Towards the production of mushroom-derived sesquiterpenoids in a light-driven microbial consortium

Synthetic microbial consortia exhibit extended metabolic capacities compared to monocultures and therefore have a great potential for biotechnological applications. Here, we envision to combine the photoautotrophic features of cyanobacteria with the versatile production potential of the fungal chassis Ustilago maydis to produce mushroom-derived sesquiterpenoids from CO₂ and light. Mushroom-forming basidiomycetes are a rich source of sesquiterpenoids, but their slow growth and low isolation yields limit the access to those valuable compounds. Moreover, successful expression of the underlying biosynthetic pathways in traditional heterologous expression platforms can be challenging. U. maydis, a basidiomycete itself, constitutes a promising alternative.

While modified cyanobacteria will serve as the "breadwinner" of our consortium, supplying U. maydis with sucrose, we aim to engineer U. maydis to efficiently produce fungal sesquiterpenoids, such as α‑cuprenene and its oxygenated antimicrobial derivative lagopodin B. Our approach involves the transplantation of heterologous sesquiterpenoid biosynthesis pathways, as well as metabolic engineering to increase sesquiterpenoid yields. To facilitate this, we are currently implementing a modular cloning (MoClo) system for U. maydis while also expanding our genetic engineering toolbox with e.g. additional strong and/or inducible promoters.

Initial metabolic engineering efforts to enhance the yield of the sesquiterpene α-cuprenene indicate that the expression strength of the heterologous α-cuprenene synthase gene, cop6, represents a bottleneck. To address this issue, we are now focusing on single- or multi-copy integration of cop6 using different promoters. Additionally, through the heterologous expression of two cytochrome P450 monooxygenase genes, we have successfully produced oxygenated α-cuprenene derivatives, bringing us closer to the valuable sesquiterpenoid lagopodin B. Furthermore, initial proof-of-concept co-cultivation experiments with sucrose-producing cyanobacteria are promising. Thus, optimized production strains will next be tested for their performance in co-culture.