Benchmarking of Fluorescent Proteins in Trichoderma reesei Using Flow Cytometry

Fungi are the subject of extensive research due to their significant impact on human life. However, the development of tools based on fluorescent proteins has been slow in these species. Trichoderma reesei, a filamentous fungus known for its high secretion capability, shows great potential for producing heterologous proteins. However, the limited range of available fluorescent proteins for T. reesei hinders progress in its biological study and industrial applications. The goal of this study is to expand the panel of fluorescent markers for T. reesei by testing six new fluorescent proteins. To enable accurate comparisons, the method previously designed for eGFP was optimized to insert coding genes as a single copy by replacing the native pTEF promoter with the Neurospora crassa equivalent. The benchmarking assay identified one yellow-fluorescent protein and two green-fluorescent proteins that outperformed eGFP for transformant screening applications and a red fluorescent protein whose screening performance is comparable to that of eGFP, which would be valuable for colabeling purposes. These results could not have been predicted from the values reported in the Fluorescent Protein Database and the relative excitation of each protein at 488 nm accounts for only part of the difference. Additionally, the data obtained on fluorescent proteins and the approach used may be beneficial for investigating other types of fungi.