Delineation of gilvocarcin, jadomycin, and landomycin pathways through combinatorial biosynthetic enzymology

The exact sequence of events in biosyntheses of natural products is essential not only to understand and learn from nature's strategies and tricks to assemble complex natural products, but also for yield optimization of desired natural products, and for pathway engineering and muta-synthetic preparation of analogues of bioactive natural products. Biosyntheses of natural products were classically studied applying in vivo experiments, usually by combining incorporation experiments with stable-isotope labeled precursors with cross-feeding experiments of putative intermediates. Later genetic studies were dominant, which consist of gene cluster determination and analysis of gene inactivation experiments. From such studies various biosynthetic pathways were proposed, to a large extent just through in silico analyses of the biosynthetic gene clusters after DNA sequencing. Investigations of the complex biosyntheses of the angucycline group anticancer drugs landomycin, jadomycin and gilvocarcin revealed that in vivo and in silico studies were insufficient to delineate the true biosynthetic sequence of events. Neither was it possible to unambiguously assign enzyme activities, especially where multiple functional enzymes were involved. However, many of the intriguing ambiguities could be solved after in vitro reconstitution of major segments of these pathways, and subsequent systematic variations of the used enzyme mixtures. This method has been recently termed ‘combinatorial biosynthetic enzymology’.

► The exact sequence of biosynthetic events is essential for the preparation of bioactive natural product analogues. ► Biosynthetic investigation of landomycin and gilvocarcin using classical methods led to many ambiguities. ► The combinatorial biosynthetic enzymology approach turned out to be the superior way to delineate the complex post-polyketide tailoring steps of landomycin, gilvocarcin and jadomycin biosyntheses. ► Previously impossible unambiguous assignments could be made for the function of many of the involved enzymes. ► Many of the earlier drawn hypotheses and conclusions were revised.