Engineering Novel Polyhydroxyalkanoates Using Polyketide Synthases in Pseudomonas putida

ACS Synthetic Biology, vol. 15, pp. 3712–3726

Abstract

Abstract Polyhydroxyalkanoates (PHAs) are bio-based polyesters with the potential to replace petroleum-based products. However, their thermal and mechanical properties are limited by the composition of the monomers used to make them. Current approaches to expanding monomer diversity require individualized pathway engineering for each targeted monomer, limiting the scalability of PHA design. In this work, we establish polyketide synthases (PKSs) as a modular and programmable platform for expanding PHA monomer diversity with variable chain length and branching. Using Pseudomonas putidaas a host, we integrate four engineered PKSs and their variants with heterologous CoA-activating enzymes and short-chain-length PHA polymerases to produce diverse (R)-3-hydroxyacid monomers with defined substitution and branching at the ɑ- and β-positions and enable their in vivo polymerization. We also apply active learning-assisted directed evolution to improve the heterodimeric PHA polymerase CapPhaEC, achieving a 3.6-fold increase in incorporation of α-branched monomers. Leveraging the modularity of PKSs, we demonstrate the biosynthesis and polymerization of linear, branched, and aromatic monomers by exchanging acyltransferase (AT) and ketoreductase (KR) domains to control chain length, branching, and stereochemistry. This study illustrates the utility of using a flexible, programmable PKS platform for rapidly designing and testing new-to-nature PHAs.

Authors 10

1. Leah S. Keiser

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

2. Aalini Jain

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

3. Panarai Primrose Gatenil

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

4. Matthias Schmidt Aachen

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute · RWTH Aachen University

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   RWTH Aachen University , , ,

   University of California , , , ,

5. Yolanda Zhu

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

6. Cholapat Varongchayakul

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

7. Amarrah Crysti Len

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

8. Kyrellos E. Ibrahim

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   Lawrence Berkeley National Laboratory , , , ,

   University of California , , , ,

9. Elias Englund

   Joint BioEnergy Institute · KTH Royal Institute of Technology

   Affiliation as printed

   Joint BioEnergy Institute , 5885 Hollis Street , , ,

   KTHRoyal Institute of Technology , , ,

10. Jay D. Keasling

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Joint BioEnergy Institute , 5885 Hollis Street , , ,

    Lawrence Berkeley National Laboratory , , , ,

    University of California , , , ,