducible promoter region
Selection of native constitutive promoters
The Pgap and Pnar* promoters were examined using GFP as a reporter (see Additional file 2: Figure S1). Non-mutated native Pnar, as a reference, was also tested. After transformation with recombinant plasmids expressing GFP under the control of each promoter (Pgap, Pnar* and Pnar), C. amalonaticus Y19 was cultured to 1.6 O.D. and measured for fluorescence. The specific fluorescences were (AU, arbitrary units): 5.7 × 104 AU/OD for Pgap, 4.3 × 104 AU/OD for Pnar*, and 5.3 × 103 AU/OD for Pnar. The strength of Pnar* was about 75% that of Pgap, or about eightfold higher than that of Pnar; thus, Pnar* was chosen as a suitable promoter for the CO-hyd operon. As in the case of E. coli, mutation in Pnar greatly improved its strength and, subsequently, GFP fluorescence in C. amalonaticus Y19.
Construction of Y19-PR1 and constitutive transcription of CODH and CO-hyd
Characterization of promoter-engineered strain Y19-PR1
With glucose as the carbon source (Fig. 6), Y19-PR1 also showed better cell growth and H2 production than did wild-type Y19. The negative effect of CO on cell growth was similar to that when cells were grown on maltose (Fig. 5). In wild-type Y19 growing on glucose, CO was marginally utilized and H2 production was not increased by CO (Fig. 6a, b). By contrast, in Y19-PR1, CO was actively metabolized, and more H2 was accumulated when CO was added. This confirmed that the expression of the CODH and CO-hyd operons in Y19-PR1 is free from CCR. The growth advantage of Y19-PR1 over wild-type Y19 can be well appreciated by comparing Figs. 5b and 6c. Under these different but WGSR-activating growth conditions, the recombinant Y19-PR1 showed, compared with the wild type, an ~30% higher specific cell growth rate and >40%-improved cell density after 9 h cultivation.
Specific activities in whole-cells and broken-cell extracts
Whole-cells CO-dependent H2 production (mmol g−1 h−1)
Formate-dependent H2 production (mmol g−1 h−1)
Crude-cell lysates CODH (μmol min−1 mg−1)
Evolving hydrogenase (μmol min−1 mg−1)
Uptake hydrogenase (μmol min−1 mg−1)
Cultivated on maltose
Y19 WT (no CO)
16.2 ± 0.7
1.6 ± 0.1
9.8 ± 0.5
Y19 WT (20% CO)
22.1 ± 1.1
12.0 ± 0.6
32.1 ± 6.1
2.5 ± 0.2
7.9 ± 0.4
Y19-PR1 (no CO)
23.8 ± 1.2
15.2 ± 0.7
49.3 ± 6.7
2.8 ± 0.2
8.9 ± 0.5
Y19-PR1 (20% CO)
22.9 ± 1.2
10.7 ± 0.5
45.6 ± 6.7
2.3 ± 0.1
8.2 ± 0.4
Cultivated on glucose
Y19 WT (no CO)
18.2 ± 0.8
1.9 ± 0.2
12.4 ± 0.7
Y19 WT (20% CO)
3.7 ± 0.2
14.0 ± 0.7
2.1 ± 0.1
3.2 ± 0.1
9.1 ± 0.4
Y19-PR1 (no CO)
19.8 ± 0.9
17.2 ± 0.9
36.3 ± 1.8
3.6 ± 0.2
11.9 ± 0.6
Y19-PR1 (20% CO)
20.7 ± 1.1
15.3 ± 0.7
41.9 ± 2.7
3.3 ± 0.2
8.9 ± 0.4
Efforts to improve CO-dependent H2 production
Despite successful construction of Y19-PR1 strain, its CO-linked H2-production activity was not improved relative to that of its wild-type counterpart. According to RT-PCR analyses (Fig. 4) and activity measurement (Table 2), the expression and enzymatic activity of CO-Hyd were not improved. We speculated that CO-Hyd activity is rate limiting, and thus we decided to improve its expression in Y19-PR1.
