Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Espoo, Finland.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0109224. doi: 10.1128/aem.01092-24. Epub 2024 Aug 12.
Methanogens are the main biological producers of methane on Earth. is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds.
Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for . Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.
产甲烷菌是地球上甲烷的主要生物生产者。是研究最为深入的产甲烷菌之一，具有强大的基因组编辑遗传工具。为了更详细地研究这种产甲烷菌的生理学特性，并在大规模项目中有效地平衡其工程化代谢途径的通量，需要进行受控基因表达，这就需要有经过良好表征的启动子和核糖体结合位点（RBS）。在这项研究中，我们构建了一个由 33 个启动子-RBS 组合组成的文库，其中包括 13 个野生型和 14 个杂交组合，以及 6 个经过理性工程设计的 5'-非翻译区（5'UTR）组合变体。通过在最常用的两种生长基质甲醇（MeOH）或三甲胺（TMA）存在的情况下诱导β-葡萄糖醛酸酶报告基因在 细胞中的表达，计算每个组合的表达强度。在这项研究中，所构建的文库涵盖了相对较宽的范围（140 倍），从最弱到最强的启动子-RBS 组合都有覆盖，并且呈现出稳定的增长，允许不同水平的基因表达。还通过对所有 33 个启动子-RBS 组合的三个不同生长阶段进行测量，评估了它们对基因表达强度的影响。我们的启动子-RBS 文库可有效实现 中基因表达的精细调控，可用于生理研究和代谢工程设计项目，例如，旨在实现一碳化合物的生物技术增值。
产甲烷古菌是温室气体甲烷的主要生产者，因此对全球变暖有很大的贡献。在受控条件下，这些微生物可以催化生物气的产生，生物气是一种可再生燃料，可能有助于对抗全球变暖及其影响。要使 的初级代谢更好和更有用，需要进行可控基因表达，但目前只有少数经过良好表征的启动子和 RBS 可用。我们的研究通过提供一个由 33 个不同的启动子-RBS 组合组成的文库，解决了这一问题，文库中的表达强度具有 140 倍的动态范围。未来的代谢工程项目可以利用这个文库，将这些启动子-RBS 组合作为一种高效、可调的基因表达系统，用于 。此外，我们在这项研究中开发的方法也可用于构建其他类型产甲烷菌的启动子文库。
