Data Sheet 1_Multi-omics characterization of β-myrcene-evolved Pseudomonas sp. M1 reveals convergent FleQ mutations and altered catabolic efficiency.xlsx

β-Myrcene is a high-value monoterpene precursor whose high hydrophobicity limits microbial biotransformation. In aqueous medium, β-myrcene forms droplets that Pseudomonas sp. M1 accesses through chemotaxis mediated by the genomic island (GI)-encoded methyl-accepting chemotaxis protein MyrS. To identify genetic targets for strain improvement, we subjected M1 to adaptive laboratory evolution (ALE) for 600 generations under β-myrcene selection and characterized two evolved isolates, M2C19 and M3C22, using comparative genomics, quantitative proteomics, and metabolite profiling. Both lineages independently acquired mutations in the AAA + ATPase domain of FleQ, the master regulator of flagellar biosynthesis, resulting in loss of polar flagella and Tad pilus proteins, and strong reduction of chemotaxis signal transduction (CheA, CheW), putatively impacting response to β-myrcene chemoattractant signal. Despite identical growth rates during exponential phase, evolved strains achieved ∼33% higher final OD600 than wild-type M1. Metabolite analysis indicated enhanced pathway flux: M2C19 accumulated myrcenoic acid 10.6-fold above wild-type, while M3C22 accumulated 3.5-fold, and upstream intermediates (myrcen-8-ol, myrcenal) were depleted in both strains. Proteome profiling revealed distinct temporal dynamics of GI induction: M2C19 showed early upregulation of GI proteins, whereas M3C22 displayed delayed induction at early exponential phase with recovery by late exponential phase. Beyond the GI, both evolved strains converged on reduced motility/chemotaxis systems and extensive membrane remodeling, while core metabolic processes diverged. M2C19 broadly upregulating respiration and β-oxidation components, and M3C22 showing systematic downregulation of these pathways at early growth stages. Overall, the results identify FleQ as a major adaptive target during β-myrcene-driven evolution and reveal distinct proteometabolic strategies that improve monoterpene processing under laboratory selection.