Microbial synthesis of polyhydroxyalkanoate blends with engineered Pseudomonas putida

Abstract

Polyhydroxyalkanoates (PHAs) are biopolymers naturally produced by various microorganisms and offer a sustainable alternative to fossil fuel-derived plastics. They can be synthesized from diverse feedstock, including waste biomass such as lignocellulose, municipal waste, sludge, and industrial by-products. To tailor their properties for specific applications, PHAs are typically blended post synthesis. An alternative approach is the direct synthesis of PHA blends in a single fermentation, which can reduce the need for multiple separate fermentations and extractions. In this study, we engineered Pseudomonas putida to synthesize PHA blends composed of poly-3-hydroxybutyrate [P3(HB)] and medium-chain-length PHA (mcl-PHA). Through using different promoters, blends with 3HB monomer content ranging from 17.9 mol% to 99.6 mol% were produced. Optimizing cultivation conditions yielded a maximum PHA production of 1.48 ± 0.15 g/L, with a PHA content of 52.2 ± 4.3 wt% of cell dry weight. A combination of gel permeation chromatography, nuclear magnetic resonance and diffusion ordered spectroscopy were employed to determine the molecular weight and confirm the identity of the PHA blend, revealing in all cases, a higher molecular weight P(3HB) than mcl-PHA. The blends produced had thermal properties comparable to PHA blends produced by post synthesis melt compounding. This work demonstrates the microbial synthesis of PHA blends in P. putida and is the first instance of blend composition control via promoter selection, paving the way for the one-step biomanufacturing of customizable PHA blends.