Abstract
The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9-C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.
Highlights
Type III polyketide synthases (PKSs) from fungi produce a wide variety of polyketides
When the E. coli BL21(DE3) transformant harboring pET22-csyB was cultured at 18 °C for 20 h in the induction medium (Fig. 2A), we noticed the production of compounds in the culture medium, which were not detected in the control transformant (Fig. 3A)
ACP thioesters of unsaturated fatty acids such as palmitoleate (C16:1, ⌬9) and cis-vaccenate (C18:1, ⌬11) are thought to be used by CsyB to form 3-acetyl-4-hydroxy-6alkenyl-␣-pyrones, the position of the double bond in the unsaturated aliphatic chain of compounds 2a–2d was not confirmed yet. From these results together with the data obtained from the acetate feeding experiments [15], we assumed that CsyB catalyzes the condensation of fatty acyl-ACP starter with malonyl-CoA to form the -ketoacyl intermediate followed by further condensation with acetomalonyl-CoA or acetoacetylCoA
Summary
Type III polyketide synthases (PKSs) from fungi produce a wide variety of polyketides. Results: CsyB catalyzes the formation of 3-acetyl-4-hydroxy-6-alkyl-␣-pyrone (AcAP) from a fatty acyl-CoA, a malonyl-CoA, and an acetoacetyl-CoA. The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. Synthetic N-acetylcysteamine thioester of -ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-␣-pyrone by CsyB These results confirmed that CsyB catalyzed the synthesis of -ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-␣-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl4-hydroxy-6-alkyl-␣-pyrone by catalyzed the coupling of two -ketoacyl-CoAs. Polyketides are secondary metabolites produced by various organisms including bacteria, fungi, and plants. We show that CsyB catalyzes condensation of two diketo acyl-CoAs, -keto fatty acyl-CoA and acetoacetyl-CoA, to form AcAP
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