Enzyme cascade catalysis has critical problems in obtaining the high concentrations of products, such as the low stabilities and activities of biocatalysts and the inhibition by hydrophobic reactants at high concentrations to biocatalysts. Here, we performed multilayer engineering of enzyme cascade catalysis to produce C11 nylon monomers at commercially viable concentrations from ricinoleic acid. The catalysis was driven by engineered Escherichia coli-based whole-cell biocatalysts and cell-free enzymes (i.e., lipases). Stabilities and activities of the biocatalysts were improved by engineering the bottleneck enzyme Baeyer− Villiger monooxygenase and introducing a cofactor regeneration system. The inhibitory effects of the byproducts n-heptanoic acid and pyruvate on the cascade enzymes were overcome, and the products were simply recovered in situ by engineering of reactions with the addition of an adsorbent resin. We obtained 248 mM undecanedioic acid or 232 mM 11-aminoundecanoic acid as a nylon monomer from 300 mM ricinoleic acid by multilayer engineering. The concentration was 500-and 640-fold higher than those produced by nonengineering, respectively. We have also simply isolated the C11 nylon monomers via solvent extraction to a rather high purity. This study will contribute to the industrial enzyme cascade synthesis of nylon monomers in an environmentallyfriendly route from renewable biomass.

Medium‐chain α,ω‐dicarboxylic acids produced from renewable long‐chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer–Villiger monooxygenases are used in the production of ω‐hydroxycarboxylic acids from long‐chain fatty acids, the engineering of biocatalysts involved in the production of α,ω‐dicarboxylic acids from ω‐hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω‐dicarboxylic acids from ω‐hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40‐fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium‐chain α,ω‐dicarboxylic acids from renewable long‐chain fatty acids.