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

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.