(86b) Inhibition Of Hydrogen Uptake In Escherichia Coli By Expressing The Hydrogenase From The Cyanobacterium Synechocystis Sp. Pcc 6803

Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. The reversible (bi-directional) hydrogenase (hoxEFUYH) of Synechocystis sp. PCC 6803 is a [NiFe]-type enzyme that produces hydrogen via the reaction 2H⁺ + 2e^- ↔ H₂ (g); the source of the two electrons is NADH. Hydrogenase enzymes in E. coli are involved in two distinct modes of hydrogen metabolism: hydrogen production via hydrogenase 3 (encoded by hycABCDEFGHI) and hydrogen uptake by hydrogenase 1 (encoded by hyaABCDEF) and hydrogenase 2 (encoded by hybOABCDEFG). All three enzymes are [NiFe]-hydrogenases and are maturated by the auxiliary proteins HypABCDEF. Using these three hydrogenases, E. coli produces hydrogen from formate by the formate hydrogen lyase system (FHL) that consists of hydrogenase 3 and formate dehydrogenase-H (encoded by fdhF); FHL enzymes catalyze the reaction HCOOH ↔ H₂ + CO₂. Expressing the cyanobacterial hydrogenase (HoxEFUYH) in E. coli led to a 41-fold increase in hydrogen production after 18 h compared to E. coli cells without the bidirectional enzyme. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles were clearly visible from the cultures. Based on this significant increase in hydrogen production, we tried to determine how the cyanobacterial locus increases hydrogen production. First we expressed different components of the cyanobacterial locus and found that overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In contrast, TG1/pBS(Kan)HoxFUY and TG1/pBS(Kan)HoxUYH produced 81 ± 22% and 113 ± 31% of the hydrogen produced by TG1/pBS(Kan)Synhox, respectively; therefore, hydrogen production in TG1/pBS(Kan)HoxUYH was somewhat better than that in TG1/pBS(Kan)Synhox indicating the importance of proteins HoxU, HoxY, and HoxH. Furthermore, hydrogen production in E. coli mutants with defects in the native FHL system (lacking E. coli hydrogenase 3 of the FHL, the maturation machinery required for assembling hydrogenases in E. coli, the transcriptional activator FhlA for FHL, or formate dehydrogenase-H) shows that the cyanobacterial hydrogenase depends on the native E. coli hydrogenase 3 as well as on its maturation proteins. A hydrogen time course experiment showed the maximum in hydrogen production by E. coli TG1 cells with or without HoxEFUYH was within 1.5 h and that from 1.5 h to 6 h, hydrogen produced in the absence of HoxEFUYH decreased 2.1 ± 0.4-fold more rapidly than that from cells with HoxEFUYH; hence, the hydrogen formed in the presence of HoxEFUYH was more stable. This suggested that hydrogen uptake is inhibited by expression of active HoxEFUYH. The results of a methylviologen (MV)-based hydrogen assay clearly indicated three different wild-type E. coli cells expressing HoxEFUYH had 2.3-3.3 times less hydrogen uptake compared to those without the cyanobacterial hydrogenase. These hydrogen uptake results were corroborated by using a plate assay for reversible hydrogenase activity which showed 10-fold less hydrogen uptake upon expressing HoxEFUYH and by a GC-based hydrogen uptake assay which indicated that hydrogen uptake activity in TG1/pBS(Kan)Synhox is 2.1 ± 0.2-fold less than TG1/pBS(Kan) over 0 to 6 h. Hence, the active cyanobacterial enzymes (HoxEFUYH) inhibit hydrogen uptake consistently in E. coli. The effect of inhibition of hydrogen uptake by HoxEFUYH was still detectable in a hycE or hycG mutation for hydrogenase 3; however, deleting both hydrogenase 1 and hydrogenase 2 neutralized the inhibition of hydrogen uptake by cyanobacterial bi-directional hydrogenase; therefore, the active cyanobacterial locus inhibits hydrogen uptake by inhibiting native E. coli hydrogenase 1 and hydrogenase 2 activities but not by inhibiting hydrogenase 3 activity. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system (hydrogenase 1, 2, and 3); instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. In conclusion, E. coli TG1 cells with pBS(Kan)Synhox enhanced 41 times more hydrogen yield than those with empty vector pBS(Kan) due to active HoxEFUYH from Synechocystis sp. PCC 6803 (primarily through active HoxUYH). The mechanism for this enhanced hydrogen production is that hydrogen is formed first by hydrogenase 3 (so the HoxEFUYH effect relies on active hydrogenase 3 and its maturation proteins), then HoxEFUYH inhibits hydrogen uptake by E. coli native hydrogenase 1 and hydrogenase 2. In effect, a novel way to reduce reversible hydrogen formation has been discovered using a cyanobacterial locus. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host.