Biosynthesis of poly(6-hydroxyhexanoate) [poly(ε-caprolactone)] using engineered polyhydroxyalkanoate synthetic...

Poly(ε-caprolactone) (PCL), also known as poly(6-hydroxyhexanoate) [P(6HHx)], is a biodegradable polyester characterized by excellent flexibility, processability, and marine degradability, making it a promising alternative to conventional plastics. However, current chemical syntheses of PCL rely on metal-catalyzed ring-opening polymerization of ε-caprolactone, raising concerns about metal contamination and environmental sustainability. Here, we report a biological method to synthesize PCL [P(6HHx)] using an engineered polyhydroxyalkanoate (PHA) system in Escherichia coli. An artificial PHA synthase (PhaC), FcPhaC4, designed via a full-consensus design algorithm to enhance structural stability and broaden substrate specificity, was employed for polymer production. E. coli expressing FcPhaC4 and cultivated with the supplementation of 6HHx synthesized polymer, confirmed to be PCL by ¹H/¹³C Nuclear Magnetic Resonance and Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry analyses. The F313Y mutant of FcPhaC4 further improved PCL yield. In addition, using these enzymes, random copolymer P(3-hydroxybutyrate-co-6HHx) was synthesized at desired monomer compositions. In vitro assays demonstrated that FcPhaC4 and its mutant exhibited activity toward 6HHx-CoA as a sole substrate, being consistent with their homopolymer-producing capacity. These results indicated that FcPhaC4 is the first enzyme capable of biologically synthesizing PCL homopolymer.