Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in <i>Pseudomonas putida</i>
Synthetic biology generates vast combinatorial designs, yet high-throughput analytical methods to screen them are poorly matched to interrogate this search space. We address this challenge by developing a biosensor-driven, growth-coupled selection strategy in <i>Pseudomonas putida</i> for isoprenol, a potential aviation fuel precursor. We found and characterized a noncanonical signaling pathway, revealing a functional and physical complex between a hybrid histidine kinase and an alcohol dehydrogenase, whose activity is tuned by heterodimerization. Leveraging this biosensor in a pooled CRISPRi library selection, we identified key host limitations. Iterative combinatorial strain engineering derived from these hits yielded a 36-fold titer increase to ~900 milligrams per liter. Integrated omics analysis revealed that metabolic rewiring toward amino acid catabolism was crucial for this improvement. This observation was found to be beneficial by technoeconomic analysis. Our modular workflow provides a powerful strategy for optimizing complex heterologous pathways and uncovering emergent host biology.
Authors
- Jennifer Gin (ORCID: https://orcid.org/0000-0001-5636-7563)
- Nawa Raj Baral (ORCID: https://orcid.org/0000-0002-0942-9183)
- Corinne D. Scown (ORCID: https://orcid.org/0000-0003-2078-1126)
- Christopher J. Petzold (ORCID: https://orcid.org/0000-0002-8270-5228)
- Aindrila Mukhopadhyay (ORCID: https://orcid.org/0000-0002-6513-7425)
- Paul D. Adams (ORCID: https://orcid.org/0000-0001-9333-8219)
- Alex Rivier (ORCID: https://orcid.org/0009-0004-9477-6201)
- Emine Akyüz Turumtay (ORCID: https://orcid.org/0000-0002-1504-4810)
- Edward E. K. Baidoo (ORCID: https://orcid.org/0000-0001-5787-1219)
- Ian K. Blaby (ORCID: https://orcid.org/0000-0002-1631-3154)
- Ian Sofian Yunus (ORCID: https://orcid.org/0000-0002-2395-0821)
- Taek Soon Lee (ORCID: https://orcid.org/0000-0002-0764-2626)
- Javier Menasalvas (ORCID: https://orcid.org/0000-0002-4003-4999)
- Thomas Eng (ORCID: https://orcid.org/0000-0002-4974-3863)
- Megan E. Garber (ORCID: https://orcid.org/0000-0003-2886-8808)
- Yan Chen (ORCID: https://orcid.org/0000-0002-1125-313X)
- S Kulakowski
- Morgan A. Apolonio (ORCID: https://orcid.org/0000-0002-1458-3708)
Institutions
- Lawrence Berkeley National Laboratory (US)
- Joint Genome Institute (US)
- Energy Biosciences Institute (US)
- Joint BioEnergy Institute (US)
- University of California, Berkeley (US)
Publication Details
- Journal
- Science Advances
- Published
- 2025-10-24
- DOI
- https://doi.org/10.1126/sciadv.ady2677
- Citations
- 5
- Primary Topic
- Microbial Metabolic Engineering and Bioproduction
- Type
- article
- Field-Weighted Citation Impact
- 4.03