High-Level Production of Short- and Medium-Chain-Length Polyhydroxyalkanoates from Glucose Using Metabolically...

The growing demand for bio-based and biodegradable plastics has intensified interest in producing polyhydroxyalkanoates (PHAs). Short- and medium-chain-length (SCL-MCL)-PHA copolymers are particularly attractive because of their enhanced flexibility and desirable thermal properties. However, their high-level production from inexpensive carbon sources, such as glucose derived from lignocellulosic biomass, remains challenging, largely due to limited precursor supply and inefficient polymerization. Here, we report high-level de novo production of SCL-MCL-PHAs from glucose in metabolically engineered Escherichia coli. We employed a modular metabolic engineering strategy comprising three modules: 1) construction of the 3-hydroxybutyryl-CoA monomer pathway; 2) enhancement of fatty acid biosynthesis to strengthen MCL-fatty acyl-CoA supply; and 3) screening of PHA synthases with broad substrate specificity. PHA synthase (PhaC) variants from Pseudomonas sp. MBEL 6-19 were identified to efficiently polymerize both SCL and MCL monomers. Fed-batch cultures of the engineered strains achieved two distinct outcomes: one strain produced 82.88 g L^-1 PHA with 5 mol% MCL fraction, while another accumulated 17.35 g L^-1 PHA with 19.52 mol% MCL fraction. Notably, these values fall within the 5-20 mol% MCL fraction range allowing good polymer applications, underscoring the industrial relevance of our results. This modular approach provides a versatile framework for tunable, sustainable production of SCL-MCL-PHAs.