Control of Secondary Metabolite Congener Distributions via Modulation of the Dissolved Oxygen Tension

Many secondary metabolites, including various polyketides, require complex enzymatic pathways for modification into their final biologically active forms. Limitation of the dissolved oxygen supplied during cultivation of various microbial strains can decrease the activity of cytochrome P-450 monooxygenases required for the processing of pathway intermediates into their final forms, resulting in the accumulation of these intermediates as the primary products. Here, a generalized oxygen-limited cultivation strategy is specifically demonstrated with a myxobacterial strain engineered to heterologously express the epothilone polyketide synthase (PKS) gene cluster under either an excess (the dissolved oxygen tension is maintained at 50% of saturation) or a depleted (no residual dissolved oxygen detected) level of oxygenation during cultivation. Cultivation of this myxobacterial strain with excess oxygenation resulted in the production of epothilones A and B as the primary products, while cultivation of this same strain under depleted oxygenation resulted in the production of epothilones C and D as the primary products. Additionally, the peak cell density in the oxygen-depleted cultivations was 60% higher than that observed in oxygen-excess cultivations. Finally, an active EpoK epoxidase was found to catalyze the production of a novel epothilone (Epo506) with an unexpected structure during the cultivation of another myxobacterial strain expressing a genetically modified epothilone PKS under excess oxygenation. The structure of Epo506 was determined by high-resolution mass spectrometry and one- and two-dimensional NMR.