Engineering fungal de novo fatty acid synthesis for short chain fatty acid production

Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker's yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker's yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C₆-FA) and octanoic acid (C₈-FA), of 464 mg l⁻¹ in total. Furthermore, we succeed in the specific production of C₆- or C₈-FA in extracellular concentrations of 72 and 245 mg l⁻¹, respectively. The presented technology is applicable far beyond baker's yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.