The growth benefits and toxicity of quinone synthesis are balanced by a dual regulatory mechanism and substrate limitations

Quinones play a central role in maintaining redox balance and conserving energy but can trigger oxidative stress at high levels. However, the mechanisms by which microbes regulate quinone levels remain poorly0020understood, hindering effective metabolic engineering to modulate microbes for quinone production. Here, we show that the synthesis of the menaquinone precursor DHNA in the lactic acid bacterium Lactococcus lactis is regulated by a combined genetic, enzymatic, and metabolic mechanism. Using synthetic biology approaches, we found that enzymes MenF and MenD both contribute to DHNA regulation, with MenD playing a more prominent role in controlling DHNA concentrations. A mathematical model elucidates a two-phase regulatory pattern resulting from the interplay of reversible flux and allosteric feedback inhibition, where either MenF or MenD can serve as the regulatory enzyme, depending on their relative expression ratio. Additionally, the overproduction of DHNA is constrained by substrate availability, ensuring a sufficient but not excessive DHNA level to benefit cell growth while mitigating cytotoxicity. Collectively, these mechanisms maintain a fine-tuned physiological quinone level and suggest that modulating substrate supplement and MenF-to-MenD ratio could be keys for engineering DHNA production