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Biosynthesis of rapamycin
Rapamycin is a macrocyclic polyketide isolated from Streptomyces hygroscopicus that has been shown to exhibit antifungal, antitumor, and immunosuppressant properties. The biosynthesis of the rapamycin core is accomplished by a type I polyketide synthase (PKS) in conjunction with a nonribosomal peptide synthetase (NRPS). The domains responsible for the biosynthesis of the linear polyketide of rapamycin are organized into three multienzymes, RapA, RapB and RapC which contain a total of 14 modules (See figure 1). The three multienzymes are organized such that the first four modules of polyketide chain elongation are in RapA, the following six modules for continued elongation are in RapB, and the final four modules to complete the biosynthesis of the linear polyketide are in RapC. Then the linear polyketide is modified by the NRPS, RapP, which attaches L-pipecolate to the terminal end of the polyketide and then cyclizes the molecule yielding the unbound product, prerapamycin. The core macrocycle, prerapamycin is then modified (See figure 3) by an additional five enzymes which lead to the final product, rapamycin. First the core macrocycle is modified by RapI, SAM-dependant O-methyltransferase (MTase), which O-methylates at C39. Next, a carbonyl is installed at C9 by RapJ, a cytochrome P-450 monooxygenases (P-450). Then, RapM, another MTase, O-methylates at C16. Finally, RapN, another P-450 installs a hydroxyl at C27 immediately followed by O-methylation by Rap Q, a distinct MTase, at C27 to yield rapamycin.

The biosynthetic genes responsible for rapamycin synthesis have been identified. As expected, three extremely large open reading frames (OFRs) designated as rapA, rapB and rapC encode for three extremely large and complex multienzymes, RapA, RapB, and RapC respectively. The gene rapL has been established to code for a NAD+ dependant lysine cycloamidase which converts L-lysine to L-pipecolic acid (See figure 4) for incorporation at the end of the polyketide. A gene rapP, which is embedded between the PKS genes and translationally coupled to rapC encodes for an additional enzyme, a NPRS responsible for incorporating L-pipecolic acid, chain termination and cyclization of prerapamycin. Additionally genes rapI, rapJ, rapM, rapN, rapO, and rapQ have been identified as coding for "tailoring" enzymes which modify the marcrcyclic core to give rapamycin (See figure 3). Finally, rapG and rapH have been identified to code for enzymes which have a positive regulatory role in the preparation of rapamycin through the control of rapamycin PKS gene expression.

Biosynthesis of this 31-membered macrocycle begins as the loading domain is primed with the starter unit, 4,5-dihydroxocyclohex-1-ene-carboxylic acid, which is derived form the shikimate pathway. Interestingly, the cyclohexane ring of the starting unit is reduced during the transfer to module 1. The staring unit is then modified by a series of Claisen condensations with malonyl or methylmalonyl substrates which are attached to an acyl carrier protein (ACP) and extend the polyketide by two carbons each. After each successive condensation, the growing polyketide is further modified according to enzymatic domains which are present to reduce and dehydrate the polyketide thereby introducing the diversity of functionalities observed in rapamycin (See figure 1). Once the linear polyketide is complete, L-pipecolic acid which is synthesized by a lysine cycloamidase from an L-lysine is added to the terminal end of the polyketide by an NRPS. Then the NSPS cyclizes the polyketide giving prerarpmycin, the first enzyme free product. The macrocyclic core is then customized by a series of post-PKS enzymes through methylations by MTases and oxidations by P-450s to yield rapamycin.