(4af) Biosynthesis of Fungal Resorcylic Acid Lactones

Resorcylic acid lactones (RALs) are macrocyclic polyketides that display diverse biological activities. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. This tandem enzymatic working mode was investigated based on hypothemycin biosynthetic system. Using purified Hpm8 and Hpm3 recombinantly expressed from Saccharomyces cerevisiae, we successfully reconstituted the >30 steps required for the synthesis of DHZ from malonyl-CoA in vitro. Kinetic analyses of the tandem IPKS system revealed that synthesis of the reduced hexaketide by Hpm8 is the rate-limiting step, and Hpm3 is only required at substoichiometric concentrations. The SAT domain of Hpm3 was also confirmed to mediate the communication between the Hpm8 and Hpm3 and is critical for the transacylation of the hexaketide intermediate between the two IPKSs. Biochemical study on PKS13, a non-reducing PKS (NRPKS) from zearalenone biosynthetic pathway revealed that it displayed broad starter-unit specificities towards fatty acyl-CoAs ranging in sizes between C6 and C16. Interestingly, PKS13 can also synthesize numerous alkyl pyrones and alkyl resorcylic esters in Escherichia coli, which is demonstrated to be interacting with E. coli fatty acid biosynthetic machinery. Heterologous reconstitution of Rdc1 and Rdc5 in S. cerevisiae led to the production of monocillin II, the precursor to radicicol. The generation of monocillin II via precursor feeding into Rdc1 culture further suggested that the chain length control by Rdc1 and Rdc5 differed from the former two systems (Hpm8 and Hpm3, PKS4 and PKS13).