| Summary: | The ever-increasing global energy demand, juxtaposed with critical concerns about greenhouse gas emissions, emphatically underscores the urgency to pivot toward sustainable and eco-friendly energy alternatives. Tapping into microbial metabolism for clean energy generation stands out as a particularly promising avenue in this endeavor. Given this backdrop, we delved deeply into the metabolic engineering potential of <i>Saccharomyces cerevisiae</i>, thereby aiming for the bioconversion of formate and acetate—both CO<sub>2</sub> derivatives—into free fatty acids (FFAs) as precursors for biofuel production. Our study not only elucidated the metabolic pathways within <i>S. cerevisiae</i> that are tailored for efficient formate and acetate utilization but also shone a light on the meticulous optimization strategies that amplify FFA synthesis. The engineered strains, under refined conditions, exhibited up to an 8-fold increase in an FFA titer, thus reaching a production level of 6.6 g/L, which showcases the potential of microbial metabolism in clean energy generation. Our findings offer a promising step toward harnessing microbial metabolism for sustainable energy production, thereby bridging the gap between waste carbon utilization and greener fuel alternatives.
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