Genome Mining and Assembly-Line Biosynthesis of the UCS1025A Pyrrolizidinone Family of Fungal Alkaloids

Abstract

UCS1025A is a fungal polyketide/alkaloid that displays strong inhibition of telomerase. The structures of UCS1025A and related natural products are featured by a tricyclic furopyrrolizidine connected to a trans-decalin fragment. We mined the genome of a thermophilic fungus and activated the ucs gene cluster to produce UCS1025A at a high titer. Genetic and biochemical analysis revealed a PKS-NRPS assembly line that activates 2S,3S-methylproline derived from l-isoleucine, followed by Knoevenagel condensation to construct the pyrrolizidine moiety. Oxidation of the 3S-methyl group to a carboxylate leads to an oxa-Michael cyclization and furnishes the furopyrrolizidine. Our work reveals a new strategy used by nature to construct heterocyclic alkaloid-like ring systems using assembly line logic.

Figure 1

Structures of fungal pyrrolizidinone natural products are shown in A (pyrrolizidinone highlighted in blue) and a proposed biosynthetic route by a PKS-NRPS is shown in B.

Figure 2

The ucs cluster is responsible for the biosynthesis of 1. (A) the cluster is found in three organisms; KS, ketosynthase; MAT, malonyl-CoA transferase; DH, dehydratase; MT, methyltransferase; KR, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; T, thiolation; R, reductase; (B) Genetic analysis of the cluster found in M. thermophila. Shown are chromatograms of extracts from: i) wild type strain; ii) MT18: the ucsR overexpression strain; iii) MT18/ΔucsA; iv) MT18/ΔucsH; (C) 2 is a shunt product formed by the overreduction of the acyclic pyrrolizidinone 3. Similar enereduction by unidentified reductase also affords shunt products such as 5.

Figure 3

Functional assignment of oxygenases in the ucs cluster. (A) Characterization of P450 UcsK: i) Extractedion chromatogram (EIC) of 5 produced by MT18/ΔucsK; ii) and iii) LC-MS analysis of the biochemical assays of UcsK using yeast microsomal fractions; (B) The α-KG dependent enzyme UcsF catalyzes the oxidation of L-isoleucine to yield (4S, 5S)-4-methylpyrroline-5-carboxylate 10; i) and ii) Analysis of UcsF assays using Fmoc-Cl as the derivatization agent to detect 8; iii) and iv) Analysis of UcsF assays using o-aminobenzaldehyde (o-AB) as the derivatization agent to detect 10.

Figure 4

Proposed biosynthetic pathway of UCS1025A based on biochemical and genetic evidences present in this work.