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In this study, whole-cell biotransformation was conducted to produce nonanedioic acid from nonanoic acid by expressing the alkane hydroxylating system (AlkBGT) from Pseudomonas putida GPo1 in Escherichia coli. Following adaptive laboratory evolution, an efficient E. coli mutant strain, designated as MRE, was successfully obtained, demonstrating the fastest growth (27-fold higher) on nonanoic acid as the sole carbon source compared to the wild-type strain. Additionally, the MRE strain was engineered to block nonanoic acid degradation by deleting fadE. The resulting strain exhibited a 12.8-fold increase in nonanedioic acid production compared to the wild-type strain. Six mutations in acrR, P crp , dppA, P fadD , e14, and yeaR were identified in the mutant MRE strain, which was characterized using genomic modifications and RNA-sequencing. The acquired mutations were found to be beneficial for rapid growth and nonanedioic acid production., (© 2021 Wiley-VCH GmbH.)