(341g) Exhaustive Screening of 3985 Isogenic Escherichia Coli Mutants for Enhanced Bacterial Hydrogen Production

Hydrogen is a promising fuel since it is renewable, efficient, and clean. Use of biological methods for hydrogen production should significantly reduce energy costs as these processes do not require extensive heating or electricity. We have used metabolic engineering with Escherichia coli to enhance hydrogen production by manipulating the key enzymes of the formate hydrogen lyase system (FHL) consisting of hydrogenase 3 and formate dehydrogenase-H (Maeda et al., Microb. Biotechnol. 1:30-39, 2008). To date, we have constructed metabolically-engineered strains that produce 141-fold higher hydrogen from formate by inactivating genes that encode enzymes for hydrogen uptake (hyaB and hybC), by inactivating the gene encoding the FHL repressor (hycA), by inactivating the gene for formate depletion (fdoG), and by producing an essential activator of FHL, FhlA. In addition, we have achieved 5-fold higher hydrogen production from glucose by inactivating the succinate synthesis pathway (frdC), the lactate synthesis pathway (ldhA), and pyruvate-consuming activity (aceE) (Maeda et al., Appl. Microbiol. Biotechnol. 77:879-890, 2007). Furthermore, our studies using random mutation approaches such as chemical mutagenesis and transposon mutagenesis have succeeded in generating several mutants in which hydrogen productivity has been improved which implies other unknown pathways are important for increasing hydrogen production. Here, we conducted an exhaustive search of all E. coli pathways for their impact on hydrogen production by screening the entire Keio mutant library using both glucose and formate as substrates; this search identifies mutations which alter hydrogen production while simultaneously identifying the key pathway. The screen was facilitated by use of chemochromic membranes (GVD Corp., Cambridge, MA) formed by a thin film of WO₃ covered with a catalytic layer of palladium; these plates were used to detect directly the amount of hydrogen gas by a colorimetric response from colonies grown anaerobically. We previously utilized this screen to engineer FhlA for improved hydrogen production (Sanchez-Torres et al., Appl. Environ. Microbiol. 75:5639?5646, 2009) and to perform the first protein engineering of hydrogenase 3 to improve hydrogen production (Maeda et al., Appl. Microbiol. Biotechnol. 79:77?86, 2008). As expected, using this powerful screen and the Keio Collection, deleterious mutations were identified in genes related to hydrogen production such as hydrogenase 3 (hyc genes), hydrogenase maturation proteins (hyp genes), formate dehydrogenase-H (fdhF), and the FHL activator (fhlA). In addition, hydrogen production was decreased by inactivating genes related to nickel transport (nikABCDE), selenocysteine synthesis and incorporation (selAB), molybdopterin biosynthesis (moeAB and moaABCDE), and ATP synthesis (atp genes). In contrast, several beneficial mutations were identified including those that inactivated several cold shock proteins, those in uncharacterized proteins, as well as a mutation in a metal ion transporter (mntH). Hence, this approach identifies genes which may be deleted as well as those that may be overexpressed to improve hydrogen production.