Kuschmierz Laura, Wagner Alexander, Schmerling Christian, Busche Tobias, Kalinowski Jörn, Bräsen Christopher, Siebers Bettina
Molecular Enzyme Technology and Biochemistry (MEB), Environmental Microbiology and Biotechnology (EMB), Centre for Water and Environmental Research (CWE), Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany.
Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany.
Front Microbiol. 2024 Nov 25;15:1443342. doi: 10.3389/fmicb.2024.1443342. eCollection 2024.
, a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the promoter (P), providing low basal activity and high pentose-dependent induction. However, the available expression vector does not include a 5'-terminal untranslated region (5'-UTR), a typical element found in bacterial expression vectors that usually enhances protein production in bacteria. To establish as a production strain in biotechnology in the long term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration of 5'-UTRs into P expression plasmids. Using the esterase Saci_1116 as a reporter protein, we observed a four-fold increase in soluble and active protein yield upon insertion of the () 5'-UTR. Screening of four additional 5'-UTRs from other highly abundant proteins (, , ) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within the 5'-UTR revealed its significance for protein synthesis. Ultimately, the 5'-UTR optimized expression vector improved the expression of various proteins, including six glycosyltransferases and one hydroxyacyl-CoA dehydratase from , and a malto-oligosyltrehalose trehalohydrolase from , demonstrating its applicability. Our results show that the integration of SD-motif containing 5'-UTRs significantly enhanced plasmid-based protein production in . This advancement in recombinant expression not only broadens the utility of as an archaeal expression platform but also marks an important step toward potential biotechnological applications.
作为热变形菌门（前嗜泉古菌门）的嗜热嗜酸古菌，是基因缺失研究和重组蛋白生产中广泛使用的模式生物。先前的研究已经证明了启动子（P）的有效性，其具有低基础活性和高戊糖依赖性诱导作用。然而，现有的表达载体不包括5'-末端非翻译区（5'-UTR），这是细菌表达载体中常见的典型元件，通常可提高细菌中的蛋白质产量。为了长期将其确立为生物技术中的生产菌株，优化其工具和能力以提高生产效率具有内在相关性。在这里，我们表明将5'-UTR整合到P表达质粒中可提高蛋白质产量。使用酯酶Saci_1116作为报告蛋白，我们观察到插入（）5'-UTR后可溶性和活性蛋白产量增加了四倍。从其他高度丰富的蛋白质（，，）中筛选另外四个5'-UTR，发现靶蛋白产量持续提高。此外，5'-UTR内Shine-Dalgarno（SD）基序的定点诱变揭示了其对蛋白质合成的重要性。最终，5'-UTR优化的表达载体改善了各种蛋白质的表达，包括来自的六种糖基转移酶和一种羟酰基辅酶A脱水酶，以及来自的麦芽寡糖基海藻糖海藻糖水解酶，证明了其适用性。我们的结果表明，含有SD基序的5'-UTR的整合显著提高了基于质粒的蛋白质在中的产量。重组表达的这一进展不仅拓宽了作为古菌表达平台的用途，也标志着朝着潜在生物技术应用迈出了重要一步。
