Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

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Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli Balaji Sundara Sekar a, Subramanian Mohan Raj b, Eunhee Seol a, Satish Kumar Ainala a, Jungeun Lee a, Sunghoon Park a,* a

Department of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, Republic of Korea b Centre for Research and Development, PRIST University, Thanjavur, TN 613 403, India

article info

abstract

Article history:

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized

Received 18 April 2014

for the production of hydrogen (H2). CO-dependent H2 production is catalyzed by a unique

Received in revised form

enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-

16 July 2014

dependent hydrogenase (COeH2ase), both of which contain metal cluster(s). In this

Accepted 27 July 2014

study, CODH and the required maturation proteins from the novel facultative anaerobic

Available online xxx

bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like

Keywords:

protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E.

Carbon monoxide dehydrogenase

coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein
1)

Citrobacter amalonaticus Y19

compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein
1). Purified heterologous CODH

Wateregas shift reaction

from the soluble cell-free extract of the recombinant E. coli showed a specific activity of

Recombinant Escherichia coli

170.6 U mg protein
1. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 COeH2ase for CO-dependent H2 production. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Hydrogen (H2) is considered an important alternative fuel of the future due to its high energy content and non-polluting nature. Much research has been conducted to develop biological H2 production technologies using different carbon substrates [1e6]. Some microorganisms utilize toxic carbon monoxide (CO) as a carbon and energy source and produce H2 by a water-gas shift reaction (WGSR); CO þ H2O 4 CO2 þ H2

(DG ¼
20 kJ mol
1) [7]. The key enzymes involved in a WGSR are CO dehydrogenase (CODH) and CO-dependent hydrogenase (COeH2ase), which are known to form a membraneebound complex in the cell. CODH oxidizes CO to CO2 and produces protons and electrons, while COeH2ase generate H2 by reducing protons using the electrons transferred from CODH [8]. The production of H2 through the WGSR has been well documented in photoheterotrophic bacteria such as Rhodospirillum rubrum [9], thermophilic bacteria including

* Corresponding author. Tel.: þ82 51 510 2395; fax: þ82 51 510 2716. E-mail address: [email protected] (S. Park). http://dx.doi.org/10.1016/j.ijhydene.2014.07.148 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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Carboxydothermus hydrogenoformans [10], and thermophilic archaea such as Thermococcus onnurineus NA1 [11]. These microorganisms generally grow slowly and/or require either light or high temperatures, which limit their industrial application for H2 production. Most of these limitations can be overcome if a recombinant Escherichia coli expressing CODH and COeH2ase is developed. However, this development is challenging because CODH and COeH2ase are multi-subunit, membrane-associated or membrane-embedded enzymes. Furthermore, both enzymes and most of their subunits contain complex metal clusters, and their functional expression, particularly the synthesis of metal clusters and their incorporation into apo proteins, require long and exotic pathways that have not been fully elucidated yet [12]. Thus far, few studies on the heterologous expression of CODH, including CODH II (involved in NADPþ regeneration) of C. hydrogenoformans [13,14], have been reported in the literature. Previously, we have isolated a non-photosynthetic, facultative anaerobic bacterium Citrobacter amalonaticus Y19 that can perform the WGSR [15]. According to genome analysis, C. amalonaticus Y19 had the coo regulon, which encompasses the CODH and COeH2ase gene clusters [16]. Y19 was similar to E. coli in many aspects; it grew quickly to high cell density under both aerobic and anaerobic conditions, could utilize many carbon sources such as glucose, maltose, sucrose, etc. for growth, and produced H2 through formate-hydrogen lyase (FHL) and WGSR. Furthermore, the 16S rDNA gene sequence of C. amalonaticus Y19 showed more than 90% similarity to that of E. coli. We hypothesized that, due to the similar nature of C. amalonaticus Y19 and E. coli, the CODH and COeH2ase in Y19 can be cloned and functionally expressed in E. coli rather easily. In an effort to establish the WGSR in E. coli, we first investigated the functional expression of the Y19 CODH in E. coli. The genes of the Y19 coo regulon were cloned in E. coli individually and in combination, and CODH activity was examined. In addition, with the CODH-expressing E. coli recombinant, CO-dependent H2 production was attempted. E. coli has the Ech-type H2ase 3 which is structurally similar to the Y19 COeH2ase [17e19], and thus it is expected that the recombinant E. coli, even without the expression of COeH2ase, produces H2 from CO oxidation if the Y19 CODH can form an active complex with the native Ech H2ase 3 in vivo. This study demonstrates that, among the six genes composing the two CODH operons, only cooF (ferredoxin-like protein) and cooS (CODH), no other maturation proteins, are needed for the functional expression of CODH in E. coli.

