Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 8012
© Khetkorn et al.; licensee BioMed Central Ltd. 2012
Received: 14 June 2012
Accepted: 7 October 2012
Published: 1 December 2012
Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase.
An engineered strain lacking a functional uptake hydrogenase (∆hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain.
Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.
The N2-fixing cyanobacterium Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand has been reported to have a high potential for hydrogen production with the ability to utilize sugars as substrate to produce hydrogen . In Anabaena, there may be three enzymes directly involved in hydrogen metabolism. 1) Nitrogenase, a multiprotein enzyme complex consisting of two proteins, dinitrogenase (MoFe protein), encoded by nifD and nifK, and the dinitrogenase reductase (Fe protein), encoded by nifH. This enzyme catalyzes the reduction of atmospheric N2 to ammonia as well as the reduction of proton (H+) to hydrogen [2, 3]. In the absence of the substrate N2, nitrogenase may exclusively catalyze hydrogen production. 2) Uptake hydrogenase, a heterodimeric enzyme with at least two subunits, HupS (small subunit) and HupL (large subunit). The large subunit, encoded by hupL, contains the active site, consisting of four conserved cysteine residues that are involved in the coordination of the metallic NiFe at center of the active site. The small subunit, encoded by hupS, contains three FeS clusters which have a function in transferring electrons from active site of HupL to the electron transport chain. The physiological function of the uptake hydrogenase is recycling of hydrogen produced by nitrogenase [2–5]. 3) Bidirectional hydrogenase, a heteropentameric, NAD+-reducing enzyme, encoded by hoxEFUYH. It consists of two protein complexes; hydrogenase (HoxY and HoxH) and a diaphorase unit (HoxE, HoxF and HoxU). The bidirectional hydrogenase is commonly found, though not universal, in both N2-fixing and non-N2-fixing cyanobacteria and catalyzes both consumption and production of molecular hydrogen [2, 3, 6].
In A. siamensis, an enhanced hydrogen production is mainly achieved through the nitrogenase enzyme . However, the net hydrogen yield is lost due to the activity of the uptake hydrogenase. To overcome this, we engineered a hydrogen uptake deficient strain by interrupting hupS with an antibiotic resistance cassette. Previous studies have reported that N2-fixing cyanobacteria such as Nostoc punctiforme, Anabaena sp. strain PCC 7120, Anabaena variabilis and Nostoc sp. strain PCC 7942 with inactivated uptake hydrogenases show an ability to produce hydrogen at higher rate when compared to their corresponding wild type strains [8–12]. Interestingly, previous reports mainly focused on HupL inactivation since the active site of uptake hydrogenase is located in the large subunit. Therefore, we focused on HupS in A. siamensis TISTR 8012. The structural hupS and hupL genes of A. siamensis have been identified and sequenced . hupS is located upstream of hupL and the predicted gene products for hupS and hupL consist of 320 and 531 amino acids, respectively. Their deduced amino acid sequences show higher than 90% and 80% similarity for HupS and HupL, respectively when compared to other cyanobacteria . RT-PCR analysis revealed that hupS and hupL were co-transcribed with an enhanced transcription when the cells were grown under N2-fixing condition . HupS and HupL of A. siamensis and other cyanobacteria need to go through a maturation process to become a fully functional enzyme .
Thus, in the present study we engineered a strain lacking a functional uptake hydrogenase (∆hupS) with the aim to enhance hydrogen production in A. siamensis TISTR 8012. In addition, the nitrogenase activity and transcript levels of genes involved in hydrogen metabolism and photosynthetic pathways in the ∆hupS strain were investigated. As expected, the ∆hupS strain was more efficient in hydrogen production under long term of light exposure than the wild type strain and the production could be prolonged for more than 72 h under light conditions.
