Construction Of A Recombinant Escherichia Coli Containing [NiFe] Hydrogenase Genes And Hydrogen Production Performance

Biohydrogen production is based on the principle of biochemical reactions, which using the metabolic system of microorganisms or genetic engineering to modify metabolic system to produce hydrogen at normal temperature and pressure by consuming organic and free water. Biohydrogen production is a sustainable way of producing hydrogen because it is more environmentally friendly and less energy consumption than other ways of hydrogen production at room temperature and atmospheric pressure.Recombinant Escherichia coli(E.coli) was constructed, which contains [NiFe] hydrogenase genes from Rhodopseudomonas palustris (R.palustris), and then hydrogen production ability was investigated in this paper.Rpalustris was separated and purified through concentration method with RCVBN medium and plate screening method. Gene sequence of R.palustris was identified with 16S rDNA molecular identification. We designed PCR primers to amplify hupS and hupL gene according to the gene sequences of [NiFe] hydrogenase genes in R.palustris. The PCR products were recovered after gel electrophoresis, and then been linked to the cloning plasmid pBM19-T vector which were transferred into E.coli DH5a. We attained the recombinant vectors which contain hupS and hupL gene. Gene sequencing results showed that the [NiFe] hydrogenase genes of R.palustris had been successfully amplified, which compared the gene sequences in GenBank. Moreover, we designed PCR primers which contained sites of restriction enzyme and amplified [NiFe] hydrogenase genes, and then PCR products were linked to expression vector pETDuet-1. As result, we successfully built two promoters expression vector pETD-SL which contains [NiFe] hydrogenase genes.E.coli BL21(DE3) was chosen as recombinant host bacteria, and expression vector pETD-SL was transferred into E.coli BL21(DE3), so we got recombinant strains. PCR and restriction enzyme analysis were carried out to testify the cloning genes and gene sequencing results guaranteed the uniformity of hupL and hupS gene of [NiFe] hydrogenase in R.palustris. Subsequently, E.coli BL21(DE3) which contain pETDuet-1 was set as the control group, recombinant strain E.coli BH20 was induced by 1 mM IPTG to produce recombinant proteins, which were detected by SDS-PAGE electrophoresis. At the same time, we collected the gas sample of E.coli BH20 and control group through NaOH displacement in inverted cylinder, and then the gas samples were analyzed by gas chromatography to testify whether recombinant strain E.coli BH20 can produce hydrogen or not. At last, hydrogen production ability of recombinant strain E.coli BH20 was investigated the in different conditions (glucose concentration, temperature, and pH).Several conclusions were drawn as following:(1) A wild type strain of R.palustris was achieved with 16S rDNA molecular identification, and construction of R.palustris was observed through microscopy. R.palustris is a gram-negative bacterium which has rod-shaped, flagellated under the microscope. The cell colonies are brownish red, their circular diameter is about 0.4-1.3 mm, and they are of moist surface smooth and neat edges on solid medium. Characteristic absorption peaks of R.palustris are 380,592,806 and 875 nm by spectrum.(2) Primers of hupS and hupL were designed, and 1400 bp and 2000 bp fragment gained by PCR reaction were linked to pBM19-T vector. Gene sequencing of hupS and hupL showed recombinant protein HupS and HupL are about 42.7 kDa and 65.5 kDa. Sequenced genes are hupS and hupL of [NiFe] hydrogenase genes by homologous sequence analysis, which belong to R.palustri. Subsequently, hupS and hupL containg sites of restriction enzyme were linked to pETDuet-1 vector, plasmid pETD-SL was successfully constructed.(3) Recombinant plasmid pETD-SL was transferred into E.coli BL21 (DE3), recombinant strain E.coli BH20 was screened, which can express [NiFe] hydrogenase of R.palustris and produce hydrogen. E.coli BH20 cultured in the anaerobic bottle will produce pupples, and E.coli BL21 (DE3) as the control group can’t produce pupples, hydrogen is confirmed by gas chromatography.(4) The results of hydrogen production efficiency of E.coli BH20 at different temperature showed that hydrogen yield is 115.8±1.0 mL, glucose consumption rate is about 61.9%. At different pH, hydrogen yield is 115.8±1.0 mL and the glucose consumption rate is about 63.0% when pH is equal to 6.5. At different concentrations of glucose, hydrogen yield is 105.3±2.4 mL and glucose consumption rate is about 63.1%. Therefore, optimum conditions of E. coli BH20 for hydrogen production were at 35 ℃, pH 6.5, glucose concentration of 2%. Under optimum conditions, E.coli BH20 can produce 122.9±2.4 mL hydrogen, total glucose utilization was 5.3%.The hupS and hupL gene of [NiFe] hydrogenase genes from R.palustris were transferred into E.coli BL21(DE3) by use of pETDuet-1 vector, recombinant strain E.coli BH20 was achieved. Hydrogen production experiment results showed that recombinant E.coli BH20 can produce hydrogen. This experiment proved that recombinant vector pETD-SL can improve the hydrogen production ability of microorganisms which rely on the formate metabolic system. This has good application value for the future of hydrogen production capacity of microorganisms containing formic acid metabolism system.