A Toolbox for Biomanufacturing of Functionalised PHA Nanoparticles with C. necator

Microbes have the potential to manufacture plastics from sustainable feedstocks while enabling novel material properties and functions that are not easily accessible through conventional chemical synthesis. Realising this potential requires a comprehensive genetic and process engineering framework that spans chassis and bioprocess optimisation, polymer property control, and downstream functionalisation. Here we develop such a platform in Cupriavidus necator, with a focus on high-value polyhydroxyalkanoate (PHA) nanoparticles. To this end we first optimise the transformation protocol for the organism. Next, we create a library of PhaC synthase variants from C. necator, Aeromonas caviae and Brevundimonas sp. in a {Delta}phaC background, demonstrating that they allow customisation of the material properties of produced PHA particles. Our results combine data from Flow cytometry, Transmission Electron Microscopy (TEM), Fourier Transform InfraRed Spectroscopy (FTIR), and Differential Scanning Calorimetry (DSC) to show that it is possible to generate materials ranging from highly crystalline PHAs to softer P(3HB-co-3HHx) copolymers and that an A. caviae PhaC variant can double the yield of large PHA granules. To improve bioprocess sustainability, we coupled C. necator with B. subtilis in sucrose-fed co-cultures, using tetracycline tolerance differences and inoculation ratios to enhance PHA production from inexpensive, sugar-rich feedstocks. Finally, we add function to the produced PHA nanoparticles by using the molecular protein-fusion technology SpyTag-SpyCatcher, showing it is possible to efficiently capture SpyCatcher-GFP on PHA granules as a proof of concept for PHA's use as a customisable bio-based nanoparticle. Together, our work offers an innovation to produce bio-PHA nanoparticles in a customisable way, with potential applications in sustainable biomanufacturing, biosensing, drug delivery and future bioremediation technologies.