Modular Pathway Engineering of an Escherichia coli Platform for De Novo Synthesis of Steviol Glycoside Precursors from Glucose

Abstract Steviol glycosides (SGs) are high-potency, zero-calorie sweeteners widely used in the food industry. Their aglycone backbone, steviol (STV), is a highly oxidized diterpenoid and a crucial precursor for the synthesis of various SGs. Traditional extraction from plants suffers from low yield and cumbersome purification. In contrast, microbial synthesis offers a sustainable alternative. An Escherichia coli platform was established for de novo biosynthesis of STV from glucose. The synthetic pathway was constructed by integrating three modules: precursor synthesis, terpene synthesis, and P450 oxidation. Protein fusion tags enhanced geranylgeranyl diphosphate synthase (GGPPS) expression, while N-terminal truncation enhanced P450 expression. Plasmid copy number modulation balanced the expression of each module. Finally, the STV titer reached 110.78 mg/L after 132 h of fed-batch fermentation, with a productivity of 0.84 mg/L/h.