Fungal Enzyme Study Reveals Stereochemistry Limits Cytochalasan Biocatalyst Versatility

Cytochalasans, a large family of fungal metabolites prized for their ability to inhibit actin polymerization and display cytotoxic, antimicrobial, antiviral and immunosuppressant activities in various assays, have long tempted chemists seeking efficient ways to build their intricate structures. Now a team led by Elizabeth Skellam, with authors including Lei Li, Tahir Ali and Christine Beemelmanns, reports in the journal Fungal Biology and Biotechnology a systematic attempt to harness the enzymes that decorate these molecules, and the work delivers a clear if sobering message: the stereochemistry of functional groups around the cytochalasan backbone, not the size of its macrocycle, is what restricts which cytochrome P450 monooxygenases can be transplanted onto non-native substrates.

Cytochalasans are built by a polyketide synthase/non-ribosomal peptide synthetase that fuses an amino acid-derived moiety to a polyketide chain, with a Diels-Alderase and an alpha,beta-hydrolase helping forge the tricyclic core. Tailoring enzymes then diversify the scaffold: P450s epoxidize the cyclohexene ring or hydroxylate the macrocycle, sometimes iteratively introducing two hydroxyl groups at adjacent carbons; Baeyer-Villiger monooxygenases insert oxygen into the macrocycle to form esters or carbonates; and oxidoreductases and transferases make further adjustments. Because chemical synthesis of cytochalasans remains challenging, requiring many protection and deprotection steps and careful stereochemical control at late stages, the authors see chemoenzymatic routes as attractive, provided the tailoring enzymes tolerate a broad range of substrates.

To map that tolerance, the researchers first mined fungal genomes using the Diels-Alderase sequence from the pyrichalasin H cluster as a query. This identified cryptic cytochalasan biosynthetic gene clusters in six species: Aspergillus heteromorphus CBS 117.55, Aspergillus sclerotioniger CBS 115,572, Colletotrichum spinosum CBS 515.97, Colletotrichum sidae CBS 518.97, Metarhizium brunneum ARSEF 3297 and Metarhizium robertsii ARSEF 23. Each cluster encoded the four core enzymes plus two P450s corresponding to macrocycle and cyclohexene regioselectivities, but varied in other tailoring genes, transcription factors and transporters. Comparative genomics also uncovered a thioredoxin-like gene, dubbed ccsX, that co-occurs with Baeyer-Villiger monooxygenase genes across more than a dozen diverse fungi, leading the team to speculate, pending confirmation in a follow-up study, that it may scavenge peroxide radicals released during uncoupling of the monooxygenase.

Since none of these fungi except A. sclerotioniger had previously been reported to produce cytochalasans, the team cultivated A. heteromorphus across a panel of media and confirmed expression of the PKS-NRPS gene in four of five media and the thioredoxin-like gene in all five. High-resolution LC-MS/MS analysis then revealed a low-abundance metabolite in four of the five media, detected at m/z 496.2291 in positive ion mode with a retention time clearly distinct from a cytochalasin E standard. In silico spectral matching gave its highest similarity score to cytochalasin E, supporting its classification within the cytochalasan family, and the authors propose from the fragmentation pattern that it derives from a C18 polyketide lacking methyl groups at C-16 and C-18. Low abundance has so far prevented isolation and full structural elucidation, so the assignment remains putative.

Phylogenetic analysis of the P450 sequences added a predictive layer. The enzymes clade according to their regiochemistry, separating cyclohexene-acting from macrocycle-acting P450s, and the macrocycle sub-clades reflect structural features of the final molecules, including polyketide chain length, the type of amino acid incorporated and the presence of specific tailoring enzymes. Multiple sequence alignments confirmed conserved heme-binding and structural motifs, and AlphaFold modelling showed highly conserved folds, suggesting all the P450s under study should be functional proteins, even where their activity on foreign substrates later failed.