Materials and methods Bacterial strains, plasmids and chemicals E. coli BL21 (DE3) (Novagen, Hornsby, Australia) (designated as EB henceforth) and E. coli BW25113 (K-strain derivative) (designated as EW henceforth) strains were used for the expression of recombinant CODH and accessory proteins. pGEM-T (Promega, Fitchburg, Wisconsin, USA), pACYCDuet-1, pCDFDuet-1, pRSFDuet-1 (Novagen, Hornsby, Australia) and pBAD/Myc-His C (Life Technologies, Korea) vectors were used

for the cloning and expression of Y19 genes in E. coli. Luria Bertani broth and Terrific broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) were used to culture recombinant E. coli. Ampicillin (100 mg/L), streptomycin (50 mg/ L), chloramphenicol (25 mg/L), and kanamycin (50 mg/L) were used in culture media. Isopropyl-b-D-thiogalactopyranoside (IPTG) (BioBasic, Canada) or L-arabinose was used for the induction of recombinant protein expression. All other chemicals used in this study were purchased from SigmaeAldrich Co. unless stated otherwise.

Identification and analysis of coo operon The contig sequences of C. amalonaticus Y19 were analyzed, and open reading frames (ORFs) were identified using an inhouse automatic annotation system, Ensoltek [20]. The identified ORFs were annotated using the GenBank database, clusters of orthologous groups, gene ontology, and Pfam databases [21]. The contigs containing the Y19 CODH and COeH2ase gene clusters were deposited in GenBank (GenBank accession no. KJ150728).

Cloning of CODH genes in E. coli The bacterial strains and plasmids used in this study are presented in Table 1. The cooFS operon encoding a ferredoxinlike subunit and CODH were amplified from the genomic DNA of C. amalonaticus Y19 and cloned into the pACYCDuet-1 vector. The recombinant plasmid harboring cooF and cooS was transferred to E. coli BL21 (DE3), and the resulting transformant is referred to as EB-FS. To understand the role of the maturation proteins and their influence on CODH activity, the genes cooC, cooT, cooJ and hypB, which encode maturation proteins, were amplified from the genomic DNA of C. amalonaticus Y19. The genes cooC and cooT were cloned into the pCDFDuet-1 vector, while the genes cooJ and hypB were cloned into the pRSFDuet-1 vector. The plasmids harboring cooC, cooT, cooJ and hypB were transformed into the EB-FS strain and the resulting strain was named EB-FSCTJH. To determine whether native E. coli H2ases can form a complex with recombinant C. amalonaticus Y19 CODH, the genes cooF and cooS were sub-cloned in the pBAD Myc-His-C vector. E. coli BL21(DE3) do not produce H2 due to the absence of H2-producing metabolism [22], while the E. coli BW25113 strain does not express T7 RNA polymerase, so both cooF and cooS were cloned into the pBAD Myc-His-C vector under the araBAD promoter. To avoid H2 consumption and improve the expression of the H2ase 3, deletion mutants (SH1, SH2 and SH3) from our previous study [23] were used. The mutant strains lack the genes for HycA (repressor of FHL, which converts formate to H2 and CO2), HyaAB and HybBC (uptake hydrogenases that can reversibly oxidize H2 to protons).

Expression of CODH and gel electrophoresis All recombinant strains were grown anaerobically in sealed 125 mL serum bottles containing 50 mL of Terrific broth at 37  C and 200 rpm. Terrific broth was supplemented with 100 mM FeSO4, 20 mM NiCl2, and 2 mM L-cysteine. The cells were

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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Table 1 e List of strains and plasmids used in this study. Strains and plasmids Strains Y19 EB EB-F EB-S EB-FS EB-CT-JH EB-FS-CT-JH EB-pA-pC-pR EW EW-FS SH1-FS SH2-FS SH3-FS Plasmids pGEM-T pACYC-FS pBAD-F pBAD-S pCDF-CT pRSF-JH pBAD-FS

