Adaptive Laboratory Evolution Rewires Pseudomonas putida for Resource‐Efficient Acetate Assimilation

Microbial Biotechnology, vol. 19

Abstract

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 13 C‐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.

Authors 19

1. Nicolás Gurdo

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

2. Aparajitha Srinivasan

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA

   Joint BioEnergy Institute Emeryville California USA

3. Tommaso Tagliani

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

4. Melanie Filbig Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology RWTH Aachen University Aachen Germany

5. Nicolas T. Wirth

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

6. Josefin Johnsen

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

7. Garret W. O'Connell

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

8. Stefano Donati

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

9. Enrico Orsi

   Technical University of Denmark

   Affiliation as printed

   BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

10. Maria V G Alván-Vargas

    Technical University of Denmark

    Affiliation as printed

    BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

11. Yan Chen

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA

    Joint BioEnergy Institute Emeryville California USA

12. Christopher J. Petzold

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA

    Joint BioEnergy Institute Emeryville California USA

13. Matthew James Blow

    Joint Genome Institute

    Affiliation as printed

    U.S. Department of Energy Joint Genome Institute Berkeley California USA

14. Thomas Eng

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA

    Joint BioEnergy Institute Emeryville California USA

15. Till Tiso Aachen Institute of Applied Microbiology

    Bielefeld University · RWTH Aachen University

    Affiliation as printed

    Institute of Applied Microbiology RWTH Aachen University Aachen Germany

    Systems Biotechnology, Technical Faculty Bielefeld University Bielefeld Germany

16. Lars M. Blank Aachen Institute of Applied Microbiology

    RWTH Aachen University

    Affiliation as printed

    Institute of Applied Microbiology RWTH Aachen University Aachen Germany

    WSS Research Centre “CatalAix” Aachen Germany

17. Adam M. Feist

    University of California San Diego · Joint BioEnergy Institute · Technical University of Denmark

    Affiliation as printed

    BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark

    Department of Bioengineering University of California San Diego La Jolla California USA

    Joint BioEnergy Institute Emeryville California USA

18. Aindrila Mukhopadhyay

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA

    Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley California USA

    Joint BioEnergy Institute Emeryville California USA

19. Pablo I. Nikel corresponding

    Technical University of Denmark

    Affiliation as printed

    BRiGHT, Technical University of Denmark Kgs. Lyngby Denmark