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Caldicellulosiruptor saccharolyticus has proven itself to be an excellent candidate for biological hydrogen (H2) production, but still it has major drawbacks like sensitivity to high osmotic pressure and low volumetric H2 productivity, which should be considered before it can be used industrially. A whole genome re-annotation work has been carried out as an attempt to update the incomplete genome information that causes gap in the knowledge especially in the area of metabolic engineering, to improve the H2 producing capabilities of C. saccharolyticus. Whole genome re-annotation was performed through manual means for 2,682 Coding Sequences (CDSs). Bioinformatics tools based on sequence similarity, motif search, phylogenetic analysis and fold recognition were employed for re-annotation. Our methodology could successfully add functions for 409 hypothetical proteins (HPs), 46 proteins previously annotated as putative and assigned more accurate functions for the known protein sequences. Homology based gene annotation has been used as a standard method for assigning function to novel proteins, but over the past few years many non-homology based methods such as genomic context approaches for protein function prediction have been developed. Using non-homology based functional prediction methods, we were able to assign cellular processes or physical complexes for 249 hypothetical sequences. Our re-annotation pipeline highlights the addition of 231 new CDSs generated from MicroScope Platform, to the original genome with functional prediction for 49 of them. The re-annotation of HPs and new CDSs is stored in the relational database that is available on the MicroScope web-based platform. In parallel, a comparative genome analyses were performed among the members of genus Caldicellulosiruptor to understand the function and evolutionary processes. Further, with results from integrated re-annotation studies (homology and genomic context approach), we strongly suggest that Csac_0437 and Csac_0424 encode for glycoside hydrolases (GH) and are proposed to be involved in the decomposition of recalcitrant plant polysaccharides. Similarly, HPs: Csac_0732, Csac_1862, Csac_1294 and Csac_0668 are suggested to play a significant role in biohydrogen production. Function prediction of these HPs by using our integrated approach will considerably enhance the interpretation of large-scale experiments targeting this industrially important organism.

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Microbial hydrogen production from biomass has been perceived as an imperative wellspring of renewable energy. Caldicellulosiruptor saccharolyticus has proven itself to be an excellent candidate for biological hydrogen production. Hydrogen is produced as a biological product from central carbon metabolism pathway by the utilization of various soluble sugar substrates. An organism's metabolism is a key factor in understanding its physiology, therefore, metabolic network of central carbon metabolism of C. saccharolyticus was reconstructed. The reconstructed and refined central carbon metabolism network comprises 31 reactions (excluding the efflux and influx reactions) and 40 metabolites in total. An elementary flux mode analysis of central carbon metabolism was carried out to investigate the underlying biochemical mechanism of hydrogen production by determining their intermediate fluxes, using linear programming (LP) method available in CellNetAnalyzer software. An in silico gene knockout of lactate dehydrogenase (ldh) and pyruvate kinase (pyk) of the modeled central carbon metabolism pathway allows the maximum theoretical hydrogen yield of 46.2758 mmol/gDW/hr. The single gene knockout of ldh gene tends to increase the flux rate of hydrogen to 32%. Similarly, double gene knockout of ldh and pyk genes yielded an increase in hydrogen flux rate to 35%. Ultimately, this elementary flux mode analysis study of central carbon metabolism pathway of C. saccharolyticus with glucose as substrate or carbon source would further act as a model for strain improvement.