Friedrich Stephanie, Schramm Marina, Kiebist Jan, Schmidtke Kai-Uwe, Scheibner Katrin
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, Senftenberg 01968, Germany.
Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses, Am Mühlenberg 13, Potsdam-Golm 14476, Germany.
Enzyme Microb Technol. 2025 Apr;185:110588. doi: 10.1016/j.enzmictec.2025.110588. Epub 2025 Jan 21.
There is an enormous potential for cell-free protein synthesis (CFPS) systems based on filamentous fungi in view of their simple, fast and mostly inexpensive cultivation with high biomass space-time yields and in view of their catalytic capacity. In 12 of the 22 different filamentous fungi examined, in vitro translation of at least one of the two reporter proteins GFP and firefly luciferase was detected. The lysates showing translation of a reporter protein usually were able to synthesize a functional cell-free expressed unspecific peroxygenase (UPO) from the basidiomycete Cyclocybe (Agrocybe) aegerita. For the most promising candidate Neurospora crassa, the influence of different conditions of cultivation and lysate preparation on in vitro translation of the reporter proteins was investigated and optimized. In general, the greatest improvements in the translational activity were achieved by the choice of the growth medium, the addition of organic nitrogen being most beneficial. Optimizing the culture and preparation conditions of the N. crassa platform improved protein yield of the original lysate by a factor of 25 for firefly luciferase and 17 for GFP, respectively. In addition to the reporter proteins, the aforementioned UPO as well as a functional UPO from Aspergillus niger were cell-free expressed using the different lysates from N. crassa. CFPS with fungal lysates opens the door to expressing UPOs in high throughput and in parallel, for example to optimize synthesis conditions or adapt catalyst properties. The presented method proves the general potential of fungal lysates for application in cell-free syntheses.
鉴于丝状真菌培养简单、快速且大多成本低廉，具有高生物质时空产率，且具备催化能力，基于丝状真菌的无细胞蛋白质合成（CFPS）系统具有巨大潜力。在所检测的22种不同丝状真菌中，有12种检测到了两种报告蛋白绿色荧光蛋白（GFP）和萤火虫荧光素酶中至少一种的体外翻译。显示报告蛋白翻译的裂解物通常能够从担子菌环盖菇（田头菇属）灰盖鬼伞中合成一种功能性的无细胞表达非特异性过氧酶（UPO）。对于最有前景的候选菌株粗糙脉孢菌，研究并优化了不同培养条件和裂解物制备条件对报告蛋白体外翻译的影响。一般来说，通过选择生长培养基可实现翻译活性的最大提高，添加有机氮最为有益。优化粗糙脉孢菌平台的培养和制备条件后，萤火虫荧光素酶的原始裂解物蛋白产量提高了25倍，绿色荧光蛋白提高了17倍。除了报告蛋白外，还使用粗糙脉孢菌的不同裂解物无细胞表达了上述的过氧酶以及黑曲霉的一种功能性过氧酶。使用真菌裂解物进行无细胞蛋白质合成，为高通量并行表达过氧酶打开了大门，例如优化合成条件或调整催化剂特性。所提出的方法证明了真菌裂解物在无细胞合成中应用的总体潜力。
