Two Related Pyrrolidinedione Synthetase Loci in Fusarium heterosporum ATCC 74349 Produce Divergent Metabolites

Abstract

Equisetin synthetase (EqiS), from the filamentous fungus Fusarium heterosporum ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the F. heterosporum genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, eqx, is responsible for producing equisetin.

A new enzyme involved in the control of the stereochemistry in the decalin formation during equisetin biosynthesis

“…Features of the gene products in the fsa cluster and its neighbors are summarized in Table 1 (GenBank: LC025956). The biosynthetic gene (eqx) cluster of 1 in Fusarium heterosporum ATCC 74349, which has been corrected recently [15], was very similar to the fsa cluster (Table 1).…”

Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

“…Some PKS's, however, have an inactive ER domain; thus, final reduction of carbon-carbon double bonds along the carbon skeleton are achieved though collaboration with a trans-acting ER as observed in the biosynthesis of lovastatin [6,7], betaenone [8], and equisetin [9]. After decalin formation and subsequent chain elongation and modification, the resulting decalin polyketide product can be released from the PKS by various mechanisms.…”

“…After decalin formation and subsequent chain elongation and modification, the resulting decalin polyketide product can be released from the PKS by various mechanisms. A terminal PKS reduction domain catalyzes release in the betaenone and equisetin biosynthesis pathway [8,9], while a trans-acting thioesterase (LovG) is involved in the lovastatin pathway [10].…”

“…A terminal PKS reduction domain catalyzes release in the betaenone and equisetin biosynthesis pathway [8,9], while a trans-acting thioesterase (LovG) is involved in the lovastatin pathway [10].…”

Recent advances of Diels–Alderases involved in natural product biosynthesis

“…25,26 Computational analysis of the [4+2] cycloaddition with a truncated substrate indicated that the enzyme accelerates the cycloaddition a 1000-fold at 30°C. 25,26 Homologous genes are also found in the biosynthetic gene clusters of the tetramic acid-containing adducts such as equisetin (Eqx3; 90% identity) 27 and pyrrolocin (gNR600; 37%) 28 and macrocyclic adduct cytochalasin (CcsF; 27%). 29 Frequent occurrence of DAase genes with PKS-NRPS genes suggested co-evolution of these genes.…”