Among the next-generation biofuels, isobutanol has attracted researchâ??s attention due to a superior combustion energy, a weaker corrosive action and a reduced aqueous miscibility. Moreover, it can be separated from fermentation media by less intensive energy processes. Naturally, Saccharomyces cerevisiae is able to produce isobutanol. Basically, isobutanol can be formed from pyruvate via the L-valine biosynthesis (Ilv2, Ilv5, Ilv3) and degradation (Aro10, Adh2) pathways. Nevertheless, due to a low yield in the natural occurring isobutanol production, this pathway has been focus of optimizations. In our previous results, the first three enzymes of valine biosynthesis were overexpressed as cytosolic isoforms, in order to optimize the pathway flow from pyruvate. The cytosolic isobutanol pathway could rise the isobutanol production to about 650 mg/L (Brat et al. 2012). This same strategy was incorporated into an industrial S. cerevisiae strain (Ethanol Red), resulting in an isobutanol production of just 200 mg/L in 24 hours cultivation. Then, one allele of the PDC1 gene was replaced by a bacterial transhydrogenase (UdhA), for enhancing of the NADPH supply for Ilv5, as well as Aro10 and Adh2, all under strong promoters. This strain was able to produce 300 mg/L. Interestingly, high concentrations of acetoin (200 mg/L), 2,3-butandiol (1.0 g/L) and dihydroxy-isovalerate (2.5 g/L) were found in the medium, but no keto-isovalerate or isobutanal. Drawing on these results, endorsed by a still high production of ethanol and glycerol, Ilv3 could be assigned as one of the bottlenecks of the isobutanol pathway. The dihydroxy-acid dehydratase (Ilv3) is a member of the ILVD_EDD protein family, which is known to comprise iron-sulfur cluster enzymes. However, further overexpression of either a cytosolic or a mitochondrial isoenzyme did not increase isobutanol production. These results show us that the primordial bottleneck surround the low activity of Ilv3 can be probably an inefficient build of the iron-sulfur cluster.
Sponsored by CNPq, Brazil.

Reference:

Brat, D. et al., 2012. Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae. Biotechnology for Biofuels, 5(1), p.65.