Abstract <p>Neglected tropical diseases and the increasing prevalence of antibiotic resistance in nosocomial infections represent a major challenge in medicine. Intensive research into bioactive compounds has identified the myxobacterial natural product aurachin D as promising drug candidate due to its potent antibiotic and antiprotozoal properties. The biosynthesis of aurachin D from 2-methyl-1<i>H</i>-quinolin-4-one (MQO) is catalyzed by the membrane-bound farnesyltransferase AuaA. Recently, this enzymatic conversion was reconstructed in the heterologous host <i>Escherichia coli</i>, yielding an aurachin D titer of 17&#xa0;mg&#xa0;L⁻¹. In this study, we investigated the influence of the medium on aurachin D biosynthesis in <i>E.&#xa0;coli</i> in order to improve the product titer. Our analyses revealed that the glycerol concentration has a major impact on both the growth of the production host and the biocatalytic formation of aurachin D. After switching to a glycerol-based minimal medium and adjustment of the MQO precursor concentration, an aurachin&#xa0;D titer of 124&#xa0;mg&#xa0;L⁻¹ was achieved in a microbioreactor system, highlighting the superior performance of minimal over complex medium as well as the beneficial effect of glycerol compared to glucose. Further studies were conducted to elucidate the mechanism underlying glycerol’s effects. While improved growth and substrate solubilization were found to be insignificant for increasing the product titer, it was demonstrated that glycerol supplementation positively influenced the expression of the enzyme AuaA. Using a folding reporter, we were able to show that the availability of correctly folded AuaA increased until growth defects of the host, presumably resulting from osmotic stress, began to appear.</p> Key points <p>• <i>Product toxicity is negligible</i><i> in the production of aurachin D using E. coli</i></p> <p>• <i>The media supplement glycerol improves the availability of the enzyme AuaA</i></p> <p>• <i>Aurachin D titers increase after switching to a minimal medium</i></p> Graphical abstract <p></p>