Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis

Abstract

Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSβ), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis

“…14 The TEII enzyme AlpS has been shown to be essential for kinamycin biosynthesis in vivo and its hydrolytic activity with a SNAC-substrate analogue demonstrated in vitro. 102 (Fig. 12A).…”

“…In these cases, aer the nal extension reaction has occurred the PK chain is transferred to a standalone ACP protein, followed by hydrolytic release catalysed by a TEII enzyme. 95,96,98,99 TEII enzymes also catalyse the hydrolytic chain release of non-polyethers, including zaragozic acid, 100 colibactin, 101 kinamycin (30), 102 and possibly indanomycin. 22,103 Kinamycin is noteworthy as it is a type II polyketide.…”

Unexpected Role of a Short‐Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis

“…Recently,AlpS that belongs to the a/ b hydrolase family has been identified as aproduct releasing thioesterase in the kinamycin biosynthesis and its deletion also resulted no major related metabolite accumulation. [26] In comparison, MrqD is from the metallo-b-lactamase family, which indicates that diverse mechanisms may be involved in type II polyketide chain releasing process.M etallo-b-lactamase family proteins have been found in other polyketides chain releasing process.F or example,a trochrysone carboxyl ACPt hioesterase (ACTE) that belongs to the metallo-blactamase superfamily catalyzes the product release from atrochrysone carboxylic acid synthase (ACAS), anonreducing PKS,ina trochrysone carboxylic acid biosynthesis. [25] On the other hand, it is important to investigate the mechanism of MrqM-catalyzed aldol-type cyclization.…”

Unexpected Role of a Short‐Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis

“…In fact, chain releasing in type II polyketide biosynthesis has been largely under‐explored. Recently, AlpS that belongs to the α/β hydrolase family has been identified as a product releasing thioesterase in the kinamycin biosynthesis and its deletion also resulted no major related metabolite accumulation [26] . In comparison, MrqD is from the metallo ‐ β‐lactamase family, which indicates that diverse mechanisms may be involved in type II polyketide chain releasing process.…”