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PreprintCited by 10Microbial Metabolic Engineering and BioproductionPlant biochemistry and biosynthesisGene Regulatory Network Analysis

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Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

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Menasalvas, Javier, Kulakowski, Shawn, Chen, Yan, Gin, Jennifer W., Turumtay, Emine Akyuz, Baral, Nawa Raj, Apolonio, Morgan A., Rivier, Alex, Yunus, Ian S., Garber, Megan E., Scown, Corinne D., Adams, Paul D., Lee, Taek Soon, Blaby, Ian K., Baidoo, Edward E. K., Petzold, Christopher J., Eng, Thomas, Mukhopadhyay, Aindrila·2025·Science Advances
DOI 10.1101/2025.03.18.643695PMC PMC12551699

Preprint — published as 10.1126/sciadv.ady2677

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Abstract

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida , a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putida ’s native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a ∼16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing ∼900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT:

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Metadata

Authors
Menasalvas, J., Kulakowski, S., Chen, Y., Gin, J. W., Turumtay, E. A., Baral, N. R., Apolonio, M. A., Rivier, A., Yunus, I. S., Garber, M. E., Scown, C. D., Adams, P. D., Lee, T. S., Blaby, I. K., Baidoo, E. E. K., Petzold, C. J., Eng, T., & Mukhopadhyay, A.
Affiliations
  • Lawrence Berkeley National Laboratory (US)
  • The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, USA
Corresponding author
Mukhopadhyay, Aindrila
ORCID
Venue
Science Advances
Publisher
openRxiv
Published
2025-03-18
Volume
11
Issue
43
Pages
eady2677-eady2677
ISSN
2375-2548 (ISSN-L)
DOI
10.1101/2025.03.18.643695
PMC
PMC12551699
OpenAlex
W4415525478
Cited by
10
References
1
Citations by year
2026: 8 · 2025: 2
FWCI
3.01
Type
journal article
Language
en
Open access
Licence
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
Funders
  • U.S. Department of Energy (503809)
  • Joint Genome Institute
  • Office of Science (https://ror.org/04xm1d337)
  • Biological and Environmental Research
  • Lawrence Berkeley National Laboratory
  • Pacific Northwest National Laboratory
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
Pseudomonas putida, hybrid histidine kinase, alcohol dehydrogenase, heterodimerization, strain engineering, amino acid catabolism, metabolic reprogramming
MeSH terms
Histidine Kinase, Alcohol Dehydrogenase, Bacterial Proteins, Biosensing Techniques, Signal Transduction, Pseudomonas putida, Hemiterpenes, Synthetic Biology, Metabolic Engineering

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