Modular co-culture engineering of Escherichia coli and Saccharomyces cerevisiae for de novo biosynthesis of tryptophol from glucose.

Microbial co-cultures enable modular division of labor between distinct species, providing a robust platform for complex bioconversions. Here, we established an Escherichia coli-Saccharomyces cerevisiae co-culture system that achieves efficient de novo biosynthesis of tryptophol (indole-3-ethanol, IET) directly from glucose. The upstream E. coli module was engineered to overproduce l-tryptophan (l-Trp) through coordinated overexpression of aroG, serA, and trpEDCBA, reaching 1.01 g/L l-Trp after 48 h of cultivation in LB-containing shake flasks. The downstream S. cerevisiae module was optimized via reinforcement of the Ehrlich pathway (upregulating ARO9, ARO10, and SFA1), transporter engineering (overexpressing TAT1), and genome model simulation-guided deletion of UGA1, collectively enhancing the uptake of l-Trp and the efficiency of its conversion to IET. Fluorescence-based population tracking confirmed stable coexistence and tunable competition of the populations of E. coli and S. cerevisiae. Further systematic optimization of inoculum ratio and fermentation parameters enabled balanced metabolic coupling, yielding a final IET titer of 2.14 g/L in a 5 L bioreactor in 80 h (productivity: 0.027 g/L/h), representing the highest reported titer from renewable carbon sources. This work demonstrates a rational, modular co-culture strategy that integrates metabolic engineering, computational design, and process optimization to achieve sustainable biosynthesis of aromatic alcohols.