Stability of a Mutualistic <i>Escherichia coli</i> Co‐Culture During Violacein Production Depends on the Kind of Carbon Source

The L-tryptophan-derived purple pigment violacein (VIO) is produced in recombinant bacteria and studied for its versatile applications. Microbial synthetic co-cultures are gaining more importance as efficient factories for synthesizing high-value compounds. In this work, a mutualistic and cross-feeding <i>Escherichia coli</i> co-culture is metabolically engineered to produce VIO. The strains are genetically modified by auxotrophies in the tryptophan (TRP) pathway to enable a metabolic division of labor. Therein, one strain produces anthranilate (ANT) and the other transforms it into TRP and further to VIO. Population dynamics and stability depend on the choice of carbon source, impacting the presence and thus exchange of metabolites as well as overall VIO productivity. Four carbon sources (D-glucose, glycerol, D-galactose, and D-xylose) were compared. D-Xylose led to co-cultures which showed stable growth and VIO production, ANT-TRP exchange, and enhanced VIO production. Best titers were ∼126 mg L^-1 in shake flasks. The study demonstrates the importance and advantages of a mutualistic approach in VIO synthesis and highlights the carbon source's role in co-culture stability and productivity. Transferring this knowledge into an up-scaled bioreactor system has great potential in improving the overall VIO production.