Walter Ruben Magnus, Zemella Anne, Schramm Marina, Kiebist Jan, Kubick Stefan
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
Front Bioeng Biotechnol. 2022 Nov 1;10:964396. doi: 10.3389/fbioe.2022.964396. eCollection 2022.
Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from and a long UPO from . The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from , thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof.
非特异性过氧酶（UPOs，EC 1.11.2.1）是一类真菌酶，可催化未活化烃类的氧官能化反应，使其成为有价值的生物催化剂。尽管人们对UPOs的兴趣与日俱增，已鉴定出数千个推定的UPO基因，但其中只有少数已成功表达并得到表征。目前尚无通用的表达系统来充分发掘其潜力。无细胞蛋白质合成已被证明是一种用于合成难以表达的蛋白质的精密技术。在这项工作中，我们旨在建立一个基于昆虫细胞的无细胞蛋白质合成（CFPS）平台来生产UPOs。CFPS依赖于具有翻译活性的细胞裂解物而非活细胞。因此，该系统参数可直接操控，而无需考虑细胞活力，从而使其具有高度适应性。基于昆虫细胞的裂解物含有具有转位活性的内质网衍生囊泡，称为微粒体。这些微粒体已被证明能够使蛋白质高效转运至其内腔，促进翻译后修饰，如二硫键形成和N-糖基化。在本研究中，我们探索了一种经过氧化还原优化的、基于囊泡的真核CFPS系统合成功能性UPOs的能力。针对来自[具体来源1]的短UPO和来自[具体来源2]的长UPO，评估了不同反应参数的影响以及转位对酶活性的影响。本文所述CFPS系统的能力通过成功合成来自[具体来源3]的新型UPO得以证明，从而使CFPS成为鉴定和评估新型UPOs及其变体的有前景的工具。
