Enhanced Hydrogen Production in Escherichia Coli through Chemical Mutagenesis, Gene Deletion, and Transposon Mutagenesis

Enhanced Hydrogen Production in Escherichia coli Through Chemical Mutagenesis, Gene Deletion, and Transposon Mutagenesis. (May 2010) Andrea Juliana Garzon Sanabria, B.S., Universidad Industrial de Santander at Bucaramanga Chair of Advisory Committee: Dr. Thomas K. Wood We demonstrate that hydrogen production can be increased by random mutagenesis using N-methyl-N ́-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 ± 0.5fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing hydrogenase 3 inactivated from the chromosome, and has constitutively active hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 ± 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 ± 0.50-fold. Deleting both fdoG and hycA increased hydrogen production 257 ± 0.50-fold, and overexpressing fhlA along with the fdoG hycA mutations increased hydrogen 308 ± 0.52-fold. Whole-transcriptome analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed. In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed comparative genome sequencing and identified two chromosomal loci with mutations in coding