General context

Phenazines, which are polyaromatic secondary metabolites, are known as compounds with high redox activity [1]. These secondary metabolites are produced by a variety of bacteria, especially Pseudomonas species where they play different roles. For instance, phenazines are pathogenicity factors in Pseudomonas aeruginosa infections, which lead to generation of disease symptoms in host organisms by negatively affecting host cell functions, including respiration, ciliary beating, epidermal cell growth, calcium homeostasis [2]. Bacterial virulence is enhanced by phenazine production as it interferes with normal host cell functions by generating reactive oxygen species due to the ability of phenazines to accept or donate electrons [2].

Phenazines are of particular interest in anaerobic conditions, because they allow bacteria to generate energy for growth or help to maintain redox homeostasis by acting as electron acceptors for the reoxidation of accumulating NADH [3,4].

Three endogenous phenazines are known to be produced by Pseudomonas aeruginosa: phenazine-1-carboxylic acid (PCA), 1-hydroxyphenazine and pyocyanin [3]. The combination and variety of functional groups added also determine the redox potential and solubility of these compounds, thus affecting their biological activity [1].

In P. aeruginosa, PCA is produced via two seven gene phenazine biosynthetic loci, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, with homology in other Pseudomonas species operons [5]. Each of these two biosynthetic operons from P. aeruginosa is sufficient for production of PCA from chorismate (Figure 1), which can be subsequently converted to 1-hydroxyphenazine or pyocyanin by others phenazine-modifying genes: phzM, phzS and phzO [6,7].

In our project, we want to produce an electrical signal in a Microbial fuel cell (MFC) only in response to a specific cancer biomarker detection.

To improve the electricity generation in our system, previous studies have shown that the use of Pseudomonas species producing phenazine-based metabolites in the anode chambers of MFCs can improve anodic electron transfer [8]. In an MFC, phenazines are found to enable a high electrical conductivity of the multilayered biofilm on the anode, leading to enhanced electricity generation from bacterial metabolism. Overexpression of the phz operon increases the electricity production of the Pseudomonas species-inoculated MFCs [6,9–11].

In 2007, the Glasgow iGEM team created the project called “ElectrEcoBlu”. They tried to use an “electrochemical mediator which enables electrical current to be generated in a microbial fuel cell”.

“By isolating the genes phzM, phzS and the seven gene operon phzABCDEFG which express pyocyanin in P. aeruginosa, we intend to harness the oxidation-reduction potential of pyocyanin to power a microbial fuel cell. While using P. aeruginosa in microbial fuel cells is extremely promising in fully exploiting and enhancing this technology, it is clearly necessary to either identify or engineer nonpathogenic bacteria that produce similar redox mediators. We have attempted to clone the pyocyanin producing genes from P. aeruginosa into E. coli to harvest pyocyanin and therefore electricity from a non-pathogenic organism.”

Here, we demonstrated that the initial part BBa_I723028 of the 2007 Glasgow iGEM team didn't work because of a truncated sequence. Indeed, the PhzG coding sequence is not complete and should not be able to lead to the synthesis of a functional enzyme able to catalyze the reaction. If we take a look in the pyocyanin biosynthesis operon (GenBank Acc. N° AF005404.2), the sequence between base 6307 and 6697 which represents a part of the PhzG CDS is missing in BBa_I723028 of the 2007 Glasgow iGEM team. The team noted on their Wiki that “The phenazine biosynthesis operon ... was over 7kb long and proved troublesome to clone”.

To improve the function of this truncated operon (BBa_I723028), we constructed expression cassettes of both full P. aeruginosa PhzA1-G1 and PhzA2-G2 operons (BBa_K4432040 and BBa_K4432041) and thus succeed in producing PCA in E. coli.

Design

To express both full P. aeruginosa PhzA1-G1 and PhzA2-G2 operons (BBa_K4432040 and BBa_K4432041) as well as the truncated operon (BBa_I723028) we constructed expression cassettes in the pSB1A3 backbone.

We choose to put each of these 3 operons under the control of the strong synthetic hybrid T5 promoter inducible by IPTG (BBa_K4432000). For an efficient translation efficiency, we designed PhzA1-G1 and PhzA2-G2 operons specific RBS libraries using the Salis Lab RBS Library Calculator v2.0 [12–14] and selected fortuitously during the cloning process BBa_K4432014 and BBa_K4432015. Indeed, it is important to select a promoter and an RBS of appropriate strength in order to adjust the expression level of each gene to obtain high yields of bioconversions without having a toxic effect on the cell.

Build

P. aeruginosa PhzA1-G1 and PhzA2-G2 operons (BBa_K4432040 and BBa_K4432041, respectively), as well as the truncated operon equivalent to the old part from iGEM Glasgow 2007 (BBa_I723028) were PCR amplified from genomic DNA of the P. aeruginosa PAO1 strain kindly provided by the host lab, followed by NEBuilder HiFi DNA Assembly to create the corresponding expression vectors in the high copy plasmid pSB1A3 (BBa_K4432140, BBa_K4432141, and BBa_K4432142 respectively).

