Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways.
NAD(P)H:H(2) pathways are theoretically predicted to reach equilibrium at very low partial headspace H(2) pressure. An evaluation of the directionality of such near-equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H(2) pathways; however, they are either not genetically tractable, and/or contain multiple H(2) synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin-dependent NAD(P)H:H(2) pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine-dependent H(2) synthesis under closed batch conditions. NADPH-dependent H(2) accumulation was observed with a maximum partial H(2) pressure equivalent to a biochemically effective intracellular NADPH/NADP(+) ratio of 13:1. The molar yield of the NADPH:H(2) pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace:liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H(2) consumption, was observed above a partial H(2) pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H(2), is more likely to be applicable for H(2) production.
Fujirebio Inc., Frontier Research Department, 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan.
Andrea
A
Veit
M Kalim
MK
Akhtar
Taeko
T
Mizutani
Patrik R
PR
Jones
D053898:Biosynthetic Pathways; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D009243:NAD / Q000096:biosynthesis*; D009249:NADP / Q000096:biosynthesis*; D019289:Pyruvic Acid / Q000378:metabolism; D013816:Thermodynamics
D005288:Ferredoxins; D009243:NAD; D009249:NADP; D006859:Hydrogen; D019289:Pyruvic Acid
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