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

Area of Science:

• Biotechnology
• Polymer Science
• Synthetic Biology

Background:

• Poly(ε-caprolactone) (PCL) is a biodegradable polyester with desirable properties, but its chemical synthesis involves metal catalysts, posing environmental concerns.
• Developing sustainable and metal-free production methods for PCL is crucial for its wider adoption as an alternative to conventional plastics.

Purpose of the Study:

• To establish a biological method for synthesizing poly(ε-caprolactone) (PCL) homopolymer and copolymers.
• To engineer and utilize a novel polyhydroxyalkanoate (PHA) synthase for PCL production in Escherichia coli.
• To demonstrate the feasibility of metal-free PCL synthesis using engineered enzymes.

Main Methods:

• Engineered an artificial PHA synthase (FcPhaC4) using a full-consensus design algorithm for enhanced stability and substrate specificity.
• Expressed FcPhaC4 in Escherichia coli and cultivated the bacteria with 6-hydroxyhexanoate (6HHx) supplementation for PCL synthesis.
• Confirmed PCL structure using ¹H/¹³C Nuclear Magnetic Resonance and MALDI-TOF Mass Spectrometry; assessed enzyme activity via in vitro assays.

Main Results:

• Successfully synthesized PCL homopolymer using engineered E. coli expressing FcPhaC4 and supplemented with 6HHx.
• Identified an F313Y mutant of FcPhaC4 that further enhanced PCL yield.
• Produced random copolymers of P(3-hydroxybutyrate-co-6HHx) with controlled monomer compositions.
• Demonstrated that FcPhaC4 and its mutant exhibit activity toward 6HHx-CoA as a sole substrate, enabling PCL homopolymer synthesis.

Conclusions:

• FcPhaC4 is the first reported enzyme capable of biologically synthesizing PCL homopolymer.
• This engineered PHA synthase system provides a sustainable, metal-free alternative for PCL production.
• The developed method allows for the synthesis of both PCL homopolymers and P(3-hydroxybutyrate-co-6HHx) copolymers with tunable compositions.