Protein phase separation for enhanced production of 3-hydroxypropionate and polyhydroxybutyrate by Halomonas

Microbial cell factories for sustainable bioproduction often suffer from trade-offs between cell growth and product synthesis. Spatiotemporal regulation of metabolic pathways offers a potential solution to this challenge. In this study, we engineered synthetic membraneless organelles (MLOs) based on liquid-liquid phase separation (LLPS) in the halophilic bacterium Halomonas bluephagenesis, a robust chassis for next-generation industrial biotechnology (NGIB) operated under open unsterile conditions. Using a tandem Arginine-Glycine-Glycine (RGG) domain as an intrinsically disordered protein scaffold, dynamic protein condensates were constructed capable of recruiting cargo proteins via coiled-coil peptide interactions. Compartmentalization of the 3-hydroxypropionic acid (3HP) biosynthetic enzymes AdhP and AldD into MLOs enhanced 3HP production by up to 91.3%. Furthermore, by fusing the LLPS-promoting WGR-1 peptide to the RGG scaffold, high-rigidity MLOs (hMLOs) were constructed enabling functional insulation of essential endogenous enzymes. Recruitment of bacterial fission ring protein FtsZ to hMLOs resulted in the cell elongation, while sequestration of citrate synthase (GltA) or respiratory chain enzyme IspB led to significant improvement in PHB production. The GltA- and IspB- recruited H. bluephagenesis TP03-2 and TP04-2 grown in shake flasks presented a 22.5% and 16.7% increase on PHB synthesis, a 6.5% and 6.8% increase on glucose-to-PHB conversion rate, respectively. A 5% increase on glucose conversion efficiency was observed for H. bluephagenesis TP03-2 incubated in a 7L bioreactor. A versatile MLO-based toolbox is established for engineering non-model industrial Halomonas.