Genetic modifications are considered as one of the most important technologies for improving fermentative hydrogen yield. Herein, we overexpress fhlA and pncB genes from Klebsiella HQ-3 independently to enhance hydrogen molar yield. HQ-3-fhlA/pncB strain is developed by manipulation of pET28-Pkan/fhlA Kan(r) and pBBR1-MCS5/pncB Gm(r) as expression vectors to examine the synchronous effects of fhlA and pncB. Optimization of anaerobic batch fermentations is achieved and the maximum yield of biohydrogen (1.42 mol H2/mol of glucose) is produced in the range of pH 6.5-7.0 at 33-37 °C. Whole cell H2 yield is increased up to 40 % from HQ-3-fhlA/pncB, as compared with HQ-3-fhlA 20 % and HQ-3-pncB 12 % keeping HQ-3-C as a control. Mechanism of improved H2 yield is studied in combination with metabolic flux analysis by measuring glucose consumption and other metabolites including formate, succinate, 2,3 butanediol, lactate, acetate, ethanol, and hydrogen. The results suggest that under transient conditions, the increase in the total level of NAD by NAPRTase can enhance the rate of NADH-dependent pathways, and therefore, final distribution of metabolites is changed. Combined overexpression of fhlA and pncB eventually modifies the energy and carbon balance leading to enhanced H2 production from FHL as well as by NADH pathway.

Abstract-Hydrogen (H 2 ) production from biomass is considered as the main source of renewable energy, is a characteristic feature of prokaryotes. H 2 is believed as the cleanest fuel without evolution of greenhouse gases on combustion. The main biological processes for H 2 production are: biophotolysis of water by algae and cyanobacteria, dark fermentation and photo-fermentation. Since last decades, a lot of work has been carried out for understanding and refining bio-hydrogen production and still it has to overcome some of the serious limitations so that it becomes a viable proposal. The bottlenecks include thermodynamic inefficiency, trouble in using lignocellulosics as feedstock, cost of raw material and low H 2 molar yields (HMY). To get rid of these major problems, the conventional approach is inadequate and people has to dynamically think modern bioinformatics approaches to overcome these factors. The accessibility of enormous sequenced genomes, functional genomic studies, and the progress of in-silico models at the genome level, metabolic pathway reconstruction, and synthetic biology tactics predict engineering strategies to enhance H 2 production in an organism. This review examines the current status and progressions that have been made in the area of biotechnology and bioinformatics, to understand and enhance H 2 evolution to overcome current limitations and make H 2 production from biological means, a reality in the coming future.Index Terms-Biohydrogen, in-silico metabolic engineering, functional genomics, synthetic biology.