Description

Source

Citrobacter amalonaticus Y19 wild-type Escherichia coli BL21 (DE3) wild-type EB-pBAD-F EB-pBAD-S EB-pACYC-FS EB-pCDF-CT, pRSF-JH EB-pACYC-FS, pCDF-CT, pRSF-JH EB-pACYCDuet-1, pCDFDuet-1, pRSFDuet-1 Escherichia coli BW25113 wild-type EW-pBAD-FS EWDhycA-pBAD-FS SH1DhyaAB-pBAD-FS SH2DhybBC-pBAD-FS

Jung et al. (1999) Invitrogen This study This study This study This study This study This study CGSC This study This study This study This study

Cloning vector pACYCDuet-1-cooF-cooS; Cmr pBAD-cooF; Ampr pBAD-cooS; Ampr pCDFDuet-1-cooC-cooT; Srr pRSFDuet-1-cooJ-hypB; Knr pBAD-Myc-His-C-cooF-cooS; Ampr

Promega This study This study This study This study This study This study

shifted to 25  C at 0.4 ± 0.05 OD600 and induced at 0.6 ± 0.05 OD600 with 0.05 mM IPTG or 0.05% L-arabinose. Y19 was also cultured in the above mentioned medium at 30  C and induced at 0.6 ± 0.05 OD600 with 20% CO. All cultures were induced for 6 h, harvested anaerobically, and centrifuged at 5000  g for 10 min. The cell pellets were washed with 100 mM N-morpholino propane sulfonic acid (MOPS) buffer (pH 7.5) and disrupted using a bead beater (Fastprep FP120, Qbiogene Inc., USA) at a speed of 6.0 for 20s (5 cycles). Undisrupted cells were removed by centrifugation at 5000  g for 5 min. Soluble and particulate fractions were separated by centrifugation at 16800  g for 1 h. The soluble and particulate fractions were used for SDS-PAGE and determination of CODH activity after being suspended in MOPS buffer (pH 7.5). Protein expression was examined by subjecting the fractions to SDS
PAGE under denaturing and non-denaturing conditions [24]. Coomassie Brilliant Blue was used to stain proteins. Protein purification was performed anaerobically using Ni-NTA chromatography as described previously [25]. All preparations of cell fractions were performed under anaerobic conditions in an anaerobic chamber (Coy, USA).

sealed silicon rubber septa under anaerobic conditions. The reduction of MV was determined using a molar extinction coefficient (Dε578) of 9.7  10
3 M
1cm
1 [26]. One unit (U) of CODH activity was defined as the amount of enzyme required to reduce one mmol of MVox to MVred in one minute. All CODH activities were measured in triplicate. The CO-dependent H2 production assay was performed as described previously [19,27]. The recombinant EW strains and Y19 were harvested in late exponential phase, washed with 100 mM MOPS buffer (pH 7.0), and resuspended in the same buffer to 1.0 OD600. One ml of the cell suspension was added to 8 ml serum bottle and sealed. All steps were performed inside anaerobic chamber. The headspace was purged with Ar gas. CO at 20% was added to the headspace after removing equal volume of Ar gas. The measurement was conducted for 1 h while shaking the bottle at 30  C and 250 rpm in an orbital incubator shaker. Y19 and E. coli BW25113 wildtype were used as positive and negative controls, respectively. The gas in the headspace was measured using gas chromatography.

Activity assays

The recombinant EB strains and Y19 strain were induced with 0.05 mM IPTG and 20% CO, respectively, and harvested during the late exponential growth phase. RNA extraction and real time PCR was performed as mentioned in Zhou et al. [28]. Briefly, the cell pellets were added with RNAprotect reagent (Qiagen Inc., USA) to avoid RNA degradation and RNA was extracted using the Nucleospin® RNA isolation kit (MachereyNagel, Germany). The purified total RNA was quantified by spectrophotometer and used for cDNA synthesis. SuperScript III first-strand synthesis system (Invitrogen, USA) was used for cDNA synthesis. The PCR efficiencies of all primers were determined by performing PCR with the genomic DNA or recombinant plasmid before performing RT-PCR. RT-PCR analysis was performed in duplicates using SYBR Green method in

