S30: Understanding and engineering the biosynthetic pathway of an anticancer tetracycline

Tetracycline antibiotics are one of the most well known classes of natural products.  Recent biosynthetic studies over the last decade have elucidated many of the enzymatic steps and solved several longstanding mysteries of tetracycline biosynthesis.  To further enhance our understanding of tetracycline biosynthesis and identify new tetracycline tailoring enzymes, we have identified and sequenced the gene cluster responsible for SF2575, an unusual tetracycline with novel structural properties including a C-9 glycosylation and acylation with angelic acid and a C-4 salicylic acid.  These structural differences are reflected in its bioactivity which lacks the typical antibacterial activity, but has potent anticancer activity.  Structure activity relationship studies have shown the angelic acid and salicylic acid groups to be vital to this activity and thus these positions are of high value for diversification to produce novel compounds for further SAR studies and potentially improved pharmacological properties.  We have identified two enzymes responsible for the salicylyl transfer reaction, SsfL1 a salicylyl-CoA ligase and SsfX3, a salicylyl transferase.  Both have broad substrate specificity in vitro, which has been exploited to generate an array of analogs of late stage biosynthetic intermediates.   To further investigate the nature of this important acyltransferase, we have solved the crystal structure of SsfX3 revealing a bidomain structure consisting of an SGNH hydrolase domain and a putative substrate recognition domain which sets the stage for exploring the mechanistic basis of the broad substrate specificity.  This pathway therefore offers new opportunities to diversify tetracycline scaffolds and shed further light on tetracycline biosynthesis.