Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways
Article first published online: 11 MAY 2008
© 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd
Volume 1, Issue 5, pages 382–394, September 2008
How to Cite
Veit, A., Akhtar, M. K., Mizutani, T. and Jones, P. R. (2008), Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways. Microbial Biotechnology, 1: 382–394. doi: 10.1111/j.1751-7915.2008.00033.x
- Issue published online: 18 AUG 2008
- Article first published online: 11 MAY 2008
- Received 16 October, 2007; revised 27 February, 2008; accepted 29 February, 2008.
NAD(P)H:H2 pathways are theoretically predicted to reach equilibrium at very low partial headspace H2 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:H2 pathways; however, they are either not genetically tractable, and/or contain multiple H2 synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin-dependent NAD(P)H:H2 pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine-dependent H2 synthesis under closed batch conditions. NADPH-dependent H2 accumulation was observed with a maximum partial H2 pressure equivalent to a biochemically effective intracellular NADPH/NADP+ ratio of 13:1. The molar yield of the NADPH:H2 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, H2 consumption, was observed above a partial H2 pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H2, is more likely to be applicable for H2 production.