CODH activity was determined according to the method described by Kim et al. [26] with slight modifications. Briefly, the CODH assay was performed under anaerobic conditions in sealed cuvettes (4 mL) with 2 mL of the assay mixture containing 100 mM MOPS buffer (pH 7.5) and 20 mM oxidized methyl viologen (MVox). The solution was pre-reduced with 2 mM sodium dithionite. Pure CO was bubbled into the assay solution for 5 min. The reaction mixtures in sealed cuvettes and crude cell extracts were incubated separately at 30  C for 10 min to equilibrate the temperature. The reaction was initiated by injecting an appropriate amount of crude/purified enzyme into the cuvette containing reaction mixture through

RNA extraction and real-time PCR

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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a StepOne real-time PCR system (Applied Biosystems, USA). The relative quantification of the mRNA levels was calculated using the DDCT method.

Analytical methods Cell concentrations and enzyme activity were measured in a 10-mm path length cuvette using a double-beam spectrophotometer (Lambda 20, PerkinElmer; Norwalk, CT, USA). Protein concentrations in samples were determined by Bradford method [29] on a microtiter plate reader (1420, Wallac Victor2; PerkinElmer) using bovine serum albumin as a standard. Gas measurements were performed using gas chromatography (DS6200 Donam Systems Inc., Seoul, Korea).

Results and discussion CODH and COeH2ase of C. amalonaticus Y19 Genome sequencing of C. amalonaticus Y19 revealed the presence of genes for CO-dependent H2 production (Fig. 1). They were distributed in three operons, cooFS, cooCTJHypB, and cooMKLXUH. The cooFS operon encodes a ferredoxin-like protein (CooF) and the CODH catalytic subunit (CooS). The cooCTJhypB operon, which may encode maturation proteins, was found downstream of cooFS, while cooMKLXUH, which encodes COeH2ase, were located upstream of cooFS in the opposite orientation. The gene arrangement of cooFS, cooMKLXUH and cooCTJHypB of C. amalonaticus Y19 were compared with those of two carboxydotrophic microorganisms, R. rubrum and C. hydrogenoformans (GenBank accession nos. NC007643 and NC007503, respectively) (Fig. 1). The number and order of genes in the COeH2ase operons among the three microbes were almost the same. However, maturation genes were most extensively established in Y19; hypB was not identified in the same operon in R. rubrum, and cooT, cooJ

and hypB were not found in C. hydrogenoformans. In C. hydrogenoformans, hypA instead of hypB was present, and its location, along with cooC, was within the COeH2ase operon. Another important gene, cooA, encoding a CO sensor that regulates the expression of the entire coo operon, has been identified upstream of Y19 cooMKLXUH. A putative sensor gene cooA-X was also found downstream of the Y19 cooCTJHypB operon, which was not identified in R. rubrum or C. hydrogenoformans. As CooS is the catalytic enzyme for CO oxidation, the amino acid sequence of Y19 CooS was compared with those of R. rubrum and C. hydrogenoformans (Fig. 2). The sequence homology with the CooS of R. rubrum and C. hydrogenoformans was 80%. It was noted that six residues, His262, Cys297, Cys335, Cys448, Cys478, and Cys528, were conserved in all three CooS [30]. Previous report suggested that these six residues are covalently bound to the [Nie4Fee4S] cluster, which constitutes the active site for CO oxidation [30]. This indicates that, although C. amalonaticus Y19 is physiologically much different from R. rubrum and C. hydrogenoformans, the structure and function of CooS in Y19 is not much different from those in R. rubrum and C. hydrogenoformans.

Expression of CODH in E. coli BL21 (DE3) The cooFS operon and maturation genes cooCTJhypB of Y19 were cloned in E. coli BL21 (DE3) (EB strain) individually and in combination, and the expression of the cloned genes was analyzed by SDS-PAGE (Fig. 3). Protein bands of the target proteins were not seen in soluble fraction and thus only the results for particulate fraction were shown. CooS (67.8 kDa) and CooF (21 kDa) protein bands were observed clearly in EBFS and EB-F strains, respectively, while CooJ (9 kDa) and CooT (7 kDa) were not observed probably due to their small size. As HypB (28 kDa) and CooC (27.2 kDa) are similar in size, the two proteins were lined up as a single band along with the antibiotic resistance proteins, which are in the range of

Fig. 1 e Gene arrangement of CODH and CO-dependent hydrogenase gene clusters in (A) Citrobacter amalonaticus Y19, (B) Rhodospirillum rubrum ATCC 11170 and (C) Carboxydothermus hydrogenoformans Z-2901. Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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Fig. 2 e Multiple sequence alignment of CooS. The CooS sequence of Citrobacter amalonaticus Y19, Carboxydothermus hydrogenoformans Z-2901 and Rhodospirillum rubrum ATCC 11170 were aligned using the multalin tool. The conserved residues are highlighted in grey. The metal ions of the [Nie4Fee4S] cluster are bound to the residues that are highlighted in black.