Results and discussion
The confirmation on a complete segregation of a ∆hupS strain of Anabaena siamensis
Effect of hupS inactivation on hydrogen production, growth rate and heterocyst differentiation
When analyzing hydrogen production, the wild type and ∆hupS strains of A. siamensis TISTR 8012 were grown under N2-fixing conditions (BG110 medium) for 12, 24, 48, and 72 h, respectively. Hydrogen production was then determined under continuous illumination of 40 μEm-2s-1 and anaerobic condition for 12 h. Interestingly, the ∆hupS strain produced hydrogen at a significantly higher rate than that of the wild type (Figure 4B). The maximum hydrogen production rate of the ∆hupS strain was 29.7 μmol H2 mg chl a-1h-1 when grown in BG110 medium for 72 h, which is almost 4-folds higher than that observed in the wild type under normal growth condition. These results demonstrate that inactivation of hupS is an effective strategy for improving cyanobacterial photobiological hydrogen production in A. siamensis TISTR 8012. Similar observations have earlier been made in other filamentous cyanobacterial strains [8–12].
Sustained hydrogen production and enhanced nitrogenase activity under long term of light exposure in the ∆hupS strain
Transcription levels of genes related to hydrogen metabolism and photosynthesis in wild type and ∆hupS
This may suggest that electrons and ATP needed for hydrogen production in the ∆hupS strain of A. siamensis TISTR 8012 can be obtained from the electron transport chain associated with the photosynthetic oxidation of water of photosystem II in the vegetative cells. In addition, there was no change observed in the transcription level of the psaA encoding the core protein of photosystem I (Figure 6B). It should be noted that the source of electron transfer to nitrogenase could arise from not only vegetative cells but also from within the heterocysts. Previously, proteins involved in the oxidative pentose phosphate pathway have been reported to be more abundant in heterocysts of a hydrogen uptake deficient strain of Nostoc punctiforme.
The inactivation of HupS of A. siamensis TISTR 8012 (∆hupS) resulted in a significant up-regulation of coxA encoding the cytochrome c oxidase subunit I which is present in vegetative cells only . The increase of CoxA activity would lower the level of O2 in vegetative cells resulting in less inhibition of bidirectional hydrogenase, leading to enhanced hydrogen production. Nevertheless, to further explore the effect of HupS inactivation on cell metabolism, the global protein expression level should be investigated.
Growth competition of wild type and ∆hupS strains in a mixed culture
This study further explores the potential for solar based biohydrogen production using a purpose designed engineered cyanobacterial strain, ∆hupS, using a general strategy outlined elsewhere . We demonstrate that inactivation of hupS encoding the small subunit of the uptake hydrogenase in the cyanobacterium Anabaena siamensis TISTR 8012 results in long term sustainable, light dependent hydrogen production with enhanced nitrogenase activity and only minor effects on cell growth and heterocyst differentiation when compared with the wild type strain. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture. However, the effects of HupS inactivation on general and specific metabolism as well as the long term stability of the ∆hupS strain warrant further investigations.
Materials and methods
Strain and growth conditions
The N2-fixing cyanobacterium Anabaena siamensis TISTR 8012 cells were grown in 50 mL of BG110 medium (without N-source) and BG11 medium containing 18 mM NaNO3 as N-source , both media were buffered with 20 mM HEPES-NaOH (pH 7.5). For ∆hupS strain, cells were grown in either BG11 or BG110 media containing 25 μg mL-1 neomycin antibiotic. The initial cell concentration was adjusted to an OD730 of 0.1 and cultures were incubated aerobically under continuous illumination of 40 μEm-2s-1 with cool white fluorescent lamps from two sides on a rotatory shaker at 160 rpm and 30°C. The growth rate was monitored by measuring the optical density of the culture at 730 nm with a spectrophotometer. The total amount of chlorophyll a (chl a) was determined spectrophotometrically at 665 nm in 90% (v/v) methanol extracts . For the morphological study, A. siamensis TISTR 8012 cells were observed under Scanning Electron Microscope, SEM (JEOL model JSM-5410LV, Japan).
Construct of recombinant plasmid and conjugative gene transfer
The strategy for construction of recombinant plasmid containing target gene interruption could be divided into three steps as shown in Figure 1. The first step, the hupS gene sequence information in A. siamensis TISTR 8012 was obtained from the NCBI database, accession number AY152844. hupS was amplified from extracted genomic DNA of A. siamensis TISTR 8012 cells by using specific primers, HupSF2 (gcatgc atgactaacgtactctggct) and HupSR2 (gcatgc gtctccattcccattaccta). The obtained hupS PCR product was purified and ligated into the pGEM-T easy vector (Promega), creating pGhupS plasmid. The second step, the Mlu I fragment containing a neomycin (NmR) resistant cassette gene from pUC4K vector was modified blunt-ending and then inserted into Eco RV site within the hupS gene of the pGhupS plasmid to produce pGhupSNm plasmid. In the last step, a hupSNm fragment from pGhupSNm plasmid was amplified by using specific primers and then cloned into pRL271 vector to produce pRLhupSNm plasmid. pRLhupSNm plasmid functions as a cargo plasmid suitable to be transferred into A. siamensis TISTR 8012 cell. All plasmids were checked and confirmed by sequencing.
