As archetypes of O2 tolerant hydrogenases, the R. eutropha enzymes have been extensively studied using various spectroscopic techniques and site directed muta­genesis (reviewed in [ 17, 36 ] ) . Although these studies have proven that direct manipulation of the hydrogenases is possible, it has also shown the difficulty of improving activity of the enzymes. Therefore, direct manipulation of the hydroge — nases to optimize the generation of energy and reducing equivalents for IBT produc­tion would be a significant challenge, and likely not a viable approach. In contrast, hydrogenase gene expression will be enhanced to assure that sufficient reducing equivalents will be available for optimal IBT yield.

To optimize IBT yield, it is important to balance the reducing equivalents gener­ated by the SH with the ATP synthesized by the MBH. Since carbon fixation will be maximized and the IBT synthesis pathway consumes two NAD(P)H molecules for every two molecules of pyruvate reduced to IBT, the hydrogenase activity balance will be shifted towards the SH.

3.2 Enhancement of Carbonic Anhydrase

Because of the inefficiency of the enzyme, the CO2 fixation by RuBisCO is likely the limiting step for an efficient production of IBT by R. eutropha. Enhancing expression and/or activity of CAs could help to increase the CO2 concentration in the cytosol, thereby limiting the competing oxygenation reaction by RuBisCO and increasing the CO2 flux through the CBB cycle and subsequently to IBT.

4 Outlook