25e30 kDa, of duet vectors (pACYC, pCDF, pRSF) used for expressing CooFSCTJH, and thus HypB and CooC could not be differentiated. To be functionally active, both CooS and CooF should be partitioned in the soluble fraction. Although no protein band for CooS was observed in soluble fraction of all recombinants, the presence of CODH activity in soluble fraction of some recombinant such as EB-FS and EB-FS-CT-JH suggests that some CooS proteins are produced in soluble fraction (see Fig. 5 and Table 2). When cooS was cloned without cooF, the protein band corresponding to CooS was not detected (Fig. 3). In contrast,

CooF protein was highly produced without CooS (Fig. 3). To determine whether CooF has a chaperone function for the expression of CooS, cooF and cooS were separately cloned in two compatible plasmids and introduced into a single EB strain. Interestingly, this recombinant strain containing the two plasmids could produce CooF but not CooS (data not shown). Analysis of the DNA sequences of the cooFS operon revealed that a putative RBS locus for cooS resided at the end of but within the structural cooF gene. Further studies are required to determine the role of this internal RBS in expressing CooS. Fig. 3 also shows that, when CooS and CooF

Fig. 3 e SDS-PAGE analysis of recombinant Escherichia coli (EB) proteins. The particulate fractions were resolved on a 15% SDS gel. PageRuler plus prestained protein ladder was used. The expression of CooS (68 kDa), HypB (28 kDa), CooC (27 kDa) and CooF (22 kDa) is marked. (Refer to Table 1 for strain description).

Fig. 4 e Relative mRNA levels of CooF and CooS. mRNA levels were analyzed using the total RNA from Citrobacter amalonaticus Y19 and recombinant EB strains. The mRNA levels were compared with rpoD as the reference gene. (Refer to Table 1 for strain description).

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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production of CooS protein (and lowered CODH activity; see Fig. 5) in EB-FS-CT-JH can be attributed to the reduced transcription of the corresponding gene. However, considering the mismatch between transcription and translation levels, more studies are needed for clarification (Fig. 3).

CODH activity of recombinant strains

Fig. 5 e Crude cell CODH activities of the Citrobacter amalonaticus Y19 strain (Y19) and the recombinant Escherichia coli (EB) strains. CODH activity of the Y19 strain (1.44 ± 0.07 U mg protein¡1) was taken as 100% to calculate the relative activity. (Refer to Table 1 for strain description).

were co-expressed with maturation proteins, the expression level of CooS and CooF, rather than that of the maturation proteins, decreased significantly. It is not unusual to observe that the expression level of some proteins decrease when many proteins are produced simultaneously [31,32].

Expression analysis of CooF and CooS by real-time PCR As SDS-PAGE analysis failed to confirm the expression of CooF and/or CooS in some recombinant strains, the transcription of cooF and cooS was analyzed by real-time PCR (RT-PCR) using the cells harvested from late exponential growth phase. Although the production of CooF protein in EB-FS and EB-FSCT-JH was not confirmed on SDS-PAGE, the transcription of cooF was very high in all recombinant strains harboring the gene (Fig. 4). The RT-PCR result also indicated that cooS was well transcribed in EB-S although the transcription level was much lower compared to that in EB-FS or EB-FS-CT-JH. The mismatch between the levels of protein production and mRNA synthesis suggests that the expression of CooF and CooS might be regulated in both transcriptional and translational levels. Reduced transcription levels of both cooF and cooS were observed when they were co-expressed with the maturation proteins in the strain EB-FS-CT-JH. The reduced

