Penilactones A and
B consist of a γ-butyrolactone and two
clavatol moieties. We identified two separate gene clusters for the
biosynthesis of these key building blocks in Penicillium
crustosum. Gene deletion, feeding experiments, and
biochemical investigations proved that a nonreducing PKS ClaF is responsible
for the formation of clavatol and the PKS-NRPS hybrid TraA is involved
in the formation of crustosic acid, which undergoes decarboxylation
and isomerization to the predominant terrestric acid. Both acids are
proposed to be converted to γ-butyrolactones with involvement
of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol
by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD
and its spontaneous dehydration to an ortho-quinone
methide initiate the two nonenzymatic 1,4-Michael addition steps.
Spontaneous addition of the methide to the γ-butyrolactones
led to peniphenone D and penilactone D, which undergo again stereospecific
attacking by methide to give penilactones A/B.
Agarwood is highly valued for its uses as incense, perfume, and medicine. However, systematic analyses of dynamic changes of secondary metabolites during the process of agarwood formation have not yet been reported. In this study, agarwood was produced by transfusing the agarwood inducer into the trunk of Aquilaria sinensis, and changing patterns of chemical constituents, especially 2-(2-phenylethyl)chromones (PECs), in wood samples collected from the 1st to 12th month, were analyzed by GC-EI-MS and UPLC-ESI-MS/MS methods. Aromatic compounds, steroids, fatty acids/esters, sesquiterpenoids, and PECs were detected by GC-MS, in which PECs were the major constituents. Following this, UPLC-MS was used for further comprehensive analysis of PECs, from which we found that 2-(2-phenylethyl)chromones of flindersia type (FTPECs) were the most abundant, while PECs with epoxidated chromone moiety were detected with limited numbers and relatively low content. Speculation on the formation of major FTPECs was fully elucidated in our context. The key step of FTPECs biosynthesis is possibly catalyzed by type III polyketide synthases (PKSs) which condensate dihydro-cinnamoyl-CoA analogues and malonyl-CoA with 2-hydroxy-benzoyl-CoA to produce 2-(2-phenyethyl)chromone scaffold, or with 2,5-dihydroxybenzoyl-CoA to form FTPECS with 6-hydroxy group, which may serve as precursors for further reactions catalyzed by hydroxylase or O-methyltransferase (OMT) to produce FTPECs with diverse substitution patterns. It is the first report that systematically analyzed dynamic changes of secondary metabolites during the process of agarwood formation and fully discussed the biosynthetic pathway of PECs.
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