Engineering substrate channeling with synthetic biomolecular condensates for improved ectoine biosynthesis

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Ectoine is a microbial protectant with applications in biomedicine and cosmetics.
• Heterologous ectoine production is limited by pathway efficiency in non-halophilic hosts.
• Ancestral enzyme reconstruction offers a path to improved metabolic functions.

Purpose of the Study:

• To enhance ectoine biosynthesis in engineered Vibrio natriegens.
• To overcome efficiency bottlenecks in ectoine production pathways.
• To develop a scalable platform for industrial ectoine manufacturing.

Main Methods:

• Reconstruction of ancestral metabolic enzymes (EctA, EctB, EctC) using bioinformatics.
• Molecular dynamics simulations for enzyme analysis.
• Construction of synthetic biomolecular condensates using a Pum2-ELP fusion system.
• Systematic evaluation of 27 combinatory biosynthetic pathways.
• Fermentation in a 5-L bioreactor.

Main Results:

• Optimal hybrid pathway identified with synergistic catalytic efficiency.
• Pum2-ELP system increased ectoine yield by approximately 1.7-fold via substrate channeling.
• Engineered strain achieved a final titer of ~50 g/L within 24 hours.
• High volumetric productivity of 2.08 g/(L·h) was reached.

Conclusions:

• Integrating ancestral enzymes with engineered spatial assembly optimizes metabolic flux.
• The synthetic biomolecular condensate system enhances ectoine production efficiency.
• This strategy enables rapid, high-titer industrial production of ectoine and other compatible solutes.