In situ engineering of synthetic yeast consortia for cross-species metabolic conversion of crude glycerol and byproducts into circular renewable bioenergy.

The rapid growth of biodiesel production generates large amounts of crude glycerol, a low-value byproduct with environmental and economic challenges. Here, we present an engineered yeast coculture system combining Saccharomyces cerevisiae and Yarrowia lipolytica to convert crude glycerol into isopropanol, a liquid organic hydrogen carrier. The system integrates metabolic engineering, cell surface display pairing, immobilization, and continuous cultivation in fibrous bed bioreactors. In S. cerevisiae, glycerol use was improved by transporter optimization, pathway redirection, and flux shift from ethanol to isopropanol. In Y. lipolytica, ethanol from S. cerevisiae was redirected to isopropanol by acetyl-CoA reinforcement, malonyl-CoA diversion, and NADPH availability. Optimized pairing and inoculation ratios enhanced stability and yield. The consortia achieved complete glycerol utilization and three reuse cycles over 180 h. With pure glycerol, 28.34 g/l isopropanol was produced, while crude glycerol reached 86.06% of this yield. This strategy offers a scalable, modular route to convert biodiesel byproducts into bioenergy carriers.