Part:BBa_I723028
PCA biosynthesis operon
The products of these 7 genes are responsible for the conversion chorismate into phenazine-1-carboxylic acid (PCA).
Improvement by Evry_Paris-Saclay 2022
This part is a truncated version of the Pseudomonas aeruginosa phzA1B1C1D1E1F1G1 operon. It is not functional as the phzG coding sequence is not complete and it is not be able to lead to the synthesis of a functional enzyme able to catalyze the reaction from 2,3-dihydro-3-hydroxyanthranilic acid (DDHA) to PCA.
Compared to the pyocyanin biosynthesis operon (GenBank Acc. N° AF005404.2), the sequence between base 6306 and 6697 which represents a part of the phzG CDS is missing in BBa_I723028. 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 part, 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.
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].
Figure 1. Biochemical pathway of phenazine-1-carboxylate (PCA) and of closely related compounds in Pseudomonas sp. based on the available information in the MetaCyc database.
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 culture.
Build
P. aeruginosa PhzA1-G1 and PhzA2-G2 operons (BBa_K4432040 and BBa_K4432041, respectively), as well as this part 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 of our 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 our 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 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 presence of PCA in the extract, as expected.
Finally, the extracts from the strains transformed with the plasmid containing truncated phzA1-G1, we can see a very strong reduction of the intensity of the PCA specific peak compared to 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 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.
Figure 2. HPLC chromatograms of commercial PCA and phenazine, and of chloroform extractions of culture media taken after 18 hours of incubation of E. coli BL21 Star™(DE3) cells transformed with the pSB1A3 plasmid harboring the PhzA1-G1 (full and truncated) and PhzA2-G2 operons, or the negative control (an empty pSB1A3 backbone). The PCA peak with a retention time of 31.78 min is highlighted in red. The HPLC chromatograms on the right are a zoom representation (30 to 35 min) of the HPLC chromatograms on the left. Chromatogram images were produced using Shimadzu’s LabSolutions Postrun Analysis software.
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 to the strong PCA production when E. coli cells contain either the full PhzA1-G1 or PhzA2-G2 operons are present, and a more than 90% reduction of PCA concentration in the presence of the truncated operon.
Figure 3. The efficiency of PCA production in two different E. coli strains carrying the pSB1A3 plasmid harboring the PhzA1-G1 (full and truncated) and PhzA2-G2 operons or the negative control (an empty pSB1A3 backbone).
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 [15,16]. 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 activity of endogenous E. coli genes. Indeed PhzG is closely related to the pyridoxine-5’ -phosphate oxidase, the product of the E. coli pdxH gene product, 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 this part (BBa_I723028) that had a truncated coding sequence.
We see from the Results page of their Wiki that the Glasgow 2017 iGEM team had trouble in cloning these large clusters. 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 of our wiki)
References
[1] Chen J-J, Chen W, He H, Li D-B, Li W-W, Xiong L, Yu H-Q. Manipulation of Microbial Extracellular Electron Transfer by Changing Molecular Structure of Phenazine-Type Redox Mediators. Environmental Science & Technology (2013) 47: 1033–1039.
[2] Pierson LS, Pierson EA. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Applied Microbiology and Biotechnology (2010) 86: 1659–1670.
[3] Price-Whelan A, Dietrich LEP, Newman DK. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics. Nature Chemical Biology (2006) 2: 71–78.
[4] Wang Y, Kern SE, Newman DK. Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer. Journal of Bacteriology (2010) 192: 365–369.
[5] He Q, Feng Z, Wang Y, Wang K, Zhang K, Kai L, Hao X, Yu Z, Chen L, Ge Y. LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa. Journal of Microbiology and Biotechnology (2019) 29: 1299–1309.
[6] Feng J, Qian Y, Wang Z, Wang X, Xu S, Chen K, Ouyang P. Enhancing the performance of Escherichia coli-inoculated microbial fuel cells by introduction of the phenazine-1-carboxylic acid pathway. Journal of Biotechnology (2018) 275: 1–6.
[7] Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. Journal of Bacteriology (2001) 183: 6454–6465.
[8] Rabaey K, Boon N, Höfte M, Verstraete W. Microbial Phenazine Production Enhances Electron Transfer in Biofuel Cells. Environmental Science & Technology (2005) 39: 3401–3408.
[9] Bosire EM, Rosenbaum MA. Electrochemical Potential Influences Phenazine Production, Electron Transfer and Consequently Electric Current Generation by Pseudomonas aeruginosa. Frontiers in Microbiology (2017) 8: 892.
[10] Pham TH, Boon N, De Maeyer K, Höfte M, Rabaey K, Verstraete W. Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation. Applied Microbiology and Biotechnology (2008) 80: 985–993.
[11] Jayapriya J, Ramamurthy V. Use of non-native phenazines to improve the performance of Pseudomonas aeruginosa MTCC 2474 catalysed fuel cells. Bioresource Technology (2012) 124: 23–28. [12] Reis AC, Salis HM. An automated model test system for systematic development and improvement of gene expression models. ACS synthetic biology (2020) 9: 3145–3156.
[13] Farasat I, Kushwaha M, Collens J, Easterbrook M, Guido M, Salis HM. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Molecular Systems Biology (2014) 10: 731.
[14] Ng CY, Farasat I, Maranas CD, Salis HM. Rational design of a synthetic Entner-Doudoroff pathway for improved and controllable NADPH regeneration. Metabolic Engineering (2015) 29: 86–96.
[15] Bank RPD. RCSB PDB - 1T9M: X-ray crystal structure of phzG from Pseudomonas aeruginosa. https://www.rcsb.org/structure/1T9M .
[16] Parsons JF, Calabrese K, Eisenstein E, Ladner JE. Structure of the phenazine biosynthesis enzyme PhzG. Acta Crystallographica. Section D, Biological Crystallography (2004) 60: 2110–2113.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 927
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Illegal PstI site found at 5103 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 927
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Illegal BamHI site found at 276
Illegal BamHI site found at 1482
Illegal XhoI site found at 220
Illegal XhoI site found at 379 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 927
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Illegal PstI site found at 4554
Illegal PstI site found at 5103 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 927
Illegal EcoRI site found at 3549
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Illegal NgoMIV site found at 142
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Illegal AgeI site found at 5392 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5535
Illegal BsaI.rc site found at 2234
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