During their life cycle, many filamentous fungi grow from small spores into intricate networks of branching mycelium, the latter of which can be used to form novel biomaterials, sustainable meat analogues, and bioremediation agents. Conventional flow cytometry has been used to analyze fungal spores, but this method is not suitable for mycelia due to their size and tendency to fuse with neighboring hyphae, limiting the ability to characterize genetic parts in the more biotechnologically pertinent morphology. Here, we quantify the strength of several constitutive promoters in both the spores and mycelium of the industrially relevant fungus Fusarium venenatum. A library of F. venenatum variants, each encoding a fluorescent protein downstream of a different constitutive promoter, is generated via Agrobacterium-mediated transformation. Agrobacterium-mediated delivery of DNA with large (>1 kb) homology arms achieved highly efficient site-specific integration of genetic constructs, enabling consistent evaluation of the tested sequences. Fluorescence microscopy is used to measure gene expression in the macroconidia of the variants. To evaluate promoter strengths in the mycelium, spores of each of the variants are encapsulated in alginate beads and grown into microcolonies hundreds of microns in length, and fluorescent protein expression is measured using large-particle flow cytometry. These results provide the first systematic characterization of genetic parts in F. venenatum across multiple life-cycle stages, enabling more precise fungal engineering for sustainability-focused applications.
