Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, FI-02150 Espoo, Finland.
ACS Synth Biol. 2022 Jul 15;11(7):2496-2503. doi: 10.1021/acssynbio.2c00137. Epub 2022 Jun 22.
The rapid-growing and genetically tractable methanogen is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in . This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of . For the purpose of demonstrating a large deletion, the flagellum operon (∼8.9 kbp) was replaced by the β-glucuronidase gene. To facilitate metabolic engineering and flux balancing in , the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.
快速生长且遗传操作简单的产甲烷菌是一种很有前途的宿主生物，可用于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。通过代谢工程来扩展其产物范围，需要可靠且高效的遗传工具，特别是对于影响初级代谢和细胞生长的基因组编辑。在这里，我们利用 ND2006（LbCas12a）中的 Cas12a 与内源性同源定向修复机制相结合，设计了一个基因组编辑工具盒。尽管 高度多倍体化，但该工具盒仍能以高达 95%的成功率删除靶基因。为了演示大片段缺失，我们用 β-葡萄糖醛酸酶基因替换了约 8.9 kbp 的鞭毛操纵子。为了便于 在 中进行代谢工程和通量平衡，我们在两种常见的生长基质甲酸或二氧化碳和氢气存在的情况下，量化了 15 个不同启动子的相对强度。该 CRISPR/LbCas12a 工具盒可作为一种在产甲烷菌中进行基因组编辑的可靠且快速的方法。
