Coproduction of Phase-Separated Carotenoids and β-Farnesene as a Yeast Biomass Valorization Strategy

that had previously been engineered to produce high concentrations (>100 g/L) of extracellular β-farnesene was further engineered to simultaneously produce intracellular carotenoids, both products being isoprenoids. Thus, a single fermentation generates two valuable products, namely, β-farnesene in the liquid phase and carotenoids in the solid biomass phase. Initial attempts to produce high levels of canthaxanthin (a ketocarotenoid used extensively in animal feed) in a β-farnesene production strain negatively impacted both biomass growth and β-farnesene production. A refined approach used a promoter titration strategy to reduce β-carotene production to a level that had minimal impact on growth and β-farnesene production in fed-batch fermentations and then engineered the resulting strain to produce canthaxanthin. Further optimization of canthaxanthin coproduction used a bioprospecting approach to identify ketolase enzymes that maximized conversion of β-carotene to canthaxanthin. Finally, we demonstrated that β-carotene is not present in the extracellular β-farnesene at a significant concentration and that which is present can be removed by a simple distillation, indicating that β-farnesene (the primary fermentation product) purity is unaffected by coproduction of carotenoids.