Novel system for efficient isolation of clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration

Mohab A. Al-Hinai, Alan G. Fast, Eleftherios T. Papoutsakis*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

86 Citations (Scopus)

Abstract

Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.

Original languageEnglish
Pages (from-to)8112-8121
Number of pages10
JournalApplied and Environmental Microbiology
Volume78
Issue number22
DOIs
Publication statusPublished - Nov 2012

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

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