Authors
Gurdo, N., Srinivasan, A., Tagliani, T., Filbig, M., Wirth, N. T., Johnsen, J., O'Connell, G. W., Donati, S., Orsi, E., Alvan-Vargas, M. V. G., Chen, Y., Petzold, C. J., Blow, M., Eng, T., Tiso, T., Blank, L. M., Feist, A., Mukhopadhyay, A., Nikel, P. I.
Abstract
Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its 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, 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 recovered 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 EDEMP 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.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 22 Aug 2026.
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