High‐Level Production of Short‐ and Medium‐Chain‐Length Polyhydroxyalkanoates from Glucose Using Metabolically Engineered <i>Escherichia coli</i>

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.