Starter unit flexibility for engineered product synthesis by the nonreducing polyketide synthase PksA

Abstract

Nonreducing polyketide synthases (NR-PKSs) are unique among PKSs in their domain structure, notably including a starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain that selects an acyl group as the primer for biosynthesis, most commonly acetyl-CoA from central metabolism. This clan of mega-enzymes resembles fatty acid synthases (FASs) by sharing both their central chain elongation steps and their capacity for iterative catalysis. In this mode of synthesis, catalytic domains involved in chain extension exhibit substrate plasticity to accommodate growing chains as small as two carbons to 20 or more. PksA is the NR-PKS central to the biosynthesis of the mycotoxin aflatoxin B1 whose SAT domain accepts an unusual hexanoyl starter from a dedicated yeast-like FAS. Explored in this paper is the ability of PksA to utilize a selection of potential starter units as substrates to initiate and sustain extension and cyclization to on-target, programmed polyketide synthesis. Most of these starter units were successfully accepted and properly processed by PksA to achieve biosynthesis of the predicted naphthopyrone product. Analysis of the on-target and derailment products revealed trends of tolerance by individual PksA domains to alternative starter units. In addition, natural and un-natural variants of the active site cysteine were examined and found to be capable of biosynthesis, suggesting possible direct loading of starter units onto the β-ketoacyl synthase (KS) domain. In light of the data assembled here, the predictable synthesis of unnatural products by NR-PKSs is more fully defined.

Figure 1

Acyl-SNAC starter units and their products (A) Potential acyl-SNAC (N-acetylcysteamine) starter units assayed in this study, and their observed naphthopyrone products. (B) Starred products that do not conform to the template shown in (A) due to unusual number of ketide extensions (also see Supporting Information).

Figure 2

Relative production of on-target naphthopyrones. Product numbers are indicated on the y-axis and starter units are indicated on the right side of each bar. In the case of starter units 8 and 27, where two naphthopyrone products of different chain lengths were produced, the peak areas of both products have been combined for this chart.

Figure 3

Core structures of observed derailment products. (A) Derailment product cores were determined based on exact mass and known chromophores. (B) Major product observed in control reactions containing malonyl-SNAC as the sole substrate.

Figure 4

Comparison of hexyl-naphthopyrone (4) production by PksA-SAT active site mutants.

Scheme 1

Biosynthetic reactions catalyzed by PksA. (A) Early steps of aflatoxin B1 biosynthesis by HexA, HexB and PksA in Aspergillus parasiticus. (B) In vitro assays of alternative and native starter units performed in this study. SAT: starter-unit:acyl-carrier protein (ACP) transacylase, KS: β-ketoacyl synthase, MAT: malonyl:ACP transacylase, PT: product template, TE: thioesterase