Hydrogen Metabolic Regulation By Hydrogenase-3 And The Preliminary Exploration Of NADH Pathway

With the deterioration of energy crisis, global climate change and environmental pollution, the exploration of green energy is becoming more and more urgent. As a renewable, clean and green alternative energy, hydrogen is attracting increasing attention. Among the existing hydrogen preparation technologies, anaerobic fermentation hydrogen production technology has many advantages,such as mild reaction conditions, simple reactor design, and wide range of substrates(organic waste). However, its relative low efficiency of hydrogen productivity still blocks its industrialization. Thus,breeding high effective strains is a vital work for industrial application of anaerobic fermentation hydrogen production technology. Currently, genetic engineering and coenzyme engineering are emerging strategies for recombining high efficient hydrogen-producing stains.Under this context, facultative anaerobe hydrogen-producing bacterium Enterobacter aerogenes CCTCC AB91102 was chosen as the study strain, for it grows very fast, fits a wide range of substrate, and needs short fermentation period. Such advantages make it one of the potential bacteria for industrial hydrogen-production. However, anaerobic fermentation is a relatively complex metabolic process, including formate hydrogen lyase pathway and NADH pathway. Homologous expression of hydrogenase-3 gene may directly enhance hydrogenase activity, resulting in improvement of hydrogen production. In addition, response surface method(RSM) was employed to optimize fermentation conditions in order to further increase hydrogen production. Meanwhile, another pathway(NADH pathway) was also focused, and an exploration of its metabolic mechanism was preliminarily conducted. The main researches and results were summarized belows.1. The genes of subunit Hyc E and Hyc G of hydrogenase-3 were homologously expressed in E. aerogenes AB91102, resulting in high-effective strains AB91102-hyc E and AB91102-hyc G. Firstly, the genes of large subunit(Hyc E), membrane-bound proteins(Hyc F) and the small subunit(Hyc G) of hydrogenase-3 were cloned from the genomic DNA of E. aerogenes AB91102 by using the degenerate primers. Then, the expression vectors p ET28-Pkan-hyc E and p ET28-Pkan-hyc G were constructed by ligation the genes of Hyc E and Hyc G with the vector p ET28-Pkan and transformed into the wild strain E. aerogenes AB91102 to obtain recombinant AB91102-hyc E and AB91102-hyc G. According to the analysis of hydrogen productin, the hydrogen production of recombinant AB91102-hyc E and AB91102-hyc G was respectively enhanced 11.51% and 6.77% than that of the original strain. Based on the analysis of broth ingredients and hydrogen contribution, the hydrogen production from formate pathway was increased 5.4% and 1.9%, while that from NADH pathway increased 54.8% and 40.6%, indicating that over-expression of hydrogenase-3 not only can improve the hydrogen yield from formate pathway, but also can enhance the yield from NADH pathway.2. The key parameters were optimized for the batch fermentation by using central composite design(CCD) together with single factor analysis. The single-factor analysis showed that initial inoculum, temperature and initial p H were the significant factors, and the optimal conditions were initial inoculum 10%, fermentation temperature 35 °C and initial p H 6.0. Then, a three-level-three-factor CCD-RSM was further performed and the results were analyzed via software SAS 9.0. The optimized conditions were initial inoculum 11.6%, fermentation temperature 34.6 °C and initial p H 6.07. In the confirmatory experiments, the obtained maximum production of hydrogen was 1.175 mol H2/mol glucose, which was well coincided with the predicted value 1.169 mol H2/mol glucose, indicating the predicted model was precise and dependable.3. The function of genes hyc E, hyc F and hyc G encoding the key subunits of hydrogenase-3 were further examined. In order to do this, genes hyc E, hyc F and hyc G were successfully respectively knocked out from E. aerogenes AB91102. The three genes respectively deleted from the chromosome DNA of E. aerogenes AB91102 by using Red recombinase were confirmed by PCR analysis, antibiotic resistance experiments and metabolites analysis. The resultant mutant strains were respectively named AB91102-?hyc E, AB91102-?hyc F and AB91102-?hyc G. Anaerobic chemostat experiment results of the mutant strains AB91102-?hyc E, AB91102-?hyc F and AB91102-?hyc G showed that the total NAD(H) concentration changed very little, however, NADH/NAD+ ratio increased from 1.06 to 1.39,1.45 and 1.41, respectively. At the same time, the glucose consumption decreased by 47.85%, 46.49% and 48.95% compared to that of wild strain. Anaerobic batch fermentation experiments showed that hydrogen yield of the mutants decreased to nil, and the glucose consumption respectively decreased by 35.1%, 34.5% and 36.0%. Moreover, according to the analysis of hydrogen production and emzyme activities, the hydrogen production from NADH pathway was zero and the activity of NADH-mediated hydrogenase was undectected, indicating that the knockout of hydrogenase-3 genes blocked or seriously inhibited the formate pathway and NADH pathway, suggesting hydrogenase-3 is a key enzyme for hydrogen production of E. aerogenes AB91102.