Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

Abstract Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9‐membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate‐processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine‐binding proteins.

Areinvestigation of the metabolites produced by B. fulva led to the identification of the probable monomer 10,w hich undergoes facile decarboxylation to 9.D ecarboxylation is also proposed to produce the exo-methylene putative intermediate 11 (Scheme 1), [10] which can then undergo cyclization with asecond molecule of 10 to produce 1.Another mode of dimerization of 10 (head-to-side dimerization) explains the biosynthesis of agnestadride A(8). [10] Barton and Sutherland also hypothesized that monomer 9 is am odified product of the citric acid cycle,p roduced from apolyketide-derived hexanoate with oxaloacetate. [3] Isotopic feeding studies supported this hypothesis [11] and suggested that acandidate maleidride biosynthetic gene cluster (BGC) should contain ah ighly reducing polyketide synthase (hrPKS), [12] as well as ag ene encoding ac itrate-synthaselike enzyme. [13]  Based on this hypothesis,w es equenced the genomes of two B. fulva strains,IMI 40021 and IMI 58422, both of which are linked to the original strain investigated by Raistrick and Smith. [1] Theformer reliably produces byssochlamic acid, but the latter is an unreliable and generally poor producer. BLAST [14] analyses were utilized to search the genomes of both B. fulva strains for likely maleidride BGCs,which led to the identification of ah ighly homologous maleidride-type BGC in each genome ( Figure 2, Genbank accession KU928136). Each BGC contains an hrPKS,acitrate-synthase-like enzyme,a nd several other interesting genes, including am ethylcitrate dehydratase.T ranscriptomic analysis of B. fulva IMI 40021 under byssochlamic acid producing and non-producing conditions confirmed that the putative BGC is highly differentially expressed ( Figure 2). Thet wo BGCs are more than 97 %i dentical but show differences in the bfR4 promoter region, which might explain the low productivity we observed in the IMI 58422 strain (see the Supporting Information).
BLAST [14] searches of genome sequences deposited at the NCBI, utilizing bfpks1 and the citrate synthase-like gene (bfL2)a sq ueries,r evealed several further likely maleidride BGCs in different fungi. TwoB GCs were identified from Talaromyces stipitatus.M any Talaromyces species are known to produce glaucanic acid (2), as well as the more complex rubratoxin B (5). [15] Athird BGC was identified in a Cochliobolus species. Cochliobolus species are synonymous with Helminthosporium,which are the original producers of heveadride (3). [5,16] The bfpks1 gene that encodes the PKS was knocked out by using the bipartite method [17] to produce B. fulva bfDpks1 strains, which no longer produced byssochlamic acid (1)o ra gnestadride A(8; Figure 3A and 3B). This is consistent with heptadride 8 being formed from the same pathway as byssochlamic acid.
In arecent investigation of the biosynthesis of phomoidride A (4), Oikawa and co-workers also identified aB GC containing an hrPKS,ac itrate-synthase-like enzyme,a nd am ethylcitrate dehydratase,a sw ell as the two maleidride-type BGCs from Talaromyces stipitatus. [18] Heterologous expression experiments utilizing the three genes from one of the T. stipitatus BGCs allowed the isolation of al onger-chain decarboxylated monomer.T hese experiments confirm the polyketide/oxaloacetateorigin of maleidride monomers. [18] However the proteins responsible for the key cyclisation reaction necessary for maleidride biosynthesis remain unkown.
From comparison of the two B. fulva BGCs and the other BGCs identified above,f our highly conserved genes were chosen for heterologous expression experiments in Aspergillus oryzae NSAR1: [19,20] the hrPKS bfpks1;t he citratesynthase-like gene bfL2;t he methylcitrate dehydratase bfL3;a nd the hydrolase 341 bfL1,w hich encodes ap rotein homologous to Ty pe II thiolesterases such as RifR, [21] which are involved in the release of abberant ACP-bound polyketide intermediates during modular polyketide biosynthesis, as well as to LovG,w hich releases the lovastatin nonaketide from its PKS. [22] Expression of these four core genes in A. oryzae NSAR1 (strains AO-BF-PMCH 1-7) led to the production of both 9 and 10 ( Figure 3D). Compound 9 is already known to be the decarboxylation product of the highly volatile and unstable 10. [10] We also expressed bfpks1, bfL2,a nd bfL3 in the absence of bfL1,w hich encodes the hydrolase.U nder these conditions, 9 and 10 were not produced (see the Supporting Information), thus strongly suggesting that the hydrolase is essential in the byssochlamic acid system.
