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New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories


New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

Esteban Martínez-García et al. Front Bioeng Biotechnol.


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.

Keywords: Escherichia coli; Pseudomonas putida; central metabolism; chromosomal integration; metabolic engineering; polyhydroxyalkanoates; transposon mini-Tn5.


Figure 1
Figure 1
Functional features of the pBAMD1-x delivery vectors. (A) Schematic representation of the pBAMD1-x plasmid series. The functional elements of each plasmid include the relevant restriction sites used for assembling the vectors, the antibiotic-resistance markers (Ap, ampicillin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin; and Gm, gentamicin), the hyper-active TnpA transposase encoded by tnpA, a conditional origin of replication [ori(R6K)] dependent of the π protein, an origin of transfer (oriT), two mosaic elements (termed ME-I and ME-O), the transcriptional terminators T0 and T1 located just outside the transposon module, and a multiple cloning site (MCS) compatible with any plasmid belonging to the Standard European Vector Architecture (SEVA) initiative (Silva-Rocha et al., ; Durante-Rodríguez et al., 2014). Note that the antibiotic-resistance gene determines the full name of each plasmid. (B) Restriction enzymes targets within the multiple cloning site of the pBAMD1-x delivery vectors (x = 2, KmR; x = 4, SmR/SpR; and x = 6, GmR). The MCS starts with the unique AvrII/SfiI recognition sites, and two NotI recognition sites (highlighted in red) were included in the MCS sequence to enable the consecutive assembly of different cargos from the SEVA collection. The sequence of ME-I is indicated by a purple box.
Figure 2
Figure 2
Functional characterization of the pBAMD1-x delivery vectors in P. putida KT2440. (A) Sequential insertion of different mini-Tn5 modules from pBAMD1-x plasmids carrying all the three possible antibiotic-resistance determinants. The flowchart shows the procedure followed for the combinatorial integrations, starting from the wild-type strain KT2440. The names given to the intermediate strains reflect the order in which each antibiotic was delivered into the recipient bacteria (K, kanamycin; S, streptomycin/spectinomycin; and G, gentamicin). The exact chromosomal localization of the insertions in these strains is given in Table 3. Plasmids used in each round of integration are indicated in red. (B) Assessment of the possible sequence preference in the target DNA during the insertion process of the pBAMD1-x delivery vectors in P. putida KT2440. The WebLogo 3.4 software was used to identify the DNA signature (if any) in which the transposon lands in the chromosome of recipient bacteria. The software was fed with the 9-bp DNA sequence targeted by mini-Tn5 in independent trials (Table 3). Note the slight preference for G/C pairs at both ends of the target DNA motif (i.e., in positions 1 and 9).
Figure 3
Figure 3
Construction of an E. coli cell factory expressing the phaC1AB1 gene cluster from C. necator as a chromosomal insertion. (A) Poly(3-hydroxybutyrate) (PHB) biosynthesis pathway. Three enzymes are necessary for de novo synthesis of PHB in C. necator: a 3-ketoacyl-coenzyme A (CoA) thiolase (PhaA), a NADPH-dependent 3-acetoacetyl-CoA reductase (PhaB1), and a PHB synthase (PhaC1). PhaA and PhaB1 catalyze the condensation of two molecules of acetyl-CoA to 3-acetoacetyl-CoA and the reduction of acetoacetyl-CoA to R-(–)-3-hydroxybutyryl-CoA, respectively. PhaC1 polymerizes these monomers to PHB, whereas one CoA-SH molecule per monomer is released. The resulting PHB polymer is stored as water-insoluble granules in the cytoplasm of the cells. (B) Organization of the functional elements borne by plasmid pBAM1-6-pha and transferred into the chromosome of the recipient E. coli strain. The transcriptional terminators included in the plasmid backbone, which flank the gentamicin resistance (GmR) determinant (accC1), are depicted as T500 and T32. Note that the elements in this outline are not drawn to scale. (C) Exploring the landscape of PHB synthesis in E. coli transconjugants. The phaC1AB1 gene cluster from C. necator was randomly integrated into the chromosome of E. coli JW2293-1 (Δpta), and 24-h cultures of individual colonies were analyzed for PHB accumulation by fluorimetry after staining the cells with Nile red (see Materials and Methods for details). Several colonies, identified by numbers in the heat-map, were kept and further analyzed to establish the precise site of mini-Tn5(phaC1AB1) insertion (Table S2 in the Supplementary Material). AFU, arbitrary fluorescence units.
Figure 4
Figure 4
Biochemical characterization of E. coli TA2293P as a microbial cell factory for PHB synthesis. (A) In vitro determination of the specific (Sp) 3-ketoacyl-coenzyme A thiolase (PhaA) activity. Cells were harvested after growing them for 24 h in M9 minimal medium added with 30 g l−1 glucose as the sole carbon source, and the activity of PhaA was determined in the cell-free extract as detailed in the Section “Materials and Methods.” (B) Poly(3-hydroxybutyrate) (PHB) accumulation. The PHB content (expressed as a percentage of the cell dry weight) was assessed by flow cytometry after growing the cells for 24 h in M9 minimal medium added with 30 g l−1 glucose as the sole carbon source. In all cases, each bar represents the mean value of the corresponding enzymatic activity ± SD of triplicate measurements from at least two independent experiments. The strains used to explore these biochemical traits were E. coli BW25113 (wild-type strain), E. coli JW2293-1 (Δpta), E. coli JW2293P (Δpta, carrying the phaC1AB1 gene cluster in a multi-copy plasmid), and E. coli TA2293P [Δpta, ykgH:mini-Tn5(phaC1AB1)]. See Table 1 for further details about the genotype of each E. coli strain. The relevant features of each strain are indicated at the bottom of the figure.

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