Dalmanol biosyntheses require coupling of two separate polyketide gene clusters

Abstract

Polyketide-polyketide hybrids are unique natural products with promising bioactivity, but the hybridization processes remain poorly understood. Herein, we present that the biosynthetic pathways of two immunosuppressants, dalmanol A and acetodalmanol A, result from an unspecific monooxygenase triggered hybridization of two distinct polyketide (naphthalene and chromane) biosynthetic gene clusters. The orchestration of the functional dimorphism of the polyketide synthase (ChrA) ketoreductase (KR) domain (shortened as ChrA KR) with that of the KR partner (ChrB) in the bioassembly line increases the polyketide diversity and allows the fungal generation of plant chromanes (e.g., noreugenin) and phloroglucinols (e.g., 2,4,6-trihydroxyacetophenone). The simultaneous fungal biosynthesis of 1,3,6,8- and 2-acetyl-1,3,6,8-tetrahydroxynaphthalenes was addressed as well. Collectively, the work may symbolize a movement in understanding the multiple-gene-cluster involved natural product biosynthesis, and highlights the possible fungal generations of some chromane- and phloroglucinol-based phytochemicals.

Fig. 1. Representative frameworks of chromane-related polyketides. (A) Dalmanol A (1) and acetodalmanol A (2) signify hybridizations between the chromane and naphthalene biosynthetic pathways. (B) The biosynthetic network of fungal pentaketides 5–7 that are skeletally identical with noreugenin (8) from plant (Rheum palmatum). DPB, 1-(2,6-dihydroxyphenyl)butane-1,3-dione. PBEO (4), 1-(2,6-dihydroxyphenyl)but-2-en-1-one.

Fig. 2. Polyketide productivity comparison among the wild-type (WT) and mutants of D. eschscholzii. (A) The two gene clusters encoding naphthalene and chromane. The WT strain and mutants (Δ4HNR, ΔlacTL, ΔTF1, ΔpksTL, ΔChrA, and ΔChrB) were compared through the LC-HR/MS profiling for the production of: (B) dalmanol A (1) and acetodalmanol A (2) (peak areas integrated (C)) and (D) 5–7, 9 and 10, with authentic samples co-analyzed as references. Data shown as mean ± SD (n = 3). ** indicated P < 0.01, by Student's t test.

Fig. 3. Proposed biosynthetic pathways for dalmanol A (1) and acetodalmanol A (2). The dalmanol carbon skeleton formation requires the naphthalene and chromane biosynthetic gene clusters, which interact intra- and intercellularly with the presumable participation of an unspecific fungal monooxygenase expressible in D. eschscholzii and A. oryzae. UMO*, unspecific monooxygenase. Dotted and solid red arrows in 17b and 17c indicate the electron transfer in the intramolecular addition and aldol reactions, respectively.

Fig. 4. Metabolic network of polyketides encoded by the chromane gene cluster in D. eschscholzii. ChrA is a partially reducing polyketide synthase (PR-PKS) with the domains of β-ketoacylsynthase (KS), acyltransferase (AT), dehydratase (DH), C-methyltransferase (MT⁰, inactive), ketoreductase (KR, active/inactive) and acyl carrier protein (ACP). ChrB is a ketoreductase partner that catalyzes the selective 3-ketone reduction of acyclic pentaketide with acetonyl or allyl side-chain terminals. UER*, unspecific enoyl reductase expressible in D. eschscholzii and A. oryzae.

Fig. 5. Co-expression of KR-mutated ChrA and ChrB in A. oryzae (AO). HPLC traces for metabolites produced by AO ((i), as a blank reference), the transformants [with ChrA (ii), ChrA-M1 for K¹⁸²⁹D (iii), ChrA-M2 for S¹⁸⁵³A (iv), and ChrA-M3 for Y¹⁸⁶⁶A (v)], and the co-transformants with ChrA and ChrB (vi), ChrA-M1 and ChrB (vii), ChrA-M2 and ChrB (viii), and ChrA-M3 and ChrB (ix).

Fig. 6. Production of 1 and 2via heterologous co-expression and co-cultivation. Co-expression of pksTL, ChrA, and ChrB in A. oryzae gave a co-transformant (pksTL/ChrA/ChrB-AO) capable of producing 1 and 2, which were afforded as well by co-culturing the transformant (pksTL-AO) with the co-transformant (ChrA/ChrB-AO). However, none of 1 and 2 was generated if culturing separately the pksTL-AO transformant and the ChrA/ChrB-AO co-transformant.