Two additional recombinants were constructed. First, the five genes of the CO-hyd operon, cooKLXUH, were homologously overexpressed, episomally, using the low-copy plasmid pDK7-p15A (under the control of Pnar* promoter pHyd-CO), while the cooM gene was moderately up-regulated from the chromosome by engineering of 5′UTR (the untranslated region). The CooM is a large (3789 bp) and membrane-embedded protein with 36 trans-membrane helices (as predicted by the TMPred tool ); as such, there has been concern that its overexpression is potentially fatal to cell viability. Various synthetic 5′UTR (including RBS) were designed for CooM using the UTR Library Designer (Additional file 4: Table S3) [21, 22]; two of them, which were expected to provide ~threefold (designated as ‘PR2′) and ~sevenfold (designated as ‘PR3’) higher translations relative to those of PR1 (where the Pnar* promoter with native RBS was employed), respectively, were chosen for further studies. When analyzed by GFP, the new RBS showed proper strength: 8.7 × 104 AU with PR2 and 1.5 × 105 AU with PR3, respectively. This corresponds to ~2.1- and ~4.0-fold higher expressions, respectively, relative to that of PR1. The new promoters with improved 5′UTR were integrated into the chromosome of Y19-PR1 to replace the Pnar* promoter using the pKOV system, and two new host strains, Y19-PR2 and Y19-PR3, were developed. Then, to these two new hosts and the original Y19-PR1 strain, the recombinant plasmid containing cooKLXUH genes (pHyd-CO) was introduced. Expression of the CO-hyd subunits was analyzed in the new recombinant strains on SDS-PAGE after growing them on both maltose and glucose (Additional file 5: Figure S2). In the Y19-PR2/pHyd-CO and Y19-PR3/pHyd-CO strains, CooM (136 kDa), CooK (33.9 kDa) and CooH (40.2 kDa) were detectable in insoluble fractions (due to their membranous nature), whereas neither CooX (22.1 kDa) nor CooU (19.4 kDa) nor CooL (15.5 kDa) was detected in either soluble or insoluble fractions. In the Y19-PR1/pHyd-CO strain, CooK (33.9 kDa) and CooH (40.2 kDa), but not CooM (136 kDa), were detectable in insoluble fractions. In wild-type Y19 without pHyd-CO, no protein band corresponding to any of the CO-hyd genes was evident.
The three recombinant Y19 were cultured and their CO-dependent H2 production activity were measured (Additional file 6: Table S4). New hosts Y19-PR2 and Y19-PR3 and their recombinants containing pHyd-CO showed defect in cell growth. The hosts Y19-PR2 and Y19-PR3 overexpressing the membrane-embedded protein CooM (Y19-PR2 and Y19-PR3) showed up to 23% reduced cell growth relative to the Y19-PR1 host (Additional file 4: Table S3). When pHyd-CO was introduced, Y19-PR2/pHyd-CO and Y19-PR3/pHyd-CO, even under the best conditions (on glucose without CO), grew to only 1.04 and 0.81 OD600, respectively, by 12 h (vs. 1.7 OD600 for Y19-PR1). Furthermore, the whole-cell CO-dependent H2-production activities of Y19-PR2/pHyd-CO and Y19-PR3/pHyd-CO were greatly reduced to 16.5 and 15.3 mmol H2 g−1 cell h−1, respectively, which levels were 28 and 33% lower, respectively, than that of Y19-PR1. On the other hand, although Y19-PR1/pHyd-CO did not show any problems in its cell growth characteristics (relative to those of Y19 WT), its CO-dependent H2 production capabilities were not enhanced. This indicates that improvement of CO-Hyd activity is highly challenging, and cannot be achieved simply by overexpression of the CO-hyd operon.
In another effort to improve CO-Hyd activity, the increase of the free membrane space enabling accommodation of additional membrane proteins such as CooM was attempted (Additional file 7: Text S1, Additional file 8: Figure S3, Additional file 9: Table S5). The inner membrane protein, PS003556, which was considered non-essential for cell growth but was expressed at a high level (according to RT-PCR), was removed from Y19-PR2, and the strain was examined for cell growth and H2 production capability both as is and after pHyd-CO introduction. No improvement in cell growth or H2 production capability in the host or its recombinant was observed relative to Y19-PR2 or its recombinant. These experiments indicated that improvement of CO-Hyd activity is highly challenging and will require further and more intensive investigation.
For faster cell growth on glucose and convenient expression of CO-linked H2 production activity, the promoter-replaced strain C. amalonaticus Y19-PR1 was developed. The engineered Y19-PR1, when grown on glucose, could constitutively express CO-linked H2-production activity at a high level without dependence on CO. However, the CO-linked H2-production activity in Y19-PR1 was not higher than that in the wild-type counterpart. Some efforts to improve CO-dependent H2-production activity, including overexpression of CO-Hyd enzymes and removal of an unnecessary membrane protein, were attempted but were not successful. Further studies to uncover the unique regulatory mechanisms of CODH and CO-Hyd expression at the molecular level and further improve the expression of CO-Hyd are under way.
carbon monoxide dehydrogenase
carbon monoxide-dependent hydrogenase
formate hydrogen lyase
SKA and SP designed the research, and SK performed the experiments. SKA, ES, JRK, and SP analyzed the data and contributed to valuable discussions. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This study was supported by a grant from the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016M3D3A1A01913248). Additionally, the authors are grateful for the financial assistance from the Brain Korea 21 Plus Program for Advanced Chemical Technology at PNU.
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