Oikawa and co-workers suggested that the longer-chain decarboxylated monomer isolated from their heterologous expression experiments is produced by an adventitious native decarboxylase. [18] Our experiments have previously identified the natural product 10 by comparing its LCMS characteristics with data collected for the synthetic homologue,w hich was Scheme 1. Proposed general pathway to maleidrides.

Angewandte Chemie
Communications also observed to decarboxylate spontaneously to give 9.T his demonstrates that, at least for the shorter-chain maleic anhydrides,noe nzymatic decarboxylation is necessary. [10] To investigate the dimerization process,s everal further genes were selected for heterologous expression. Analysis of the BGC comparisons from several organisms (Figure 2) revealed that in addition to the core genes,two further types of genes are common to each BGC,namely either one or two genes encoding putative ketosteroid isomerase (KI)-like proteins and either one or two putative phosphatidylethanolamine-binding proteins (PEBP).
Co-expression of the two KI-like genes (bfL6 and bfL10) with the core genes (bfpks1, bfL1, bfL2,a nd bfL3)i nt he heterologous host (strains AO-BF-PMCH + KIs 1-21) led to the production of byssochlamic acid (1)a nd the decarboxylated intermediate 9 in several transformants ( Figure 3E). Agnestadride A( 8)a nd the intermediate 10 were also detected, but in low titer (see the Supporting Information). This result shows that in the A. oryzae NSAR1 background, the four core "monomer" genes and the KI-like genes are sufficient to form both nonadrides and heptadrides.
To determine whether the presence of both KI-like genes is necessary for dimerization, or alternatively,w hether each KI-like gene controls ad ifferent dimerization mode (nonadride/ heptadride), the core "monomer" genes were coexpressed with either KI1 (bfL6)orKI2 (bfL10)alone to give strains AO-BF-PMCH + KI1 1-7 and AO-BF-PMCH + KI2 1-7. Both sets of strains only produced 9 and 10,w ith no evidence for dimerized products (see the Supporting Information). Reverse-transcription PCR (RT-PCR) confirmed that both KI1 and KI2 genes were expressed in each experiment, therefore it can be concluded that within the A. oryzae NSAR1 background, the core "monomer" genes and KI1 or KI2 alone are not sufficient to catalyze dimerization.
TheBGC comparisons also revealed that genes encoding proteins similar to PEBPs are common to all of the maleidride BGCs ( Figure 2). Thetwo PEBP genes (bfL5 and bfL9)were   (8); H) Purified byssochlamic acid (1). Compounds 12, 12' ',and 13 are cometabolites of 1 and have been previously identified and characterized. [10] Angewandte Chemie Communications thus co-expressed with the core genes and the KI-like genes in A. oryzae NSAR1 to create strains AO-BF-PMCH + KIs + PEBPs 1-8. No new compounds were identified from extracts from these strains.However,direct quantitative comparisons between strains containing the PEBP genes in addition to KI genes showed an over 20-fold increase in the biosynthesis of byssochlamic acid (to 44 mg L À1 ,F igure 3F), as well as acorresponding increase in the production of the heptadride 8.Byssochlamic acid (1)isolated from these experiments was chromatographically and spectroscopically identical to 1 isolated from wild-type (WT) B. fulva (see the Supporting Information). Compounds 12 and 12' ' were also observed in these experiments:t hese are most likely formed in vitro through partial hydrolysis of 1 during extraction and analysis. However,the reduction product 13 is not formed in A. oryzae, thus suggesting that 13 arises in B. fulva through the activity of an adventitious enzyme.
