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'.