Maximizing microbial bioproduction from sustainable carbon sources using iterative systems engineering

Thomas Eng, Deepanwita Banerjee, Javier Menasalvas, Yan Chen, Jennifer Gin, Hemant Choudhary, Edward Baidoo, Jian Hua Chen, Axel Ekman, Ramu Kakumanu, Yuzhong Liu Diercks, Alex Codik, Carolyn Larabell, John Gladden, Blake A. Simmons, Jay D. Keasling, Christopher J. Petzold, Aindrila Mukhopadhyay*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.
Original languageEnglish
Article number113087
JournalCell Reports
Issue number9
Number of pages25
Publication statusPublished - 2023


  • Pseudomonas putida KT2440
  • Growth coupling
  • Genome-scale metabolic models
  • Indigoidine
  • Strain engineering
  • CRISPR/recombineering
  • ALE
  • Proteomics analysis
  • Bioproduction
  • Lignin


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