- Eng, Thomas;
- Banerjee, Deepanwita;
- Menasalvas, Javier;
- Chen, Yan;
- Gin, Jennifer;
- Choudhary, Hemant;
- Baidoo, Edward;
- Chen, Jian Hua;
- Ekman, Axel;
- Kakumanu, Ramu;
- Diercks, Yuzhong Liu;
- Codik, Alex;
- Larabell, Carolyn;
- Gladden, John;
- Simmons, Blake A;
- Keasling, Jay D;
- Petzold, Christopher J;
- Mukhopadhyay, Aindrila
Maximizing the production of heterologous biomolecules is a complex problem that can be addressed with a systems-level understanding of cellular metabolism and regulation. Specifically, growth-coupling approaches can increase product titers and yields and also enhance production rates. However, implementing these methods for non-canonical carbon streams is challenging due to gaps in metabolic models. Over four design-build-test-learn cycles, we rewire Pseudomonas putida KT2440 for growth-coupled production of indigoidine from para-coumarate. We explore 4,114 potential growth-coupling solutions and refine one design through laboratory evolution and ensemble data-driven methods. The final growth-coupled strain produces 7.3 g/L indigoidine at 77% maximum theoretical yield in para-coumarate minimal medium. The iterative use of growth-coupling designs and functional genomics with experimental validation was highly effective and agnostic to specific hosts, carbon streams, and final products and thus generalizable across many systems.