Wild-type R. eutropha will accumulate up to 80% of its dry cell mass as the carbon storage compound PHB when it encounters nutrient stress [34] . During this strin­gent response, R. eutropha stops growth and slows down central metabolism (Brigham et al., manuscript in preparation) so that most of the intracellular carbon flux is redirected towards PHB synthesis.

Eliminating the PHB synthesis pathway from R. eutropha causes an overflow of the intermediate pyruvate during nutrient starvation, which is excreted to prevent toxicity [35]. Because pyruvate feeds the BCAA synthesis pathway used to generate IBT [4], nutrient starvation could be used to maximize carbon flux towards IBT.

2 R. eutropha IBT Production Pathway

2.1 Hydrogenase Enzymes

Hydrogenases are classified in three families, the [Fe]-hydrogenases, the [FeFe]- hydrogenases, and the [NiFe]-hydrogenases, based on their catalytic-site metal cofactors [21]. Only the NiFe hydrogenases are discussed here, as all three hydro­genases present in R. eutropha belong to this family [17]. Furthermore, the RH are not discussed since it does not have a role in the hydrogen metabolism of R. eutro­pha H16 [22]. As mentioned previously, the two types of energy conserving hydro — genases, MBH and SH, produce the energy and reducing equivalents supporting autotrophic growth of R. eutropha. Because of their oxygen tolerance, these hydro­genases in R. eutropha have been studied in detail (reviewed in [17, 36]).

MBH uses extracellular hydrogen to provide reducing equivalents to the respira­tory chain, allowing the four-electron reduction of O2 to H2O. The electrons gained in oxidation of hydrogen at the NiFe catalytic site are transported through the three iron-sulfur (FeS) clusters of the electron transfer subunit to a cytochrome b in the membrane anchor [37]. From cytochrome b the electrons are directed into the qui — none pool. The proximal FeS cluster, closest to the catalytic site, is critical for oxy­gen tolerance of the MBH, which is inferred from rapid reduction of O2 bound to the catalytic site [38]. The required electrons for the three-electron reduction of the peroxide radical, bound to the active site after oxidation by O2, are transferred from the quinone pool.

The cytoplasmic SH uses intracellular H2 to directly reduce NAD+ to NADH, through electron transfer from its catalytic subunit, through an FeS-containing elec­tron transfer subunit, to a flavin containing diaphorase moiety consisting of two subunits [19]. The remaining two subunits create a binding pocket for NADPH, which is required for catalytic activation of the enzyme [39]. The autotrophic growth rate of R. eutropha decreases twofold in an SH deletion mutant, illustrating that it is necessary to support the high reducing equivalent requirement of the CBB cycle [20]. The oxygen tolerance of the SH is thought to have a different molecular basis than that of the MBH, since it cannot access the quinone pool as rapidly. The coor­dination sphere of the NiFe active site is proposed to contain two additional cyanide (CN) ligands, one coordinating each metal atom [40, 41]. However, a recent study suggests that these two CN ligands are not present in situ, suggesting an alternative, yet to be elucidated, mechanism for oxygen tolerance [42] .

In summary, both types of energy-conserving hydrogenase are oxygen tolerant at ambient concentrations and serve a distinct purpose in maintaining the energy bal­ance of the cell. Optimal IBT production requires a balance of both hydrogenase activities.