Biology subjects

Skellam, E.

Publications and source records attributed to Skellam, E..

3 recordsLinked to original sources

bioRxiv2026

Heterologous expression of lyngbyatoxin biosynthetic genes in Aspergillus oryzae reveals transcriptional barriers but enables LtxC-mediated biotransformation

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

synthetic biology↗

bioRxiv2026

Complete biosynthesis of penicillin G in Nicotiana benthamiana

Commercial penicillin production has relied on microbial fermentation for more than 80 years. Here, we engineered the plant, Nicotiana benthamiana, to produce penicillin G in its leaves by transient expression of up to seven fungal biosynthetic genes. Remarkably, all recombinant proteins localize to the analogous subcellular compartments without engineering signal peptide sequences or post-translational modification sites. Although non-ribosomal peptide synthetases occur widely in fungi and bacteria to produce a plethora of specialized metabolites, their evolutionary distribution does not extend to plants. Our results now open a new metabolic frontier for natural product synthesis, and offer possibilities to address global health concerns through an alternative biotechnology platform for fungal-derived pharmaceutical production.

synthetic biology↗

bioRxiv2026

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

BackgroundCytochalasans are a large family of fungal metabolites which inhibit actin polymerization and ultimately lead to a broad range of biological effects in different assays. Investigations into the biosynthesis of cytochalasans has revealed that the cytochrome P450 monooxygenase (P450s) tailoring enzymes possess a somewhat relaxed substrate-specificity and may accept structurally-related intermediates for oxidation, partly explaining the variety of structural variations observed in this family of molecules. In this study, we investigate a broad range of P450 enzymes via combinatorial biosynthesis to better understand their substrate scope and potential applications as biocatalysts. ResultsGenome mining enabled us to identify cryptic cytochalasan biosynthetic gene clusters (BGCs) in six different species of fungi, each with at least two P450 enzymes encoded. Comparative genomics identified a cryptic thioredoxin-like enzyme encoded in cytochalasan BGCs that co-occurs with the gene encoding a Baeyer-Villiger monooxygenase. Heterologous expression of seven P450s in Magnaporthe grisea mutant strains, lacking P450s required for pyrichalasin H biosynthesis, enabled functional characterization of three P450s, two of which were previously cryptic. The experimental results, combined with phylogenetic analysis of the P450 sequences, reveal subtle information regarding the structures of the associated cytochalasans and begins to explain why some P450s are inactive on the substrates available to them. ConclusionsThe P450 enzymes involved in cytochalasan biosynthesis are known to be site-selective in their native host but also possess intrinsic promiscuity due to being able to modify structurally-related analogues. By investigating a diverse set of P450s from characterized and cryptic BGCs, we were able to identify that the stereochemistry of functional groups around the cytochalasan backbone is more restrictive than the size of the macrocycle when introducing the P450 enzyme to non-native substrates.

biochemistry↗