## 2 Genetics, Biochemistry, and Evolution of DAPG Biosynthesis
### Biosynthesis and Degradation of DAPG  
The first step in the biosynthesis of DAPG is catalysed by the type III polyketide synthase (PKS) PhlD [14; 19]. Type III PKSs are homodimeric enzymes that catalyse the iterative condensation of a starter substrate (usually an acyl-CoA) with several extender sub  
Figure 1: Organization of the 2,4-DAPG biosynthetic cluster and current understanding of the biosynthesis scheme. (**A**) Organization of the _phl_ biosynthetic gene cluster found in DAPG-producing _Pseudomonas_ spp. (**B**) Current understanding of the biosynthesis and degradation of DAPG in the genus _Pseudomonas_. MAPG ATase is an enzyme multiplex composed of PhlA, PhlB and PhlC units. Abbreviations are as follows: MAPG, monoacetylphloroglucinol; DAPG, 2,4-diacetylphloroglucinol. strate units (usually malonyl-CoA) to generate a linear polyketide, which is subsequently cyclized [20]. [PERSON] and [PERSON] [14] proposed that PhD uses acetoacetyl-CoA as the starter substrate to produce monoacetylphloroglucinol (MAPG), but it was later showed that PhD produces phloroglucinol from malonyl-CoA instead [19]. While PhD uses malonyl-CoA as a preferred substrate, it also accepts other starter substrates with an aliphatic chain of C\({}_{4}\)-C\({}_{12}\) in vitro [21]. The proposed mechanism leading to the formation of phloroglucinol is that PhD catalyses the iterative condensation of three molecules of malonyl-CoA into 3,5-diketoheptanedioate [19]. This polyketide intermediate undergoes decarboxylation and is subsequently cyclized via a Claisen condensation, leading to the formation of phloroglucinol [19; 21]. In the following steps, acetylation of phloroglucinol leads to the formation of MAPG, which is subsequently acetylated into DAPG. MAPG was identified as a putative intermediate in DAPG biosynthesis by [PERSON] and colleagues [22], who also found experimental evidence for an enzymatic acetyltransferase activeily in cell-free extracts of \(P\). _kilonensis_ F113. [PERSON] and [PERSON] [14] found that _phlACB_ was required for the biosynthesis of MAPG and DAPG, suggesting a role for PhlACB in the production and the acetylation of MAPG. [PERSON] and colleagues [19] confirmed that the product of _phlACB_ catalyses the acetylation of phloroglucinol and MAPG: The addition of phloroglucinol to the culture medium of an \(E\). _coli_ strain carrying plasmid-localized _phlACB_ led to the formation of MAPG and DAPG. Later, [PERSON] and colleagues [17] characterized the multimeric enzyme composed of PhlA, PhlC and PhlB units, which was named MAPG acetyltransferase (MAPG ATase). This enzyme, unlike most acetyltransferases described so far, was shown to catalyse C-C bond formation without the use of CoA-activated substrates [17]. The MAPG ATase catalyses the disproportionation of two molecules of MAPG, resulting in the formation of DAPG and the production of phloroglucinol. The MAPG ATase also catalyses the reverse reaction, yielding two molecules of MAPG from a molecule of DAPG and a molecule of phloroglucinol [17]. This results in an equilibrium where DAPG, MAPG and phloroglucinol are present at quasi-equimolar concentration. Recent studies have provided new insight into the catalytic properties, the mechanism, and the structure of the MAPG ATase [23; 24; 25; 26; 27]. This enzyme was shown to use various non-natural substrates as acyl-donor in in vitro experiments [26; 27; 28]. This is particularly interesting given the fact that the acyl donor involved in MAPG biosynthesis from phloroglucinol remains to be characterized. The crystal structure revealed that PhlACB subunits are arranged in a Phl(A\({}_{2}\)C\({}_{2}\))\({}_{2}\)B\({}_{4}\) composition where four PhlB units mediate the binding of two PhlA and two PhlC dimers [23]. Crystal soaking and site-directed mutagenesis experiments suggest that only PhlC units are involved in the acyl transfer reaction [23]. DAPG is degraded by the zinc-dependent hydrolase PhlG [29; 30].