Arabinose-Inducible Univariant Control System (AUCS) for Microbial Production of Proteins, Enzymes, and Metabolites

Precise control of gene expression is essential to synthetic biology and metabolic engineering, particularly for microbial production. The widely used IPTG-inducible T7lac promoter (P_T7lac) offers strong expression but suffers from metabolic burden, inclusion body formation, and induction heterogeneity. Conversely, the arabinose-inducible araBAD promoter (P_BAD) provides tight regulations but yields modest expression levels, is incompatible with glucose media, and requires high inducer concentrations (20-100 mM). We introduce the arabinose-inducible univariant control system (AUCS), a robust, tightly regulated, and low-cost expression platform designed to combine the strengths of P_T7lac and P_BAD while overcoming their drawbacks. AUCS eliminates carbon catabolite repression and minimizes induction heterogeneity via the constitutive expression of the arabinose transporter AraE. Disruption of the arabinose catabolism enables maximal protein output with only 3 μM l-arabinose, orders of magnitude lower than P_T7lac and P_BAD systems, achieving a >99% reduction in inducer cost. Leveraging a customized promoter library (P_TA1-3), AUCS enables the precise, high-yield expression of single proteins, multienzyme operons, and complex biosynthetic pathways (>10 genes). Benchmarked against P_T7lac, AUCS achieved comparable or superior yields of proteins (the egg-white protein ovalbumin), enzymes (terpene synthases, carotenoid cleavage dioxygenases), and secondary metabolites (linalool, nerolidol, and sclareol) while maintaining outstanding reproducibility and stability over 36 generations. AUCS represents a powerful advancement for precision fermentation, enabling sustainable and cost-effective production of high-value biomolecules and substantially reducing the environmental footprint of chemical manufacturing.