Systematic metabolic engineering of Photobacterium sp. TLY01 for high-yield biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate- co -4-hydroxybutyrate) (P34HB) is a biodegradable copolymer with considerable potential in biomedical and industrial applications. This study presents the first systematic metabolic engineering of Photobacterium sp. TLY01 for efficient production of P34HB. Multiple pathways for 4-hydroxybutyrate (4HB) monomer supply were engineered, including those utilizing γ-butyrolactone (GBL), 1,4-butanediol (BDO), and a de novo route from glycerol. Notably, Photobacterium sp. TLY01 demonstrated remarkable tolerance to GBL, and achieved up to 90 mol% 4HB incorporation via heterologous expression of the CoA transferase gene orfZ . The introduction of dhaT and aldD from Pseudomonas putida enabled 4HB synthesis from BDO. To establish a fully glycerol-based system, a succinate-derived 4HB pathway from Clostridium kluyveri was introduced, while three native gabD genes were deleted to redirect carbon flow toward 4HB formation, enabling P34HB production without exogenous precursors. Furthermore, deletion of gltA combined with supplementation of tricarboxylic acid cycle intermediates enhanced polymer biosynthesis, increasing P34HB titers by 3–4 times to approximately 15 g/L in shake-flasks. Fed-batch fermentation using the engineered strain TPS4O Δ gltA achieved a P34HB titer of 90.61 g/L with a yield of 0.44 g/g glycerol. These results establish a sustainable and scalable platform for P34HB production using a Vibrionaceae chassis. • First systematic metabolic engineering of Photobacterium strain for high-efficiency P34HB biosynthesis. • Diverse 4HB pathways engineered, including GBL, BDO, and a de novo route from glycerol. • Exceptional GBL conversion rate (>95%) achieved through targeted CoA transferases enhancement. • TCA cycle tuning boosted P34HB production 3–4 folds to ∼15 g/L in flasks and 90.61 g/L in fed-batch fermentation.