Adaptive Laboratory Evolution Rewires Pseudomonas putida for Resource‐Efficient Acetate Assimilation
9 affiliations
- 1BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark
- 2Joint BioEnergy Institute Emeryville California USA
- 3Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA
- 4Institute of Applied Microbiology RWTH Aachen University Aachen Germany
- 5U.S. Department of Energy Joint Genome Institute Berkeley California USA
- 6Systems Biotechnology, Technical Faculty Bielefeld University Bielefeld Germany
- 7WSS Research Centre “CatalAix” Aachen Germany
- 8Department of Bioengineering University of California San Diego La Jolla California USA
- 9Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley California USA
Abstract
Acetate is an attractive renewable two‐carbon substrate for microbial biotechnology, but toxicity limits growth and carbon‐use efficiency at process‐relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome‐reduced strain of Pseudomonas putida and combined whole‐genome sequencing, quantitative physiology, reverse engineering, transcriptomics, proteomics and 13C‐acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recapitulated most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi‐omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism and oxidative stress‐associated proteins, indicating resource reallocation away from costly stress and non‐essential programs. Fluxomics further revealed reduced carbon cycling and increased glyoxylate shunt flux, consistent with improved acetate‐carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource‐efficiency phenotype and identify gacA and fabB as actionable targets for acetate‐based bioproduction.
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BibTeX
@article{gurdo2026adaptive,
title = {{Adaptive Laboratory Evolution Rewires Pseudomonas putida for Resource‐Efficient Acetate Assimilation}},
author = {Nicolás Gurdo and Aparajitha Srinivasan and Tommaso Tagliani and Melanie Filbig and Nicolas T. Wirth and Josefin Johnsen and Garret W. O'Connell and Stefano Donati and Enrico Orsi and María V. G. Alván‐Vargas and Yan Chen and Christopher J. Petzold and Matthew Blow and Thomas Eng and Till Tiso and Lars M. Blank and Adam Feist and Aindrila Mukhopadhyay and Pablo I. Nikel},
journal = {Microbial Biotechnology},
year = {2026},
month = oct,
doi = {10.1111/1751-7915.70459},
url = {https://doi.org/10.1111/1751-7915.70459},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Adaptive Laboratory Evolution Rewires Pseudomonas putida for Resource‐Efficient Acetate Assimilation
AU - Nicolás Gurdo
AU - Aparajitha Srinivasan
AU - Tommaso Tagliani
AU - Melanie Filbig
AU - Nicolas T. Wirth
AU - Josefin Johnsen
AU - Garret W. O'Connell
AU - Stefano Donati
AU - Enrico Orsi
AU - María V. G. Alván‐Vargas
AU - Yan Chen
AU - Christopher J. Petzold
AU - Matthew Blow
AU - Thomas Eng
AU - Till Tiso
AU - Lars M. Blank
AU - Adam Feist
AU - Aindrila Mukhopadhyay
AU - Pablo I. Nikel
PY - 2026
DA - 2026/10/01
JO - Microbial Biotechnology
DO - 10.1111/1751-7915.70459
UR - https://doi.org/10.1111/1751-7915.70459
AB - Acetate is an attractive renewable two‐carbon substrate for microbial biotechnology, but toxicity limits growth and carbon‐use efficiency at process‐relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome‐reduced strain of Pseudomonas putida and combined whole‐genome sequencing, quantitative physiology, reverse engineering, transcriptomics, proteomics and 13C‐acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recapitulated most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi‐omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism and oxidative stress‐associated proteins, indicating resource reallocation away from costly stress and non‐essential programs. Fluxomics further revealed reduced carbon cycling and increased glyoxylate shunt flux, consistent with improved acetate‐carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource‐efficiency phenotype and identify gacA and fabB as actionable targets for acetate‐based bioproduction.
SN - 1751-7915
ER -