Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors

Pablo Ivan Nikel, Victor de Lorenzo

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Engineering of robust and safe microbial cell factories requires genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We took advantage of the properties of broad-host-range mini-Tn5 vectors and two regulated expression systems (LacIQ/Ptrc and XylS/Pm), together with FRT-flanked, excisable antibiotic resistance determinants, to generate a set of vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria. This arrangement of modular elements allows the cloning and subsequent markerless insertion of expression cargoes and leaves behind an antibiotic-sensitive host upon the action of the yeast Flp recombinase. We engineered a Pseudomonas putida KT2440 Pm:: gfp strain that displayed strong fluorescence upon exposure to 3-methylbenzoate, a XylS effector, and allowed us to examine the performance of the Pm promoter at the single cell level. We also reconstructed a device for sugar transport and phosphorylation in Escherichia coli independent of the native phosphoenolpyruvate-dependent phosphotransferase system by the stable implantation of genes derived from the obligate anaerobe Zymomonas mobilis. In both cases, the information carried by the implanted genes was stably inherited in the absence of any selective pressure. Deliverable expression systems such as those described here will enhance the applicability of various Gram-negative bacteria in biocatalysis and environmental bioremediation. 
Original languageEnglish
JournalJournal of Biotechnology
Volume163
Issue number2
Pages (from-to)143-154
Number of pages12
ISSN0168-1656
DOIs
Publication statusPublished - 2013
Externally publishedYes

Bibliographical note

© 2012 Elsevier B.V. All rights reserved.

Keywords

  • Biocatalysis
  • Chromosomal insertion
  • FRT
  • LacIQ/Ptrc
  • Mini-Tn5 transposon
  • Pseudomonas
  • XylS/Pm

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