The CODH activity was measured by the reduction of methyl viologen using cell-free extracts. The recombinant E. coli with cooFS (EB-FS) alone or in combination with cooCTJHypB (EB-FSCT-JH) showed CODH activity, while the recombinants harboring cooF (EB-F) or cooCTJHypB (EB-CT-JH) without cooS did not show any activity (Fig. 5). These results indicate that CooS, which encodes CODH, is the key protein responsible for CO oxidation. The result with EB-S, the recombinant harboring cooS alone, is puzzling; EB-S did not show any activity, although transcription of cooS was confirmed. It is speculated that CODH encoded by cooS is not produced in an active form when CooF is not co-expressed. EB-FS exhibited higher CODH activity (4.93 ± 0.51 U mg protein
1) than wild type C. amalonaticus Y19 or EB-FS-CT-JH (1.44 ± 0.07 U mg protein
1) (0.51± 0.04 U mg protein
1). The (over)expression of maturation proteins was expected to be essential for functional expression or at least to improve CODH maturation to a certain extent, but their expression only contributed to a decrease in CODH activity. The decrease in activity could be related with the lowered CooS expression in EB-FS-CT-JH compared to that in EB-FS in both transcription and translation levels. The expression of active CODH in E. coli, even in the absence of maturation proteins such as CooC, CooT, CooJ and HypB, indicates that the maturation proteins, which are present in coo operons and assumed to be specific to CODH and/or CODH-dependent H2-ase are not essential for the heterologous expression of CODH in E. coli. However, considering the complex nature of the synthesis and incorporation process of the metal cluster, it is unlikely that Y19 CODH is synthesized by itself in an active form in recombinant E. coli without the help of maturation or accessory proteins. It is more reasonable to assume that isozymes present in E. coli took over the role of the maturation proteins in Y19 coo operons. Specifically, CooC, CooT, CooJ and HypB assist in nickel binding to CODH and NiFe hydrogenase [12,33]. It is well documented that the proteins encoded by the hyp operon in E. coli are responsible for Ni-processing for hydrogenase (H2ase) and other similar enzymes. The amino acid similarity between Y19 HypB and E. coli HypB was 64%.

Table 2 e CODH activity from a recombinant Escherichia coli strain (EB-FS) in the crude, soluble, insoluble and purified extracts. Enzyme source Cell free crude extract Soluble fraction Insoluble fraction Purified enzymec a b c

Total proteina (mg) Total activity (Ub) Specific activity (Ub mg protein
1) Purification (Fold) Yield (%) 77.35 66.89 12.24 0.319

± 3.69 ± 2.73 ± 0.91 ± 0.06

381.8 ± 23.80 377.92 ± 14.94 15.05 ± 1.12 54.33 ± 3.58

4.93 ± 0.51 5.65 ± 0.14 1.23 ± 0.06 170.58 ± 1.18

e 1 e 30.2

e 100 e 14.4

Approximately 0.25 g cell dry weight (CDW) of cells was used. One unit (U) of activity is defined as 1 mmol of MV reduced per min. Purified to electrophoretic homogeneity from soluble fractions.

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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Purification of CODH CooS protein was purified to electrophoretic homogeneity from the soluble fraction of the EB-FS strain using a nondenaturing Ni-NTA-HP resin column (Table 2 and Fig. 6). Although most CODH proteins were expressed in the particulate fraction, higher CODH activity was observed in the soluble fraction. This suggests that soluble CooS proteins, although produced at a low level, are catalytically active. The purity enrichment determined by the specific activity of CODH of the cell-free extract was 30-fold (170.6 U mg protein
1) with a protein yield of 14%. When the purified enzyme was analyzed using native PAGE (Fig. 6), a protein band of approximately 140 kDa was observed. When the same enzyme was analyzed by denaturing SDS-PAGE, a protein band of approximately 70 kDa was obtained. These results suggest that CODH of Y19 is a homodimer of CooS proteins. Similar results were observed when CooS was purified from C. hydrogenoformans [8]. Table 2 also indicates that, in vitro, CooS alone, without the help of the ferredoxin-like protein CooF, can oxidize CO. This indicates that Y19 CODH can directly transfer electrons to oxidized methyl viologen which is in agreement with previous studies with purified CODH from R. rubrum [16] and C. hydrogenoformans [8]. For CO-dependent H2 production in vivo, however, functional expression of CooF is important. As CooF activity was not measured by the current assay method, it is not clear whether CooF was actively expressed in the recombinant E. coli strains in this study.

