Mechanism of Unexpected In-Trans Post-PKS Polyketide Reduction in Cochliodone Biosynthesis

Cochliodone A, a dimeric azaphilone-type compound, is the major product and one of many natural products produced by Chaetomium globosum, a filamentous fungus. Cochliodone A exerts antimalarial and antimycobacterial activities in addition to being cytotoxic against KB, BC1, and NCI-H187 human cancer cell lines. The potential of cochliodone A and its analogues as effective therapeutics against cancer, tuberculosis, and malaria, together with its complex dimeric chemical structure, are reasons enough for continued investigation. Here, sequence analyses of the open reading frames found in the previously identified cochliodone A biosynthetic gene cluster, together with a series of gene-knockout experiments, heterologous in vivo production of pathway intermediates in Aspergillus nidulans and in vitro assays of key enzymes allowed us to propose a biosynthetic pathway and detailed mechanisms leading to the production of cochliodones. In addition, we identified that the elimination of the dimerizing multicopper oxidase CcdJ from the ccd pathway led to pathway crosstalk between the ccd pathway and an unrelated benzaldehyde-producing biosynthetic pathway formation to generate new secondary metabolites. Most interestingly, however, through the in vitro study, we established that the sequential actions of an acetyltransferase, an acetate lyase, and an enoyl reductase achieve a full reduction of the unsaturated backbone of the polyketide (PKs) product generated by the nonreducing polyketide synthase (PKS) CcdL. This alternative mode of reducing a polyketomethylene chain could be engineered further to develop a facile chemoenzymatic modification of the polyketide backbone saturation level postpolyketide synthesis in trans.