Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases

Abstract

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.

Figure 1

Structures of (A) fungal polyketides derived from collaborative actions of two IPKSs. (B) oxaleimide 1 and TAN-1813. Bold lines show enrichment from [1,2-¹³C]-acetate.

Figure 2

Characterization of the natural products from the pox cluster in P. oxalicum. (A) The gene clusters of interest. (B) HPLC traces of the metabolic extracts from different P. oxalicum cultures: (i) POX3 (transcriptionally activated through overexpression of PoxB) (ii) wild-type, (iii) ΔpoxE of POX3, (iv) ΔpoxF of POX3 and (v) ΔpoxO of POX3. *: secalonic acid D; **: linolenic acid that overlaps with 12. (C) X-ray crystal structure of 11a, (R)-PGME-amide of 1. (D) Structures of oxaleimide 2, 3, 9 and 10. (E) LC/MS analysis of the metabolic extracts from the culture of (i) A. nidulans A1145 (An), (ii) An expressing poxF, (iii) An expressing poxF and poxM, (iv) An expressing poxF, poxM and poxL, and (v) standard of 14, containing trace of olefin isomer. Extracted LC traces corresponding to the m/z⁺ for 14 (m/z⁺ = 186), m/z⁻ for 15 (m/z⁻ = 185) and m/z⁻ for 16 (m/z⁻ = 183). (F) Chemical complementation of POX3/ΔpoxF using 14 or 15. Extracted LC traces corresponding to the m/z⁻ for 2 (m/z⁻ = 500) and 4–8 (m/z⁻ = 484) of (i) POX3/ΔpoxF, (ii) POX3/ΔpoxF supplemented with 15, (iii) POX3/ΔpoxF supplemented with 14 and (iv) POX3. (G) Proposed biosynthesis of 14.

Figure 3

Proposed biosynthetic pathway of compound 1 to 10. PKS-NRPS domain abbreviations: KS: ketosynthase; MAT: malonyl-CoA:ACP transacylase; DH: dehydratase; MT: methyltransferase; KR: ketoreductase; ACP: acyl carrier protein; C: condensation; A: adenylation; T: thiolation; R: reductase; ER: enoylreductase. To determine the biosynthetic origin of the C18 substituent, we looked for a blocked mutant that may accumulate a precursor to 1 and 9 prior to rearrangement. Among the mutant constructs, we isolated a new compound 12 upon knockout of the α/β-hydrolase poxO (Figure 2b, v). 12 is the trans-decalin containing alcohol depicted in Figure 3. The alcohol can be formed from the over-reduction of the aldehyde 13. This could occur when the Knoevenagel condensation of 13 to form 18 does not take place. Formation of similar over-reduced products from PKS-NRPKS has been observed. Hence, this suggests that PoxO is responsible for the Knoevenagel condensation. More importantly, the structure of 12, and that inferred for 13, reveal that the aliphatic chain may be derived from incorporation of a nonproteinogenic amino acid, (S, E)-2-aminodec-4-enoic acid (14), by the NRPS domain of PoxE (Figure 3). Condensation between the octaketide product of the PKS and the amino group of 14 yields an aminoacyl product 17, which can be cyclized by the Diels-Alderase PoxQ and reductively released by the reductive (R) domain of PoxE to yield 13.