Test

To demonstrate the whole-cell bioproduction of PCA, either E. coli BL21 Star™(DE3) (Thermo Fisher Scientific) or E. coli NEB® 5-alpha (New England Biolabs) cells were transformed with the pSB1A3 plasmid harboring the PhzA1-G1 (full or truncated) and PhzA2-G2 operons, along with an empty pSB1A3 backbone as negative control.

Bacterial cultures fully grown overnight in LB medium containing 100 µg/mL ampicillin and induced with 100 µM IPTG, were centrifuged (15 minutes at 4000 g, 4°C) in order to separate the biomass from the culture medium. Further, PCA was extracted from 2 mL of supernatant with an equal volume of chloroform three times. For this, samples were vortexed, centrifuged for 5 min at 4°C, 4000 g and the lower organic layer was recovered and evaporated to dryness. The dry pellets were resuspended in a total volume of 200 μL of acetonitrile and filtered using an AcroPrep™ Advance 96-Well Filter Plate (350 μl) equipped with a 0.2 μm wwPTFE membrane (Pall Corporation) by centrifugation (1 minute, 1500 g, room temperature).

Afterwards, they were directly analyzed by high pressure liquid chromatography (HPLC) using a Shimadzu Prominence LC20/SIL-20AC equipped with a Kinetex XB-C18 reversed phase column (250 mm × 4.5 mm, 5 μm) and an UV–Vis detector. Mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) were set at a flow rate of 0.4 mL/min and the following linear gradient method was established: 0–5 min: 5% B to 50% B; 5–35 min: 50% B to 100% B; 35–45 min: 100% B, 45-50 min: 100%B to 5% B; 60 min, stop (see the Measurement page on this wiki for further details). The sample injection volume was 10 μL, the column was maintained at 40°C, and the metabolites were monitored at 250 nm and 350 nm. The product quantification was done by interpolation of the integrated peak areas using calibration curves prepared with standard samples for PCA as detailed on the Measurement page on this wiki.

Learn

To assess PCA bioproduction in each strain, both qualitatively and quantitatively, the extracts were subject to a reverse phase chromatography analysis. The method we developed (see the Measurement page of this wiki) allowed us to unambiguously identify PCA and distinguish it from the closely related phenazine (Figure 2).

This chromatographic analysis revealed the production of PCA by E. coli cells when either the full PhzA1-G1 or PhzA2-G2 operons were present in the cell. Indeed, a peak with the same retention time as the commercial pure PCA was clearly visible on the chromatogram indicating its production.

In contrast, when only the plasmid backbone was transformed into the E. coli cells, we can not see any peak with the same retention time as PCA. This indicates that there is no PCA present in the extract, as expected.

Finally, in the extracts from the strains transformed with the plasmid containing the truncated PhzA1-G1 we can see a very strong reduction of the intensity of the PCA specific peak compared to those from the whole PhzA1-G1 operon. The detection of low amounts of PCA from this truncated operon indicates that it does not allow the bioconversion at an efficient rate to obtain a significant oxidation-reduction potential.

No phenazine production was observed in any strain tested. Also, no other major and unknown peak was visible on the chromatograms of extracts from the 3 tested operons compared to extracts from E. coli cells containing only the plasmid backbone.


Quantitative analysis of the PCA production by these 3 operons (Figure 3) in 2 different E. coli strains, each with at least 3 replica cultures, attest the strong PCA production when E. coli cells contain either the full PhzA1-G1 or PhzA2-G2 operons, and a more than 90% reduction of PCA concentration in the presence of the truncated operon.


Our results demonstrate that the missing part of the PhzG1 gene is important for the PCA bioproduction. Indeed, PhzG1 gene is translated into a pyridoxamine-5'-phosphate oxidase of 214 amino acids. Removing the last 391 bases of the initial sequence (corresponding to 129 amino acids) perturbs considerably the structure of the protein and consequently its function. Structural analysis reveals that this removed part allows the interaction with sulfate ion, acetic acid, and flavin mononucleotide [15]. These ligands are essential cofactors for the oxidation of the substrate molecule involved in the PCA pathway.

Our negative control shows that there is no endogenous production of either PCA, nor phenazine. The residual PCA production observed when the truncated PhzA1-G1 is expressed in E. coli may be the consequence of promiscuous activities of endogenous E. coli genes. Indeed PhzG is closely related to the pyridoxine-5’-phosphate oxidase, the product of the E. coli pdxH gene, which catalyzes the final step in pyridoxal-5’-phosphate biosynthesis.

Conclusions

We have successfully built two parts for the bioproduction of PCA in E. coli, based on the genomic sequences of the P. aeruginosa PhzA1-G1 and PhzA2-G2 operons (BBa_K4432040 and BBa_K4432041). We proved their functionality in E. coli and thus improved a previous part (BBa_I723028) that had a truncated coding sequence.

The Glasgow 2017 iGEM team had trouble in cloning these large clusters, as indicated on the Results page of their wiki. Indeed, cloning these parts was not straightforward for us too, but after numerous trials we obtained the recombinant E. coli strains for bioproduction studies. Both of them gave good bioconversion results and we used them both to improve the electricity generation in our MFC system, as described in the Proof of Concept page on this wiki.

References

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