acid and the 9C fatty acid 9-hydroxy-nonanoic acid. The possibility that the entire molecule is assembled as a single polyketide with a Baeyer-Villiger oxidation inserting an oxygen into the carbon backbone has been ruled out because C1 of monic acid and C9' of 9-hydroxy-nonanoic acid are both derived from C1 of acetate. Monic acid biosynthesis Biosynthesis of the 17C monic acid unit begins on MmpD (Figure 1). One of the AT domains from MmpC may transfer an activated acetyl group from acetyl-Coenzyme A (CoA) to the first ACP domain. The chain is extended by malonyl-CoA, followed by a SAM-dependent methylation at C12 (see Figure 2 for PA-A numbering) and reduction of the B-keto group to an alcohol. The dehydration (DH) domain in module 1 is predicted to be non-functional due to a mutation in the conserved active site region. Module 2 adds another two carbons by the malonyl-CoA extender unit, followed by ketoreduction (KR) and dehydration. Module three adds a malonyl-CoA extender unit, followed by SAM-dependent methylation at C8, ketoreduction, and dehydration. Module 4 extends the molecule with a malonyl-CoA unit followed by ketoreduction. Assembly of monic acid is continued by the transfer of the 12C product of MmpD to MmpA. Post-PKS tailoring The keto group at C3 is replaced with a methyl group in a multi-step reaction (Figure 3). MupG begins by decarboxylating a malonyl-ACP. The alpha carbon of the resulting acetyl-ACP is linked to C3 of the polyketide chain by MupH. This intermediate is dehydrated and decarboxylated by MupJ and MupK, respectively. The formation of the pyran ring requires many enzyme-mediated steps (Figure 4). The double bond between C8 and C9 is proposed to migrate to between C8 and C16. Gene knockout experiments of mupO, mupU, mupV, and macpE have eliminated PA-A production. PA-B production is not removed by these knockouts, demonstrating that PA-B is not created by hydroxylating PA-A. A knockout of mupW eliminated the pyran ring, identifying MupW as being involved in ring formation. The epoxide of PA-A at C10-11 is believed to be inserted after pyran formation by a cytochrome P450 such as MupO. A gene knockout of mupO abolished PA-A production but PA-B, which also contains the C10-C11 epoxide, remained. 9-Hydroxy-nonanoic acid biosynthesis The nine-carbon fatty acid 9-hydroxy-nonanoic acid (9-HN) is derived as a separate compound and later esterified to monic acid to form pseudomonic acid. 13C labeled acetate feeding has shown that C1-C6