The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida

Abstract

The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/P_nagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.

Keywords: Pseudomonas putida; carbazoles; chromosomal gene integration; glycolipids; synthetic biology; toolbox.

Figure 1.

Concept of the modular pYT vector series. First, a suitable vector from an existing library can be selected. Relevant elements defining the integrative YT_core sequence are depicted schematically. Details are given in Fig. S1. Orange and green regions denote sequences framing the gene cluster of interest, which is to be expressed. Vectors with different resistance markers, reporter genes and chromosomal integration modes are available. If necessary, adaptations for other required vector features can be made via standardised procedures. The integration of target genes of interest can be realised via conventional, ligase-independent or yeast recombinational cloning; positions of homing endonuclease recognition sites are indicated by black asterisks. Cloned vectors facilitate generation of expression strains via integration at different genomic positions (marked in the schematic representations of P. putida KT2440 chromosomes): Three vector series are available enabling random Tn5 transposition, recombination-based integration at pre-installed landing pads in one of the 16S rRNA-encoding genes of P. putida KT2440 rrn operons (denoted with A to G), and targeted transposon Tn7 integration at the attTn7 site.

Figure 2.

Functional validation of pYT marker and reporter modules. (A) Selective growth of E. coli DH5α cells after transformation with pYT vectors carrying different resistance markers. Marker gene indicating characters in vector names are highlighted. (B) Phenotypes of P. putida cells after random transposon Tn5 integration of pYT cassettes with different transcription reporters. Arrows indicate exemplary expressing clones. (C) Reporter signal quantified after differential salicylate induction of P. putida strains after integration of pYT cassettes at the attTn7 site. Reporter gene indicating characters in vector names are highlighted. Data represent the mean of three independent experiments with error bars indicating the corresponding standard deviations.

Figure 3.

Violacein and deoxyviolacein production of P. putida after pYT-mediated vio gene integration via Tn5 transposition or into rrn operons. (A) Cloning scheme of pYT construct as well as product titres and corresponding HPLC-PDA analyses obtained after random transposon Tn5 integration of the vioABCDE gene cluster. (B) Cloning scheme of pYT construct as well as product titres and corresponding HPLC-PDA analyses obtained after interposon integration of vioABCDE into the landing pads within rrn operons of P. putida RW16SA-F. Biosynthesis of (deoxy)violacein was verified by HPLC-PDA analyses of extracts. PDA spectra of product peaks in chromatograms (recorded at 600 nm) are shown. Titres were estimated by spectrophotometrical measurements. Data represent the mean of three independent experiments with error bars indicating the corresponding standard deviations.

Figure 4.

Evaluation of different attTn7-integrated expression modules for rhamnolipid production in P. putida. (A) Cloning schemes of pYT constructs as well as rhlB transcript levels, eYFP reporter fluorescence, and titres of mono-rhamnolipids obtained after Tn7 integration and expression of rhlAB with different promoters. The fraction (g/g) of HAA per total surfactant, i.e. the sum of mono-rhamnolipids and HAA, is shown as inset. The commonly dominant C10-C10 mono-rhamnolipid congener is depicted. (B) Correlation of reporter signals (i.e. fluorescence or enzyme activity) and mono-rhamnolipid titres depending on different salicylate inducer concentrations in strains carrying rhlAB and reporter genes under control of nagR-P_nagAa. Increasing salicylate concentrations (0, 0.001, 0.1, 2, and 5 mM) correspond to increasing mono-rhamnolipid titres with the exception of the PE-H-expressing strain (where salicylate concentrations are indicated for each data point). (C) Titre of mono-rhamnolipids and HAA fraction upon biosynthetic module expansion with rmlBDAC and algC genes for improved supply of the precursor dTDP-l-rhamnose to reduce HAA accumulation. Biosynthetic products were quantified by HPLC-CAD analyses. Data represent the mean of three independent experiments with error bars indicating the corresponding standard deviations.

Figure 5.

Comparison of different gene integration and expression modes for the production of arcyriaflavin A in P. putida. (A) Cloning schemes of pYT constructs facilitating different integration and expression modes of the arcyriaflavin A biosynthetic genes rebODCP in P. putida. (B) Arcyriaflavin A titres quantified by HPLC-PDA analyses of crude cell extracts using commercial arcyriaflavin A as reference. Data represent the mean of three independent experiments with error bars indicating the corresponding standard deviations.