Development of <i>Photobacterium</i> sp. LN01 as a Versatile Halophilic Platform for Tunable Biosynthesis of Polyhydroxyalkanoates

Poly(3-hydroxybutyrate-<i>co</i>-4-hydroxybutyrate) (P34HB) is a biodegradable polyester with tunable properties depending on its 4HB monomer content. Here, we engineered the newly isolated halophilic bacterium, <i>Photobacterium</i> sp. LN01, for efficient P34HB production. The wild-type strain naturally synthesizes poly(3-hydroxybutyrate) (PHB) from carbon sources such as sucrose and glycerol and produces P34HB when supplemented with γ-butyrolactone (GBL) as a cosubstrate. A genetic toolbox comprising conjugation and genome editing was developed for targeted metabolic engineering. Heterologous expression of the CoA transferase gene <i>orfZ</i> from <i>Clostridium kluyveri</i>, first on a low-copy plasmid and then through chromosomal integration at the <i>phaC</i> locus, increased 4HB incorporation from ∼9 to ∼30 mol %. Further replacement of the lactate dehydrogenase gene with the <i>orfZ</i> expression module suppressed lactate formation and yielded up to 16.45 g/L P34HB with 39.79 mol % 4HB in shake flasks. Finally, fine-tuning the expression of <i>orfZ</i> minimized the metabolic burden during bioreactor cultivation, achieving 103.10 g/L P34HB with 13.02 mol % 4HB. The GBL conversion rate was 71.28%, representing the highest level reported to date. This work establishes <i>Photobacterium</i> sp. LN01 as a promising chassis for efficient and tunable P34HB biosynthesis.