Repurposing a Fully Reducing Polyketide Synthase toward 2-Methyl Guerbet-like Lipids

In nature, thousands of diverse and bioactive polyketides are assembled by a family of multifunctional, “assembly line” enzyme complexes called polyketide synthases (PKS). Since the late 20th century, there have been several attempts to decode, rearrange, and “reprogram” the PKS assembly line to generate valuable materials such as biofuels and platform chemicals. Here, the first module from Mycobacterium tuberculosis (Mt) PKS12, an unorthodox, “modularly iterative” PKS, was modified and repurposed toward the formation of 2-methyl Guerbet lipids, which have wide applications in industry. We established a robust method for the recombinant expression and purification of this modified module (named [M₁*]), and we demonstrated its ability to catalyze the formation of several 2-methyl Guerbet-like lipids (C₁₃–C₂₁). Furthermore, we studied and applied the promiscuous thioesterase activity of a neighboring β-ketoacyl synthase (KS) to release [M₁*]-bound condensation products in a one-pot biosynthetic cascade. Finally, starting from lauric acid, we could generate our primary target compound (2-methyltetradecanoic acid) by coupling the Escherichia coli fatty acyl-CoA synthetase FadD to [M₁*]. This work supports the biosynthetic utility of engineered PKS modules such as [M₁*] and their ability to derive valuable Guerbet-like lipids from inexpensive fatty acids.