The functional tests used a combinatorial biosynthesis platform in which candidate P450 genes were expressed under the amyB promoter in Magnaporthe grisea mutant strains. The Delta-pyiD strain produces pyrichalasin H analogues lacking a hydroxyl group at C-18, so a compatible macrocycle P450 should restore pyrichalasin H production. The validated iterative hydroxylase CHGG_01243 from Chaetomium globosum did exactly that, restoring production in eight of eleven transformants, though it did not dihydroxylate the pyrichalasin core, showing that this known iterative enzyme does not act iteratively on a non-native substrate with a smaller macrocycle. AhCYP2 from A. heteromorphus also restored pyrichalasin H, in three of six transformants, confirming it as a functional macrocycle-associated P450. As a control, CspCYP2 expressed in the Delta-pyiG strain, which lacks hydroxylation at C-7, restored pyrichalasin H, confirming its predicted cyclohexene role.

The failures proved as informative as the successes. CcsD from Aspergillus clavatus, suspected of installing the C-17/C-18 oxidations of cytochalasin E/K, produced no new metabolites, with RT-PCR revealing that M. grisea struggled to splice its first intron, likely preventing translation of functional protein. XsCYP2 from Xylaria sp., cloned cleanly from cDNA and verified as transcribed, was also inactive for reasons that remain unknown. CspCYP1 and MrCYP1 likewise failed to restore production or generate new peaks despite confirmed transcription. The authors note that a subtle structural difference may explain part of this pattern: the methyl groups at C-16 and C-18 in pyrichalasin H are cis, whereas in cytochalasin E and the epoxycytochalasin series they are trans, with the C-18 methyl inverted. Because P450 hydroxylation typically proceeds with retention of stereochemistry, the enzymes appear unable to abstract hydrogen from substrates whose methyl stereochemistry does not match.

To test how widespread such stereochemical variation is, the team analyzed 385 recently reported cytochalasan structures. Fifty-seven percent derive from a C16 polyketide chain and forty percent from a C18 chain, with the remainder from C12, C14 or C20 chains, and the relative stereochemistry of the C-16 and C-18 methyl groups varies considerably. AlphaFold models also flagged residues near the heme that may govern substrate positioning: a conserved phenylalanine in all functional macrocycle P450s, a second phenylalanine in C16-associated enzymes replaced by a valine in C18-associated ones, a conserved glutamine versus tyrosine split along the same lines, and a conserved serine in iterative macrocycle P450s. The authors plan mutagenesis studies to pin down these roles.

The study also offers a practical shortcut. Rather than the laborious rounds of transformant screening that combinatorial biosynthesis demands, the researchers purified the intermediate 7-deshydroxypyrichalasin H from the Delta-pyiG strain and pulse-fed it, dissolved in DMSO, to the Delta-pyiD strain, which retains the functional cyclohexene P450 PyiG. LC-MS confirmed restoration of pyrichalasin H, demonstrating that exogenously added cytochalasans can penetrate the fungal cell wall and be biotransformed by native enzymes in situ. Taken together, the findings show that cytochalasan P450s do possess intrinsic promiscuity, but that matching macrocycle size alone is not enough: successful biocatalytic pairing requires attention to the stereochemistry of the functional groups decorating the oxidation site, knowledge the authors say should enable more efficient production of novel cytochalasans whose biological activities may ultimately be tuned by the methyl patterns installed by the PKS-NRPS machinery.

Subject of Research: Substrate versatility and stereochemical restrictions of cytochalasan cytochrome P450 monooxygenases investigated by combinatorial biosynthesis in fungi

Article Title: Investigating the versatility of cytochalasan cytochrome P450 monooxygenases using combinatorial biosynthesis reveals stereochemical restrictions

Article References: Li, L., Ali, T., Goralczyk, J., Jayasundara, S., Paul, A., de Amorim, M. R., Beemelmanns, C., & Skellam, E. (2026). Investigating the versatility of cytochalasan cytochrome P450 monooxygenases using combinatorial biosynthesis reveals stereochemical restrictions. Fungal Biology and Biotechnology, 13(1), Article 9. https://doi.org/10.1186/s40694-026-00214-2

Image Credits: AI Generated

DOI: 10.1186/s40694-026-00214-2

Keywords: cytochalasans, cytochrome P450, combinatorial biosynthesis, fungal natural products, biocatalysis, biosynthetic gene clusters, Magnaporthe grisea, stereochemistry, genome mining, heterologous expression, biotransformation, Baeyer-Villiger monooxygenase