Klein Maximilian, Hilts Angus S, Fennessy Ross T, Trattnig Nino, Stehrer-Polášek Thomas, Rittmann Simon K-M R, Fink Christian
Arkeon GmbH, Tulln a.d. Donau, Austria.
Archaea Physiology & Biotechnology Group, Department of Functional and Evolutionary Ecology, Universität Wien, Vienna, Austria.
Microbiol Spectr. 2025 Jun 3;13(6):e0306824. doi: 10.1128/spectrum.03068-24. Epub 2025 Apr 17.
The archaeal model microorganism has been studied for methane production for decades. However, genetic modifications are required to harness for the generation of novel archaeal cell factories for industrial-scale production of commodity and high-value chemicals. Only the development of tools for genetic engineering opens up this possibility. Here, we present the establishment of the first markerless mutagenesis system for genetic modification of . This system allows the recycling of positive selection markers and enables multiple sequential gene deletions or integrations. As a demonstration, we clarified the postulated isoleucine biosynthesis pathway directly from pyruvate via citramalate synthase (CimA). In doing so, we identified a putative CimA in and deleted the CimA coding gene, resulting in auxotrophy for isoleucine. The complementation of initiated through constitutive expression led to prototrophic growth similar to the wild type, demonstrating that is essential for pyruvate-derived isoleucine biosynthesis in . As it has been shown in before, we were able to complement isoleucine biosynthesis with the integration of a synthetic isoleucine biosynthesis pathway from threonine for the first time in a methanogenic archaeon. This was achieved via genome integration of the characterized thermostable threonine deaminase from . The successful integration of an alternative pathway for isoleucine production paves the road for future application of multi-gene biosynthetic pathways to overproduce industrially relevant chemicals.
The autotrophic, hydrogenotrophic methanogen is one of the best-studied model organisms in the field of thermophilic archaea. The fact that shows robust growth and scalability in bioreactor systems makes it a highly suitable candidate for industrial-scale bioprocesses. Additionally, the reported study provides the tools for genetic engineering that enable sequential genome modification in . Scalable bioreactor cultivation, the ability to genetically engineer, and the recent discovery of natural amino acid secretion in set the cornerstone for the generation of the first cell factories in archaeal biotechnology to economically produce carbon dioxide-derived commodity and high-value chemicals at industrial scale.
古菌模式微生物用于甲烷生产的研究已有数十年。然而，要利用其构建新型古菌细胞工厂用于工业规模生产商品和高价值化学品，还需要进行基因改造。只有开发出基因工程工具才能实现这一可能。在此，我们展示了首个用于[古菌名称]基因改造的无标记诱变系统的建立。该系统允许阳性选择标记的循环利用，并能进行多个连续的基因缺失或整合。作为示例，我们直接从丙酮酸经由柠苹酸合酶（CimA）阐明了假定的异亮氨酸生物合成途径。在此过程中，我们在[古菌名称]中鉴定出一个假定的CimA，并删除了CimA编码基因，导致异亮氨酸营养缺陷。通过组成型表达启动的[古菌名称]互补作用导致了类似于野生型的原养型生长，表明CimA对于[古菌名称]中源自丙酮酸的异亮氨酸生物合成至关重要。正如之前在[另一古菌名称]中所表明的那样，我们首次在产甲烷古菌中通过整合来自苏氨酸的合成异亮氨酸生物合成途径来补充异亮氨酸生物合成。这是通过整合来自[某来源]的已表征的耐热苏氨酸脱氨酶进行基因组整合实现的。成功整合异亮氨酸生产的替代途径为未来应用多基因生物合成途径过量生产工业相关化学品铺平了道路。
自养、氢营养型产甲烷菌[古菌名称]是嗜热古菌领域中研究最深入的模式生物之一。[古菌名称]在生物反应器系统中显示出强劲的生长和可扩展性，这一事实使其成为工业规模生物过程的高度合适候选者。此外，所报道的研究提供了基因工程工具，能够在[古菌名称]中进行连续的基因组改造。可扩展的生物反应器培养、基因工程能力以及最近在[古菌名称]中发现的天然氨基酸分泌为在古菌生物技术中生成首个细胞工厂奠定了基础，以便在工业规模上经济地生产源自二氧化碳的商品和高价值化学品。
