Journal
JOURNAL OF BIOTECHNOLOGY
Volume 163, Issue 2, Pages 143-154Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2012.05.002
Keywords
Mini-Tn5 transposon; Chromosomal insertion; FRT; Biocatalysis; Pseudomonas; LacI(Q)/P-trc; XylS/Pm
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Funding
- BIO and FEDER CONSOLIDER-INGENIO programme of the Spanish Ministry of Science and Innovation
- EU
- PROMT Project of the CAM
- Consejo Nacional de Investigaciones Cientificas y Tecnicas (Argentina)
- EMBO Long-Term Fellowship [ALTF 13-2010]
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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 (LacI(Q)/P-trc 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. (C) 2012 Elsevier B.V. All rights reserved.
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