Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria,ACS Synthetic Biology

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Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-08-17 , DOI: 10.1021/acssynbio.1c00269
Christopher M Jones ₁ , Sydney Parrish ₁ , David R Nielsen ₁

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Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO₂ requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.

中文翻译：

在蓝藻中利用多倍体进行无标记和无质粒基因组工程

在这里，我们描述了一种在蓝藻中进行无质粒基因组工程的通用方法，该方法利用其染色体的多倍体作为自然的反选择系统。而不是通过交付在复制质粒时，编码 DNA 修饰酶的基因通过等位基因交换整合到染色体上的必需基因中，这由抗生素选择促进，这一过程很容易发生，并且只有轻微的适应性缺陷。由于这些整合位点的重要性，永远不会实现完全分离，只要维持抗生素选择，菌株就会保持为亚二倍体。结果，一旦所需的基因组修饰完成，去除抗生素选择会导致编码DNA修饰酶的基因迅速从群体中消除。在快速生长的蓝藻中使用两种不同的位点特异性重组系统 CRE-lox 和 DRE-rox 提供了这种新的和可推广的策略的概念证明聚球藻属 PCC 7002，以及模型蓝藻集胞藻属中的 CRE-lox 。PCC 6803. 同时，该方法的可重复使用性通过构建集球藻属的高 CO 2_需求且无标记的 Δ ndh3 Δ ndh4 Δ bicA Δ sbtA突变体来证明。PCC 7002. 总体而言，该方法能够在蓝藻中简单有效地构建稳定且未标记的突变体，而无需开发额外的穿梭载体或反选择系统。

更新日期：2021-09-17

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