Inhibition Isobutanol

Therefore, there are already suitable isobutanol producers that could be good candidates for this pathway, with the appropriate optimisations. For example, for the production of 1 mol isobutanol, 1 mol NADPH would be consumed and 1 mol NADH produced this might lead to NADH/NADPH imbalance inhibiting isobutanol formation. This could be prevented by a transhydrogenase and by NADPH production over pentose phosphate pathway (van Leeuwen et al. 2012 Blombach et al. 2011 Sauer et al. 2004). 

CPT was determined using a radiochemical isotope forward assay as described previously. In brief, the assay system contained lOmM Tris-HCl-buffer (pH 7.6), 0,1% fatty acid-free bovine serum albumin, 1 mM dithiothreitol, 0.08 mM palmitoyl-CoA and 5 mM [ CJ-camitine in a final volume of 1 ml and the assay temperature was 30 °C. Experiments were performed either in the presence or in the absence of 0.4 mM malonyl-CoA to inhibit CPT I. Concentration of L-AC was varied between 0 and 30mM. Incubations were started by the addition of homogenate and stopped after lOmin by addition of ammonium sulfate and isobutanol. Labelled pahnitoylcamitine was measured after separation of the organic phase using a scintillation counter (Beckmann LS 6500). 

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