Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces subiculosus and Radicicol from Pochonia chlamydosporia

Abstract

Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1,2 double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7,8 double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7,8-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1,2-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.Hypothemycin and structurally related compounds are resorcylic acid lactones (RALs) that are produced by Hypomyces subiculosus and other fungi (1,7, 14,25,26,28,43). The structures of some naturally occurring RALs are shown in Fig.

Rational reprogramming of fungal polyketide first-ring cyclization

“…The cyclization chamber is proposed to feature an active site dyad (AtCURS2: H 1308 , E 1497 ; CcRADS2: H 1325 , E 1520 ), where the aspartic acid (D 1543 ) that polarizes the catalytic base (H 1345 ) in NSAS is replaced by glutamic acid. This functionally conserved replacement is present in all known RAL and DAL PT domains (14)(15)(16)38) but not clades II-V of functionally characterized PT domains (29). The same D to E replacement is, nonetheless, common in dehydratases with a fold similar to the fold of PT domains, and it was also found in some type II PKS aromatase/ cyclase enzymes (32).…”

“…After the collapse of the enolate and aldol addition to the carbonyl, the oxyanion may be stabilized by a network of water molecules that are coordinated in NSAS by S 1356 , D 1543 , T 1546 , and N 1568 . Only some of these residues are conserved in AtCURS2 (S 1319 , E 1497 , V 1500 , and V 1521 ) and CcRADS2 (S 1336 , E 1520 , V 1523 , and N 1547 ), and similar replacements are also present in all RAL PT domains (14)(15)(16)38). The second one-half of the oxyanion hole is provided by the backbone amine of a glycine in dehydratase domains and hydratases with which PT domains share a double hot dog fold and a proposed evolutionary origin (10).…”

“…Although the biosynthesis of most fungal polyketides requires a single iPKS enzyme, the assembly of the resorcylic acid lactones (RALs) involves a pair of collaborating hrPKSs and nrPKSs acting in sequence (12)(13)(14)(15)(16)(17). Fungal RALs are rich pharmacophores with estrogen agonist (zearalenone), mitogen-activated protein kinase inhibitory (hypothemycin), and heat shock response modulatory activities [radicicol and monocillin II (1)] ( Fig.…”

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

“…Circular dichroism (CD) spectra were acquired with a JASCO J-810 instrument. 1 H, 13 C, and 2D NMR (homonuclear correlation, heteronuclear single quantum coherence, and heteronuclear multiple-bond correlation) spectra were obtained in CD 3 OD or C 5 D 5 N on a JEOL ECX-300 spectrometer. See SI Materials and Methods for details.…”

“…TE domains form the BDL macrolactone using the ω-1 alcohol as a nucleophile, but may use alternative nucleophiles such as the C9 enol to yield α-pyrones or water or alcohols from the media to form acyl resorcylic acids (ARAs), acyl dihydroxyphenylacetic acids (ADAs), and their esters (18)(19)(20)(21)(22). iPKSs that produce BDLs in the RAL 14 , RAL 12 , and DAL 12 subclasses have been characterized and reconstituted both in vivo by heterologous expression in yeast and in vitro using isolated recombinant iPKS enzymes (11)(12)(13)(14)(23)(24)(25). Domain exchanges among different BDLSs were used to decipher some of the programming rules of these enzymes and yielded a limited number of structurally diverse unnatural products (3,18,20,21,26,27).…”