ArticleMicrobial cell factories2023
Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis.
Article in Microbial cell factories, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed, 9 citations in OpenAlex.
- Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacteriumMicrobiology spectrum · 2026Article
- Unlocking genome engineering inMicrobiology spectrum · 2026Article
- Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance inmSystems · 2026Article
- Development of a starch-fermenting Zymomonas mobilis strain for bioethanol production.Microbial cell factories · 2024Article
- A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species.Microbial cell factories · 2024Article
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Authors and funding
10 authors at 3 institutions in 1 country.
Funding
Abstract
backgroundZymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis.
resultsWe constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong P
conclusionsThis study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.
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