Abstract
Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.
Highlights
Over the last few years, a number of Gram-negative bacteria have become increasingly attractive chassis for a number of synthetic biology and metabolic engineering purposes
These insertion plasmids share all the advantages and several structural features of their pBAM1 predecessor. These features include (i) the narrow host-range origin of replication of plasmid R6K [ori(R6K)], dependent on the Π protein; (ii) an origin of transfer, oriT, that allows for the conjugative transfer of the plasmid from a host strain to a new bacterial recipient through RK2-mediated mobilization; (iii) the bla-encoded β-lactamase marker that confers resistance to ampicillin as a selective marker of the backbone vector; and [iv] a modified, hyper-active transposase encoded by transposase gene (tnpA) just outside a DNA segment that is flanked by the terminal sequences of Tn5
In our current study, we presented a set of new mini-Tn5-derived vectors that can be used to engineer the genome of Gram-negative bacteria
Summary
Over the last few years, a number of Gram-negative bacteria have become increasingly attractive chassis for a number of synthetic biology and metabolic engineering purposes. A number of plasmid vectors based on both wildtype and minimized versions of Tn5 (i.e. mini-transposons) had allowed the user to introduce stable insertions of foreign DNA into the chromosome of virtually any Gram-negative bacteria (de Lorenzo et al, 1990, 1998; Herrero et al, 1990; de Lorenzo and Timmis, 1994; Martínez-García et al, 2011; Martínez-García and de Lorenzo, 2012; Nikel and de Lorenzo, 2013a) Such Tn5-derived elements present clear advantages over the use of their plasmidbased counterparts for the introduction and expression of heterologous genes into several bacterial species. These features allow for the integration of more than one DNA cargo into the same genome
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