Banko, P. | Search bioRxiv

bioRxiv2026

Structural determination of human nucleosomes reconstituted by the ExACT platform

The structure and function of eukaryotic chromatin have been extensively studied using conventional salt dialysis-based nucleosome assembly, which has provided fundamental structural insights into nucleosomes as well as a mechanistic understanding of their roles in DNA replication, repair and gene expression. Recently, we developed a labor-saving and time-efficient nucleosome assembly method using a wheat germ cell-free Expression and Assembly Coupled Technology (ExACT), which is emerging as a powerful tool for chromatin research. Here we report cryo-electron microscopy (cryo-EM) structures of human H3.1- and H3.3-containing nucleosomes assembled using this approach and validated by deep-learning-based amino acid-wise model quality (DAQ) scoring. The structures of H3.1- and H3.3-nucleosomes are nearly identical to previously reported models, confirming the structural fidelity of the method. In addition, we determined the previously unreported structure of the primate-specific H3.X-containing nucleosome. Together, these findings validate the cell-free co-expression nucleosome assembly platform and establish this method as a robust framework for biochemically investigating chromatin dynamics.

molecular biology↗

bioRxiv2025

Establishment of an antimetabolite-based transformation system for the wood-decaying basidiomycete Phanerochaete chrysosporium

The model wood-decaying basidiomycete Phanerochaete chrysosporium has been extensively studied to elucidate the molecular mechanisms of wood decomposition. However, genetic studies have been limited by the lack of adequate genetic tools. Here, we established an antimetabolite-based transformation system, originally developed for ascomycetes, for use in P. chrysosporium. The transformation system utilizes pyrithiamine (PT), a thiamine antimetabolite, in combination with the pPTRII vector that contains the PT resistance gene (ptrA). PT effectively inhibited the growth of P. chrysosporium, and the introduction of ptrA conferred resistance to transformant mycelia. The transformation efficiency was comparable to that in ascomycetes, suggesting that the transformation system is also applicable to basidiomycetes. To examine the suitability of the system for heterologous gene expression, four cassettes were constructed to express GFP under the promoters of the actin1, DED, and GAPDH genes. Promoter activities were assessed via fluorescent microscopy observation of transformant mycelia and GFP quantification in crude cell extracts, revealing that the actin1 promoter drove the highest level of expression. Furthermore, truncating repeat sequences of the autonomously replicating sequence in the vector backbone improved transformation efficiency, likely due to the reduction in vector size. The transformation efficiency of the gene cassette-inserted vector in P. chrysosporium was relatively higher than that reported with alternative transformation systems in other species of wood- decaying basidiomycetes. The present transformation system could provide a platform for protein expression and genetic engineering in P. chrysosporium and potentially in other wood-decaying basidiomycetes. ImportanceWood-decaying basidiomycetes are well-recognized for their exceptional capabilities to decompose lignocellulosic biomass and oxidize a broad range of complex organic compounds. These capabilities are essential for maintaining the forest ecosystem and hold potential in biotechnological applications such as transforming recalcitrant biomass into useful compounds and degrading toxic substances in industrial effluents. However, genetic manipulation in basidiomycetes remains challenging because of the inefficiency of transformation systems. In the model lignocellulose-degrading basidiomycete, P. chrysosporium, transformation methods using dominant markers are scarce and were reported over two decades ago, necessitating the re- establishment of a functional system compatible with modern genetic tools. In this study, an efficient genetic transformation system was achieved by using an antimetabolite-based selection strategy for P. chrysosporium. This transformation system would lay the foundation for advancing our understanding of the molecular mechanisms of wood decomposition and support the targeted optimization of basidiomycetes for various biotechnological applications.

microbiology↗