Metabolic engineering of for anaerobic isobutanol production.

Background: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery.

Results: In this study, the effect of isobutanol on was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as from is required for isobutanol production in . Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the gene driven by the tetracycline-inducible promoter . In addition, we determined that overexpression of a heterologous gene and two native genes ( and ) involved in valine metabolism in a recombinant strain expressing can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, --, driven by and the gene driven by the constitutive strong promoter, , were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks.

Conclusions: This study demonstrated that overexpression of in combination with a synthetic heterologous operon, --, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in .