Hasenklever, D. | Search bioRxiv

bioRxiv2026

UstiGate: Next generation toolkit for advanced genetic engineering of the basidiomycete chassis Ustilago maydis

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.

synthetic biology↗

bioRxiv2026

A Spectrum of Possibilities: A Systematic Evaluation of Fluorescent Proteins in Cyanobacteria

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

synthetic biology↗