(274g) Metabolic Engineering To Enhance Bacterial Hydrogen Production

Hydrogen is renewable, efficient, and clean, it is a promising fuel as it has a higher energy content than oil (142 MJ/kg for H₂ vs. 42 MJ/kg for oil), and fermentative bacteria hold great promise for its generation. Escherichia coli has well-established metabolic pathways, and it is one of the easiest strains to manipulate genetically; hence, it is an attractive host for metabolically engineering hydrogen production. We have created a novel, simple technique that makes it relatively easy to introduce multiple mutations into E. coli by combining the KEIO collection (isogenic E. coli K-12 library containing all the 3985 non-lethal deletion mutations) and P1 phage transduction. This allows us to introduce unlimited metabolic mutations (each in 2 days) without having to depend on multiple antibiotic resistance markers; we have introduced as many as 9 chromosomal deletions without affecting growth to date. This tool has been used by us to direct the metabolic flux toward hydrogen production via formate in E. coli. E. coli produces hydrogen from formate by hydrogenase 3 (encoded by hycABCDEFGHI) and formate dehydrogenase-H (encoded by fdhF) which are the key enzymes of the formate hydrogen lyase system (FHL); these enzymes catalyze the reaction HCOOH ↔ H₂ + CO₂. Our strategy for metabolic engineering E. coli for enhanced hydrogen production was threefold. (i) We first prevented hydrogen consumption by the bacterium by inactivating hydrogenase 1 (hyaB; large subunit) and hydrogenase 2 (hybC; large subunit). (ii) By manipulating two FHL regulatory proteins, HycA and FhlA, we increased expression of FHL; HycA represses FHL by opposing hyc transcriptional activation resulting in a decrease of hydrogen production so hycA was deleted, and FhlA activates FHL by binding directly to the intergenic region between the hyc and hyp operons and between the hycA and hycB genes so fhlA was overexpressed. (iii) We modified formate metabolism. E. coli has three undesired pathways for eliminating formate produced by fermentation (we desire to route all formate to hydrogen): excretion of formate by putative formate transporters (FocA and its homolog FocB), degradation of formate by formate dehydrogenase-N (FdnG, α-subunit) coupled with nitrate reductase A (NarG, α-subunit), and degradation by formate by dehydrogenase-O (FdoG, α-subunit). Hence, formate transport, formate dehydrogenase-N/nitrate reductase A activity, and formate dehydrogenase-O activity may be deleted to enhance hydrogen production. Inactivation of hydrogenase 1 and hydrogenase 2 yielded a 3.2-fold decrease in hydrogen uptake activity compared to wild-type strain, and hydrogen production in the double mutant (hyaB hybC) was 3.2-fold higher than that in the wild-type strain in complex formate medium after 1 h. The addition of a hycA mutation to the double mutant (hyaB hybC) increased hydrogen production 4.8-fold compared to wild-type cells. The quadruple mutant (hyaB hybC hycA focB) slightly increased hydrogen production (5.2 times more hydrogen than the wild type strain in complex-formate medium after 1 h). Deleting fdoG gene in a hyaB hybC hycA background led to a significant increase in hydrogen production as BW25113 hyaB hybC hycA fdoG cells produced 11-fold more hydrogen than the wild-type cells. In addition, the expression of fhlA in BW25113 hyaB hybC hycA, and BW25113 hyaB hybC hycA fdoG led to a 1.9- and 1.2-fold increase in hydrogen production (BW25113 hyaB hybC hycA/pCA24N-FhlA vs. BW25113 hyaB hybC hycA/pCA24N and BW25113 hyaB hybC hycA fdoG/pCA24N-FhlA vs. BW25113 hyaB hybC hycA fdoG/pCA24N). Ultimately, BW25113 hyaB hybC hycA fdoG/pCA24N-FhlA produced 26-fold more hydrogen than the wild-type strain (BW25113/pCA24N) in complex formate medium after 1 h in the closed system. Since the accumulation of hydrogen in the headspace in the closed system will tend to reverse the hydrogen synthetic reaction, an anaerobic system that maintained low hydrogen headspace pressure (open system) was constructed. The expression of FhlA in wild-type background (BW25113/pCA24N-FhlA) led to 30-fold increase of hydrogen production rate compared to wild-type cells that do not express FhlA indicating that overexpression of FhlA is essential for enhanced hydrogen production. BW25113 hyaB hybC hycA fdoG/pCA24N-FhlA produced 141 times more hydrogen than the wild type strain with empty vector pCA24N whereas hydrogen production in BW25113 hyaB hybC/pCA24N-FhlA, BW25113 hyaB hybC hycA/pCA24N-FhlA, and BW25113 hyaB hybC hycA focB/pCA24N-FhlA were 71-, 80-, and 76-fold higher than that in the wild type strain. The hydrogen yield in BW25113 hyaB hybC hycA fdoG/pCA24N-FhlA was 1.15 ± 0.01 mol hydrogen/mol formate compared to 0.64 ± 0.01 mol-hydrogen/mol-formate for BW25113/pCA24N. This indicates that the metabolically-engineered E. coli cells (BW25113 hyaB hybC hycA fdoG/pCA24N-FhlA) more efficiently convert formate into hydrogen and that these metabolically-engineered cells obtain the theoretical yield of 1 mol hydrogen produced per mol of formate consumed. Taken together, we created a single fermentative E. coli strain with five mutations (deletion of hyaB, hybC, hycA, and fdoG and overexpression of fhlA⁺) that produces the largest amount of hydrogen to date (141-fold more hydrogen than the wild-type strain at a rate of 112 µmol/mg protein/h), that reaches the theoretical yield from formate, and which has no decrease in cell viability. The method developed here to introduce multiple stable mutations in a single strain without reducing cell viability holds much promise for continued increases in hydrogen production using E. coli as well as tremendous promise for many other applications of pathway engineering where multiple mutations are required.