Discovery of 23 natural tubulysins from Angiococcus disciformis An d48 and Cystobacter SBCb004

The tubulysins are a family of complex peptides with promising cytotoxic activity against multi-drug-resistant tumors. To date, ten tubulysins have been described from the myxobacterial strains Angiococcus disciformis An d48 and Archangium gephyra Ar 315. We report here a third producing strain, Cystobacter sp. SBCb004. Comparison of the tubulysin biosynthetic gene clusters in SBCb004 and An d48 reveals a conserved architecture, allowing the assignment of cluster boundaries. A SBCb004 strain containing a mutant in the putative cyclodeaminase gene tubZ accumulates pretubulysin A, the proposed first enzyme-free intermediate in the pathway, whose structure we confirm by NMR. We further show, using a combination of feeding studies and structure elucidation by NMR and high-resolution tandem mass spectrometry, that SBCb004 and An d48 together biosynthesize 22 additional tubulysin derivatives. These data reveal the inherently diversity-oriented nature of the tubulysin biosynthetic pathway.