CO-dependent hydrogen production in recombinant E. coli BW25113 expressing CODH of Y19 E. coli is known to have at least two uptake H2ases (H2ase 1 and 2) and one evolving H2ase (H2ase 3), the latter of which is part of formate hydrogen lyase (FHL) [34,35]. We were curious whether the recombinant CODH could form a complex with one of these H2ases and produce H2 from CO. According to the sequence analysis by basic local alignment search tool (BLAST), the large and small subunits of COeH2ase were quite similar to the subunits of H2ase 3 of E. coli (Data not shown). Because E. coli BL21 (DE3) cannot produce H2 from formate [22] and H2ase 3 is expected to be the most suitable H2ase for forming a complex with the recombinant CODH, E. coli BW25113 which can actively

Fig. 6 e A) Native PAGE and B) SDS-PAGE of purified CODH fractions. The arrows indicate CooS homodimers (~140 kDa) in native PAGE and monomeric CooS (68 kDa) in SDS-PAGE.

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produce H2 from formate was chosen for the experiment and introduced with the cooFS operon. The plasmid with T7 promoter was replaced with a plasmid with arabinose promoter for the expression of cooFS as E. coli BW25113 does not have T7 RNA polymerase. Four E. coli BW25113 (EW) strains including three mutants previously developed in our laboratory (SH1, SH2, and SH3) (Table 1), were used as hosts [23]. Briefly, SH1 represents BW25113 lacking HycA, a negative regulator of formate hydrogen lyase. SH2 is a SH1 deletion mutant lacking H2ase 1 activity and SH3 is a deletion mutant of SH2 devoid of H2ase 2 activity. As shown in Fig. 7, all four recombinant EW exhibited CODH activity in the cell-free extract. Nevertheless, no COdependent H2 production was observed in any of the four strains. This indicates that CO-dependent H2 production requires both CODH and CODH-specific H2ase. In other words, the formate-dependent H2ase in the FHL complex or the uptake H2ases cannot form a complex with CODH to receive electrons from CO oxidation to generate H2. However, the possibility that CooF might have not been expressed in an active form in the recombinant E. coli cannot be fully eliminated. The results with recombinant EW strains confirmed again that only cooFS, not CODH-specific maturation proteins, are required for functional expression of the CODH protein in E. coli. The specific activities of the four recombinants were similar (2.65 ± 0.35 U mg protein
1) and this activity was not much different from that of EB-FS (see Fig. 4). According to SDS-PAGE analysis (data not shown), protein expression of CooS in the soluble fraction of EW strains was not visible as in the case of EB strains. Additionally, the expression in the particulate fraction was much lower than that in EB strains. However, CODH activity in EW strains was not much different from that in EB strains, confirming that the soluble CODH, not the particulate CODH, is the active form of the enzyme. CODH activity in recombinant E. coli is expected to increase if its soluble expression can be improved.

Conclusion The main objective of this study was to express active CODH in E. coli to develop a highly efficient recombinant E. coli that

Fig. 7 e CODH activity and CO-dependent H2 production activity of the Citrobacter amalonaticus Y19 strain (Y19) and the recombinant Escherichia coli BW25113 (EW, SH1, SH2, SH3) strains. (See Table 1 for strain description).

Please cite this article in press as: Sundara Sekar B, et al., Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.07.148

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can perform a WGSR to produce H2 from CO. Only cooFS, not the accessory proteins such as CooC, CooT, CooJ and HypB, was required for functional heterologous expression of Y19 CODH in E. coli. However, despite the expression of active CODH, neither E. coli BL21 (DE3) nor E. coli BW25113, which are B and K type E. coli, respectively, produced H2 from CO. To achieve H2 production from CO, the additional heterologous expression of Y19 COeH2
ase in E. coli is in progress.

Acknowledgments This study was supported by the Development of Biohydrogen Production Technology through the Hyperthermophilic Archaea Program of the Ministry of Ocean and Fisheries in the Republic of South Korea (D10906313H320000120) and a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP, 2012K1A3A1A19036612). In addition, the authors are grateful to the BK21 plus program for Advanced Chemical technology at Pusan National University (21A20131800002). The authors thank Dr. Ashok Somasundar for helpful discussions. The authors are thankful to Ms. Yeounjoo Ko for providing technical assistance during anaerobic protein purification.

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