Polymers benefit from the use of biogenic resources such as fatty acids. They enable easy access to valuable monomeric building blocks, which, in comparison to their exclusively fossil counterparts, lead to products with improved physicochemical properties. Monomers of special interest are medium-chain dicarboxylic acids, which are not easy to obtain by traditional chemical means. Previously, we established an in vitro pathway that combined a 9-lipoxygenase and a 9/13-hydroperoxide lyase, which enabled the conversion of linoleic acid via a hydroperoxy intermediate into 9-oxononanoic acid, the precursor of azelaic acid. Herein, we aimed for the further development of the multi-enzyme cascade, which included the oxidation of 9-oxononanoic acid and the establishment of a suitable whole-cell catalyst. A detailed investigation of the simultaneous in vitro reaction setup revealed that both lipoxygenase activation and the subsequent hydroperoxide lyase reaction depend on the hydroperoxide reaction intermediate. For the activation of lipoxygenase, the hydroperoxide lyase activity, therefore, has to be significantly reduced. In accordance with these observations, we established a suitable dual-expression system and we further demonstrated that endogenous E. coli redox enzymes are feasible to oxidize 9-oxononanoic acid to azelaic acid. The resulting whole-cell catalyst is, therefore, able to perform the direct bioconversion of linoleic acid into azelaic acid. The use of organic solvent as the second phase improved the overall performance of the E. coli host strain. The developed one-pot, single-step process afforded 29 mg L−1 of azelaic acid within 8 h with a substrate conversion of 34 % and a selectivity of 47 %.