Sporolides A and B (1) are polycyclic macrolides from the marine actinomycete Salinispora tropica with a previously undescribed molecular architecture. Sequencing of the 5 183 331 bp S. tropica CNB-440 genome has shed light into sporolide biosynthesis, which is highlighted by an enediyne po ketide synthase and an unexpected tyrosine degradation pathway. In this paper, we describe the in vitro characterization of the biosynthetic gene spoT1, which is involved in the initial biochemical reaction to the highly functionalized cyclohexenone unit of 1.