Improving isobutanol production with the yeast by successively blocking competing metabolic pathways as well as ethanol and glycerol formation.

BACKGROUND

Isobutanol is a promising candidate as second-generation biofuel and has several advantages compared to bioethanol. Another benefit of isobutanol is that it is already formed as a by-product in fermentations with the yeast , although only in very small amounts. Isobutanol formation results from valine degradation in the cytosol via the Ehrlich pathway. In contrast, valine is synthesized from pyruvate in mitochondria. This spatial separation into two different cell compartments is one of the limiting factors for higher isobutanol production in yeast. Furthermore, some intermediate metabolites are also substrates for various isobutanol competing pathways, reducing the metabolic flux toward isobutanol production. We hypothesized that a relocation of all enzymes involved in anabolic and catabolic reactions of valine metabolism in only one cell compartment, the cytosol, in combination with blocking non-essential isobutanol competing pathways will increase isobutanol production in yeast.

RESULTS

Here, we overexpressed the three endogenous enzymes acetolactate synthase (Ilv2), acetohydroxyacid reductoisomerase (Ilv5) and dihydroxy-acid dehydratase (Ilv3) of the valine synthesis pathway in the cytosol and blocked the first step of mitochondrial valine synthesis by disrupting endogenous , leading to a 22-fold increase of isobutanol production up to 0.22 g/L (5.28 mg/g glucose) with aerobic shake flask cultures. Then, we successively deleted essential genes of competing pathways for synthesis of 2,3-butanediol ( and ), leucine ( and ), pantothenate () and isoleucine () resulting in an optimized metabolic flux toward isobutanol and titers of up to 0.56 g/L (13.54 mg/g glucose). Reducing ethanol formation by deletion of the gene encoding the major alcohol dehydrogenase did not result in further increased isobutanol production, but in strongly enhanced glycerol formation. Nevertheless, deletion of glycerol-3-phosphate dehydrogenase genes and prevented formation of glycerol and increased isobutanol production up to 1.32 g/L. Finally, additional deletion of aldehyde dehydrogenase gene reduced the synthesis of the by-product isobutyrate, thereby further increasing isobutanol production up to 2.09 g/L with a yield of 59.55 mg/g glucose, corresponding to a more than 200-fold increase compared to the wild type.

CONCLUSIONS

By overexpressing a cytosolic isobutanol synthesis pathway and by blocking non-essential isobutanol competing pathways, we could achieve isobutanol production with a yield of 59.55 mg/g glucose, which is the highest yield ever obtained with in shake flask cultures. Nevertheless, our results indicate a still limiting capacity of the isobutanol synthesis pathway itself.