Modulating ilvA encoding threonine deaminase for balanced growth and PHB synthesis by Halomonas grown in rich...

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 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) in 7 L 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, These results indicate that complete deletion of ilvA disrupts the balance between cell growth and PHB production. 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. These results suggest that controllable attenuation of ilvA, instead of full gene deletion, provides a potentially scalable approach for improving PHB production without severely compromising cell growth.