Selected PHA Synthases Exhibit Nonstrict Stereospecificity and Produce Stereoirregular Poly(3-hydroxybutyrate) In Vitro

Abstract

Poly(3-hydroxybutyrate) (P3HB) stands out as a microbial biopolymer with biodegradable properties, offering a sustainable alternative to petroleum-based plastic. However, its application is hindered by brittleness and processing challenges, partially attributed to its fully (R)-isotactic stereocenter configuration. The enzymes responsible for P3HB production, polyhydroxyalkanoate (PHA) synthases, are traditionally considered to be strictly stereospecific, causing P3HB isotacticity. In this study, we developed an in vitro assay to analyze the stereospecificity of this enzyme class. Our investigation of PHA synthases reveals that the enzymes from Allochromatium vinosum and Chromobacterium sp. USM2, despite belonging to different PHA synthase classes and having low sequence identity, share the trait of not strictly adhering to stereospecificity. Notably, they demonstrate the capability of producing stereoirregular P3HB in vitro. Furthermore, we identified two key limitations influencing the production of stereoirregular P3HB in vitro. First, PHA synthases appear to require a high (S)- over (R)-substrate ratio. Second, under (R)-limiting conditions, the resulting stereoirregular P3HB exhibits a lower molar mass compared to its isotactic counterpart. Our findings challenge the assumption of universally strict stereospecificity among the PHA synthases tested and identify factors governing stereoirregular P3HB formation under assay conditions. The developed assay may support future enzyme engineering efforts aimed at tuning P3HB stereochemistry and ultimately polymer properties.