The effect of divalent metal ions (i.e., Mn2+, Mg2+, Zn2+, Cu2+, and Co2+) on the production of anticancer ansamitocin P-3 (AP-3) by submerged cultures of Actinosynnema pretiosum in medium containing agro-industrial residues was investigated, and Mg2+ was found to be the most effective. Under the optimal condition of Mg2+ addition, the maximal AP-3 production titer reached 85 mg/L, which was 3.0-fold that of the control. The activities of methylmalonyl-CoA carboxyltransferase (MCT) and methylmalonyl-CoA mutase (MCM) were enhanced. The content of two precursors, malonyl-CoA and methylmalonyl-CoA, was lower than that of control. This work demonstrates that Mg2+ addition is a simple and effective strategy for increasing AP-3 production through the regulation of enzyme activity and pools of precursors. The information obtained can be helpful to its efficient production on large scale. (C) 2011 Elsevier Ltd. All rights reserved.