Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.
CRISPR-Cas system
Elimination of editing plasmid
Theophylline-dependent riboswitch
Zymomonas mobilis
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
received:
15
05
2023
accepted:
07
09
2023
revised:
23
08
2023
medline:
13
11
2023
pubmed:
20
9
2023
entrez:
20
9
2023
Statut:
ppublish
Résumé
Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.
Identifiants
pubmed: 37728624
doi: 10.1007/s00253-023-12783-y
pii: 10.1007/s00253-023-12783-y
doi:
Substances chimiques
Riboswitch
0
Theophylline
C137DTR5RG
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7151-7163Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
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