Sphingofungins belong to a group of structurally related sphingolipid inhibitors produced by fungi, which specifically inhibit serine palmitoyl transferases, enzymes catalyzing the initial step during sphingolipid biosynthesis. Sphingolipids are integral parts of the eukaryotic cell membrane, and disturbances in their homeostasis have been linked to various human diseases. It has been suggested that external interventions, via sphingolipid inhibitors, may represent a promising approach for alternative therapies. Here, we identified and elucidated the biosynthetic gene cluster responsible for the biosynthesis of sphingofungins B, C, and D in Aspergillus fumigatus. Moreover, in vitro analyses have shown that sphingofungin biosynthesis starts with the condensation of a C18 polyketide with the uncommon substrate aminomalonate. Furthermore, the investigations on sphingofungin E and F produced by Paecilomyces variotii pointed out that different aminomalonate derivatives are used as substrates for those chemical variants. This research boosts knowledge on the general biosynthesis of sphingolipid inhibitors in fungi.

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.

Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed.