Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, 210023, Jiangsu Province, China.
Appl Microbiol Biotechnol. 2020 Apr;104(8):3597-3609. doi: 10.1007/s00253-020-10517-y. Epub 2020 Mar 7.
Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C. glutamicum. Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C. glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C. glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five genes (up to ~ 13.0 kb) and knock-in of one DNA fragment. Furthermore, through kanamycin resistance repair, the ssDNA recombineering parameters were optimized. We hope the highly efficient method will be helpful for the studies of C. glutamicum, and potentially, to other bacteria. KEY POINTS: • Counterselection marker I-SceI-mediated C. glutamicum genome engineering • A suicide vector contains I-SceI expression elements and its recognition sites • Gene deletions and knock-in were conducted; efficiency was as high as 90% • Through antibiotic resistance repair, ssDNA recombineering parameters were optimized.
谷氨酸棒杆菌被广泛用于生产氨基酸，也是生产增值化合物的底盘。有效的基因组工程方法对于谷氨酸棒杆菌的代谢工程和合成生物学研究至关重要。在此，建立了一种基于内切酶 I-SceI 的谷氨酸棒杆菌 ATCC 13032 模型菌株的基因组工程策略。该策略采用基于 R6K 复制子的、自杀性质粒。质粒 pLS3661 包含受严格调控的 IPTG（异丙基-β-D-1-硫代半乳糖吡喃糖苷）诱导型 I-SceI 表达元件和两个 I-SceI 识别位点。将同源臂克隆到 pLS3661 中，并将重组载体转入谷氨酸棒杆菌 ATCC 13032 后，通过克隆片段与其染色体等位基因之间的同源重组，在卡那霉素选择下选择出杂合二倍体。随后，通过 IPTG 诱导 I-SceI 表达刺激的双链断裂修复，解决杂合二倍体，生成所需的突变体。该方案避免了预先生成菌株、转移第二个 I-SceI 表达质粒的步骤，且不受菌株、培养基和温度的限制。我们通过删除五个基因（最多约 13.0 kb）和敲入一个 DNA 片段验证了该方法的可行性。此外，通过卡那霉素抗性修复，优化了 ssDNA 重组酶的参数。我们希望这种高效的方法将有助于谷氨酸棒杆菌的研究，并可能有助于其他细菌。 关键点： • I-SceI 介导的谷氨酸棒杆菌基因组工程的筛选标记 • 自杀载体包含 I-SceI 表达元件及其识别位点 • 进行了基因缺失和敲入，效率高达 90% • 通过抗生素抗性修复，优化了 ssDNA 重组酶的参数。
