Localising enzymes to biomolecular condensates increases their accumulation and benefits engineered metabolic pathway performance in <i>Nicotiana benthamiana</i>

The establishment of Nicotiana benthamiana as a robust biofactory is complicated by issues such as product toxicity and proteolytic degradation of target proteins/introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered biomolecular condensates in N. benthamiana leaves using transient expression of synthetic modular scaffolds. The in vivo properties of the condensates that resulted were consistent with them being liquid-like bodies with thermodynamic features typical of multicomponent phase-separating systems. We show that recruitment of enzymes to condensates in vivo led to several-fold yield increases in one- and three-step metabolic pathways (citramalate biosynthesis and poly-3-hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced yield could be for several reasons including improved enzyme kinetics, metabolite channelling or avoidance of cytotoxicity by retention of the pathway product within the condensate, which was demonstrated for PHB. However, we also observed a several-fold increase in the amount of the enzymes that accumulated when they were targeted to the condensates. This suggests that the enzymes were more stable when localised to the condensate than when freely diffusing in the cytosol. We hypothesise that this stability is likely the main driver for increased pathway product production. Our findings provide a foundation for leveraging biomolecular condensates in plant metabolic engineering and advance N. benthamiana as a versatile biofactory for industrial applications.