Xu Yifei, Peeters Andries Ivo, Bervoets Indra, De Mey Marjan, Baes Rani, Peeters Eveline
Research Group of Microbiology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium.
Centre for Synthetic Biology, Department of Biotechnology, University of Ghent, Ghent, Belgium.
Front Microbiol. 2025 Aug 5;16:1602937. doi: 10.3389/fmicb.2025.1602937. eCollection 2025.
In eukaryotes and bacteria, it is well-established that the genomic location of ectopic gene integration influences the expression level due to replication-associated gene dosage effects as well as effects mediated by chromatin organization. In contrast, in archaea, the impact of genomic location on gene expression remained unexplored. Here, we investigated this impact in the model archaeon , a crenarchaeal species that has a chromatin architecture with mixed eukaryotic-like and bacterial-like features. We aimed to integrate a standardized β-galactosidase () reporter cassette into diverse loci in the genome of SK-1 for a comparative analysis. Nine integration mutant strains were successfully obtained, for which qRT-PCR analysis and reporter gene assays revealed significant variation in transcriptional and translational expression of the reporter, respectively, demonstrating that genomic location strongly influences gene expression in . However, variability in transcription levels and its regulation was shown to be primarily driven by transcriptional activity of neighboring genes, due to the high coding density in the genome as well as a lack of insulator elements. In conclusion, this study not only provides insights into genome context effects, but also provides inspiration for the future design of genomic knock-in constructions in .
在真核生物和细菌中，异位基因整合的基因组位置会因复制相关的基因剂量效应以及染色质组织介导的效应而影响表达水平，这一点已得到充分证实。相比之下，在古菌中，基因组位置对基因表达的影响仍未得到探索。在此，我们在模式古菌中研究了这种影响，该古菌是一种泉古菌，其染色质结构具有类似真核生物和类似细菌的混合特征。我们旨在将一个标准化的β-半乳糖苷酶（）报告基因盒整合到SK-1基因组的不同位点，以进行比较分析。成功获得了九个整合突变菌株，对其进行的qRT-PCR分析和报告基因检测分别显示报告基因的转录和翻译表达存在显著差异，表明基因组位置强烈影响中的基因表达。然而，转录水平的变异性及其调控主要由邻近基因的转录活性驱动，这是由于基因组中的高编码密度以及缺乏绝缘子元件。总之，本研究不仅为基因组背景效应提供了见解，也为未来中的基因组敲入构建设计提供了灵感。
