Biosynthesis of 12-aminododecanoic acid (ADDA) directly from biomass-derived sugars would enable a more sustainable process for manufacturing the engineering polymer Nylon 12. ADDA biosynthesis is currently hindered by the cytotoxicity of dodecanoic acid (DDA) to growing cells, and the accumulation of the overoxidized byproduct dodecanedioic acid (DDDA). In this study, these challenges were addressed by engineering an autoinducible system to better control in vivo DDA synthesis without impacting growth, and deleting aldehyde dehydrogenases and oxidases to reduce DDDA accumulation. As a result, a one-step fermentation process was established to synthesize ADDA from glucose and cellobiose. Finally, batch fermentation achieved 1035 mg/L ADDA and 5% yield, which is the highest titer and yield accomplished directly from sugar to date. This research contributes to the mechanistic understanding of microbial DDA, ADDA, and DDDA synthesis, as well as the goal of developing more sustainable processes for nylon production.