Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin, 300072, China; State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu, 610041, China.
Metab Eng. 2023 Sep;79:130-145. doi: 10.1016/j.ymben.2023.07.007. Epub 2023 Jul 24.
Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼10-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.
文库中的遗传元件特征良好，可精细调控基因表达，是生物和生物技术研究与应用的基础。产甲烷菌 Methanococcus maripaludis 生长迅速，遗传操作方便，是将二氧化碳和可再生氢气生物转化为燃料和高附加值产品的理想宿主，也是研究古菌的基础生物学的理想宿主。然而，缺乏用于基因表达的分子工具限制了它作为精细调控基因和代谢途径表达的主力菌株的应用。在本研究中，我们开发了一个遗传工具包，包括启动子文库、核糖体结合位点（RBS）文库和用于染色体整合的中性位点文库，以促进 M. maripaludis 中的精确基因表达。我们构建了一个由 81 个组成型启动子组成的启动子文库，其表达强度涵盖了约 10 倍的动态范围。重要的是，我们确定了强古菌启动子的碱基组成规则，并成功改造了弱启动子，使其活性增强了 120 倍。我们还建立了一个包含 42 种不同 RBS 序列的 RBS 文库，其翻译强度涵盖了约 100 倍的动态范围。此外，我们确定了 8 个中性位点，并开发了一种一步式、基于 Cas9 的无标记敲入方法用于染色体整合。我们成功地将鉴定出的启动子和 RBS 元件应用于显著提高重组蛋白表达 41 倍，并调节必需基因表达，从而在 M. maripaludis 中产生相应的生理变化。因此，这项工作为利用这种自养产甲烷菌作为古菌生物学和生物技术研究与应用的理想主力菌株奠定了坚实的基础。
