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

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.