Aqueous-Phase Polycondensation of Hydroxy Fatty Acids via a Whole-Cell CoA Activation–Acyltransferase Cascade

High Resolution Image Download MS PowerPoint Slide Polyester formation by polycondensation in water is limited by an esterification–hydrolysis equilibrium that strongly favors hydrolysis under mild conditions. Although aqueous-phase esterification has been demonstrated with chemical catalysts and isolated enzymes, these systems typically target small-molecule esters or rely on preactivated donors. The polyhydroxyalkanoate (PHA) pathway is the only known native metabolic route for polyester biosynthesis, but it is essentially limited to the polymerization of 3-hydroxybutyrate and related short-chain hydroxyalkanoate monomers. Consequently, realizing efficient aqueous-phase polycondensation to high-molar-mass polyesters remains a major challenge. Here, an intracellular CoA activation/acyltransferase cascade (ACOS5 At -WS2 Mh ) for poly(hydroxy fatty acid) (PHFA) biosynthesis is constructed within a whole-cell catalyst. Mechanistic analysis indicates that a mildly hydrophilic region lining the ACOS5 At substrate tunnel is critical for ω-hydroxy fatty acid (ωHFA) recognition and activation in water. The substantial steric bulk of ω-hydroxyacyl-CoA (ωHFA-CoA) hinders its entry into the WS2 Mh hydroxyl-donor channel, enforcing a hydroxyl-terminal chain-growth mode, whereas the higher diffusivity and lower steric hindrance of short oligomers underlie the low dispersity of the resulting PHFA. Chassis engineering, catalytic optimization, and a substrate-channeling fusion-protein strategy collectively increase the cascade flux and the product titer. Under optimized conditions, a PHFA titer of 1.87 g L –1 ( M n,app = 10.8 kDa; Đ = 1.05) was obtained in a 3 L bioreactor, yielding polymers with heterotelechelic hydroxyl/carboxyl chain ends. This work establishes a green, fully aqueous, whole-cell route from unactivated ωHFAs to non-PHA polyesters and provides general design principles for engineering living catalysts capable of overcoming hydrolysis-limited equilibria in condensation polymerization.