Assay of Isobutanol Production Using Biofuel Producing <em>E</em>. <em>coli </em>Strains

The increasing demand for sustainable energy has driven research into biofuels as alternatives to fossil fuels. Isobutanol, a higher-chain alcohol, presents advantages over ethanol due to its higher energy density and compatibility with existing fuel infrastructure. This study evaluates the efficiency of three alcohol dehydrogenases—YqhD from Escherichia coli, ADH2 from Saccharomyces cerevisiae, and adhA from Lactococcus lactis—in catalyzing isobutanol production in engineered E. coli strains. An IPTG-inducible expression system was used to overexpress these enzymes, and isobutanol production was quantified via a colorimetric enzyme assay. The results showed that adhA exhibited the highest isobutanol yield (112.19 mM), the greatest molecular production per cell (7.089 × 10¹³ molecules/cell), and the highest yield relative to glucose consumption (0.277 g isobutanol/g glucose). YqhD produced moderate levels, while ADH2 showed inconsistent results, likely due to misfolding and aggregation. These findings confirm adhA as the most efficient enzyme for isobutanol biosynthesis, supporting its potential for metabolic engineering applications aimed at optimizing biofuel production. Future research should focus on refining expression conditions and scaling up production to enhance the viability of isobutanol as a renewable fuel source.