UstiGate: A standardized modular cloning platform for advanced engineering of the fungal chassis Ustilago maydis

Abstract

The corn smut fungus Ustilago maydis is an established model for fungal biology and an emerging host for biotechnology. However, its broader application in synthetic biology has been constrained by a limited repertoire of standardized genetic parts and tools for multigene engineering. Here, we introduce UstiGate, a modular toolkit that combines MoClo-compatible DNA assembly with targeted genomic integration in U. maydis. The toolkit comprises 92 parts and vectors, including more than 20 promoter regions as well as terminators, selectable markers, fluorescent reporters and modules for different genomic integration sites. Quantitative reporter analyses at both, population and single-cell level, revealed a broad range of promoter strengths and characterized their behavior across growth phases, media, genomic loci and reporter contexts. We further identified a maltose-inducible, glucose-repressed promoter that enables autoinduction and established carotenoid-biosynthesis genes as integration sites supporting visual pre-selection of transformants. Finally, UstiGate enabled the assembly and simultaneous genomic integration of four transcriptional units in a single transformation. UstiGate thus provides a standardized and quantitatively characterized platform for standard applications as well as multigene engineering, enabling advanced synthetic biology applications such as pathway transplantation in U. maydis.