Search NASA⌕ Search
Home/Archive/DOE OSTI

DOE OSTI · 3006357

Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in Pseudomonas putida

Menasalvas, Javier [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0000000240034999)·Kulakowski, Shawn [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)]·Chen, Yan [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:000000021125313X)·Gin, Jennifer W. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000156367563)·Akyuz Turumtay, Emine [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000215044810)·Baral, Nawa Raj [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000209429183)·Apolonio, Morgan A. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000214583708)·Rivier, Alex [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)]·Yunus, Ian S. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000223950821)·Garber, Megan E. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)]·Scown, Corinne D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)]·Adams, Paul D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0000000193338219)·Lee, Taek Soon [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000207642626)·Blaby, Ian K. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000216313154)·Baidoo, Edward E. K. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000157871219)·Petzold, Christopher J. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000282705228)·Eng, Thomas [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000249743863)·Mukhopadhyay, Aindrila [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000265137425)
View original source ↗Read source document ↗Export metadata ↓

Abstract

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 Pseudomonas putida 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.

Explore related subjects

CRISPRiPseudomonas putidabiofuelbiosensorgRNA libraryisoprenolstrain engineeringsynthetic biologytechno-economic analysistwo-component signaling

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Menasalvas, Javier [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0000000240034999), Kulakowski, Shawn [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Chen, Yan [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:000000021125313X), Gin, Jennifer W. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000156367563), Akyuz Turumtay, Emine [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000215044810), Baral, Nawa Raj [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000209429183), Apolonio, Morgan A. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000214583708), Rivier, Alex [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Yunus, Ian S. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000223950821), Garber, Megan E. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Scown, Corinne D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)], Adams, Paul D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0000000193338219), Lee, Taek Soon [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000207642626), Blaby, Ian K. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000216313154), Baidoo, Edward E. K. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000157871219), Petzold, Christopher J. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000282705228), Eng, Thomas [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000249743863), Mukhopadhyay, Aindrila [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000265137425). 2025-10-24. Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in Pseudomonas putida. https://doi.org/10.1126/sciadv.ady2677

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related reports

DOE OSTI2025

Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance in Zymomonas mobilis

Genetically engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and transposon insertion libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis, against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable.

CRISPRi↗