title: Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors.
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 (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.
authors.affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología-CNB-CSIC, Madrid 28049, Spain.
authors.forename: Pablo I
authors.initials: PI
authors.lastname: Nikel
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000235:genetics*; D010641:Phenotype; D010766:Phosphorylation; D017355:Zymomonas / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D005947:Glucose