Modulating ilvA Encoding Threonine Deaminase for Balanced Growth and PHB Synthesis by Halomonas Grown in Rich Nitrogen Source

Bioplastic poly(3-hydroxybutyrate) (PHB) production by Halomonas bluephagenesis is typically activated under nitrogen limitation, inevitably restricting biomass formation and overall productivity. Here we identified gene ilvA encoding threonine deaminase as a flux-sensitive node linking branched-chain amino-acid synthesis to nitrogen sensing. Complete ilvA deletion or σ⁵⁴ disruption in H. bluephagenesis created a pseudo–nitrogen-limitation state that increased PHB accumulation yet concurrently suppressed microbial growth. To overcome this trade-off, two independent ilvA fine-tuning strategies were evaluated including synthetic sRNA-mediated translational repression and tunable SspB/ClpXP proteolysis. In contrast, complete ilvA deletion created a pseudo-nitrogen-limited state that elevated PHB synthesis however reduced cell growth (cell dry weight or CDW) studied in 7L bioreactors, reaching only 60 g/L CDW containing 80% PHB, far below the wild-type grown to 95 g/L CDW containing 60% PHB under same conditions, illustrating that binary ilvA removal destroyed the growth-PHB production balance. Under nitrogen rich fed-batch conditions, however, sRNA-based partial repression maintained CDW at 95 g/L while increasing PHB from 60% to 80 wt%, thereby establishing a growth-PHB production balance window. The study demonstrates that controllable attenuation rather than elimination of amino acid synthesis gene ilvA provides a robust and industrially scalable strategy for simultaneously enhancing PHB synthesis and cell growth.