Plasmids used in this study
Source / Reference
Cloning vector, Apr lacZ′, mcs
Cloning vector carrying sacB, Em and Cm
GenBank accession #L05081
Source of Nm cassette
Helper plasmid carrying metylates AvaI, AvaII and AvaIII sites
Conjugative plasmid, Km spontaneous mutant of RK2
pGem-T easy vector contained hupS
Nm cassette inserted into Eco RV site within hupS of pGhupS
Cloning vector, Apr lacZ′, mcs
Hydrogen production determination
The cells were harvested and resuspended in 5 mL medium in a 13 mL of glass vial, then sealed with a rubber septum and a proper screw lid. The vial was bubbled with argon gas for 15 min to eliminate oxygen and incubated under different conditions at 30°C before determining hydrogen production. After 12 h incubation, a 400 μL of head space gas sample was withdrawn from the vial with a gas tight syringe and the hydrogen gas was analyzed by a gas chromatograph (Peri-chrom PR2100, France) with a Molecular Sieve 5A 60/80 mesh column equipped with a thermal conductivity detector and argon as the carrier gas. The hydrogen production rate was expressed as μmol H2 mg chl a-1 h-1.
Nitrogenase activity determination
In vivo nitrogenase activity was measured using the acetylene-reduction assay. In the absence of N2, the enzyme catalyzes the conversion of acetylene (C2H2) to ethylene (C2H4) gas. The reaction was carried out in a glass vial by incubation of the cells suspension (2 mL) with 1 mL of 10% (v/v) acetylene (C2H2) balanced in argon. The ethylene (C2H4) production was detected by using a Gas Chromatograph with a Porapak Q, 50/80 mesh column equipped with a flame ionization detector (Shimadzu, Japan). Enzyme activity was expressed as μmol C2H4 mg chl a-1 h-1.
Primers used in RT-PCR reactions
Sequence 5′ to 3
Target of primer pair
PCR product, bp
Determination of the relative abundance of wild type and ∆hupS strain of Anabaena siamensis TISTR 8012 in a mixed culture
Axenic cultures of A. siamensis TISTR 8012 wild type and the ∆hupS strain were mixed in known proportions. Colony PCRs were performed using primers specific to hupS and analyzed by 0.8% agarose gel electrophoresis. The sizes of the obtained PCR product were approximately 1.0 kb and 2.2 kb representing hupS of the wild type and hupS interrupted with neomycin resistant cassette gene of the engineered strain, respectively. The intensities of the PCR bands were compared within each lane and calculated by using GeneTools program (SynGene, USA) to detect the presence and the relative abundance of the wild type and ∆hupS strain in a single sample. Using this protocol a standard curve was generated and shown as percent of ∆hupS with respect to wild type strain. For competition experiments, the axenic cultures of wild type and ∆hupS strains of A. siamensis TISTR 8012 were mixed together in a 50 mL growth flask in a ratio of 1:1 based on OD730 measurements and incubated under growth conditions. The relative abundance of the wild type versus the ∆hupS strain was then calculated during the experiment.
This work was supported by the Royal Golden Jubilee Ph.D. program (PHD/0147/2549), the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphote Endowment Fund) to W. Khetkorn and A. Incharoensakdi, the Swedish Energy Agency, and the Swedish Research Links program (project 348-2009-6486). The research grants by Commission on Higher Education, Thailand (CHE) (the university staff development consortium), the Thai government SP2 (TKK2555) and the National Research University Project, CHE (FW0659A) to A. Incharoensakdi is also acknowledged. W. Khetkorn also thanks the Graduate School of Chulalongkorn University for providing post-doctoral Fellowship.
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