Background <p>Nutrient limitation strategies are commonly applied in bioprocess development to engineered microorganisms to further maximize the production of the target molecule towards theoretical limits. Biomass formation is often limited under the limitation of key nutrients, and understanding how fluxes in central carbon metabolism are re-routed during the transition from nutrient excess to nutrient-limited condition is vital to target and tailor metabolic engineering strategies. Here, we report the physiology and intracellular flux distribution of an engineered acetol-producing <i>Escherichia&#xa0;coli</i> on glycerol under nitrogen-limited, non-growing production conditions.</p> Results <p>Acetol production in the engineered <i>E.&#xa0;coli</i> strain is triggered upon nitrogen depletion. During nitrogen limitation, glycerol uptake decreased, and biomass formation rates ceased. We applied ¹³C-flux analysis with 2-¹³C glycerol during exponential growth and nitrogen starvation to elucidate flux re-routing in the central carbon metabolism. The results indicate a metabolically active non-growing state with significant flux re-routing towards acetol biosynthesis and reduced flux through the central carbon metabolism. The acetol biosynthesis pathway is favorable for maintaining the NADPH/NADP⁺ balance.</p> Conclusion <p>The results reported in this study illustrate how the production of a value-added chemical from a waste stream can be connected to the metabolism of the whole-cell biocatalyst, making product formation mandatory for the cell to maintain its NADPH/NADP⁺ balance. This has implications for process design and further metabolic engineering of the whole-cell biocatalyst.</p>