Theb iosynthetic pathway to byssochlamic acid and agnestadride Ah as thus been fully elucidated through genome and transcriptome sequencing,g ene disruption, and heterologous expression. Putative maleidride BGCs were identified from the genomes of Talaromyces and Cochliobolus species.B oth genera are known to produce nonadrides. Comparison of the byssochlamic acid cluster to the putative maleidride gene clusters identified acore set of genes that are common to all five BGCs.Four genes were identified (bfpks1, bfL1, bfL2,a nd bfL3)t hat, when expressed in the heterologous host A. oryzae NSAR1, produce the monomer 10,a s well as its spontaneous decarboxylation product 9,b oth of which have been previously identified from WT B. fulva extracts. [10] Experiments to determine the genes involved in dimerization demonstrated that both KI-like genes coexpressed with the core genes are necessary.F urthermore, co-expression of the core genes with both KI-like genes and both PEBPs gave increased yields of dimerized products.The mechanism for the dimerization still needs to be established through in vitro experiments.
Based on previous biosynthetic investigations,o ur discovery of the byssochlamic acid pathway,a nd research conducted by Oikawa and co-workers, [18] we now propose ag eneral biosynthetic route to maleidrides (Scheme 1). In each maleidride pathway,t he initial steps are common, with potential differences including polyketide chain length and pattern of reduction. Forexample,the biosynthesis of 1-3, 6, and 8 requires triketides; 5 requires pentaketides;a nd 4 requires hexaketides.T he polyketide is presumably released from the PKS by the BfL1 hydrolase,a lthough it is not yet known whether this is as the free acid or as aCoA-thiolester. This result contrasts with the recently reported work of Oikawa, who showed that the hydrolase did not appear to be required in the case of aT alaromyces PKS. [18] However it is known that some iterative fungal PKSs,f or example the squalestatin tetraketide synthase, [23] can release polyketides without the requirement for ad edicated thiolesterase. Reaction between the polyketide and oxaloacetate,catalyzed by the citrate-synthase-like enzyme,i sf ollowed by dehydration by the methylcitrate dehydratase homologue,which then sets up the synthesis of the maleic anhydride decarboxylated monomers 9 and 11.I ti si nteresting to note that fungi have evolved at least two distinct routes to maleic anhydride moieties.O ther organisms oxidize vicinal aromatic methyl groups to form this biologically important motif. [24,25] TheK I-like enzyme(s) can then react 10 with either 9 or 11.T hese differences,a nd the orientation of the reacting species,d etermine the size,s ubstitution pattern, and stereochemistry of the central carbocyclic ring. Previous in vitro work by Baldwin and others [26] has shown that such reactions are chemically feasible,a lthough in the absence of enzymes, strong bases are required and low yields are observed. Our experiments suggest that the KI-like proteins from B. fulva act together to form heterodimeric enzymes that show "flexibility" in that they create both nonadrides and heptadrides.T oo ur knowledge, B. fulva IMI 40021 is the first fungus known to produce cross-class maleidrides (i.e., nonadride and heptadride), so this "flexibility" may be specific to the byssochlamic acid/agnestadride Apathway.Other known maleidride producers appear to have pathways that only involve one class of dimerization, although it is possible that reinvestigation of the metabolites produced may identify other minor compounds with different dimerization modes. TheP EBP-like enzymes also appear to be involved in the dimerization, and although their catalytic role is not yet clear, it is possible their known anionic binding ability may be involved. [27] One possibility may be the chaperoning of highly unstable carboxylates such as 10 in order to prevent premature decarboxylation prior to dimerization. However further understanding of the role of the KI and PEBP proteins must await in vitro experiments.G ene clusters encoding the biosynthesis of octadrides such as zopfiellin (7)h ave not yet been reported, but our results suggest that similar KI-like and PEBP-like proteins may be involved in their biosynthesis.
In conclusion, our experiments have revealed for the first time that maleidride biosynthesis can be recreated in ah eterologous host and the observations open the way for future experiments to create new compounds in this class with potentially interesting and useful biological properties through pathway engineering.F urther experiments are currently underway in our laboratories to understand the mechanisms and selectivity of the ring-forming enzymes.