Boosting Heterologous Phenazine Production in Pseudomonas putida KT2440 Through the Exploration of the Natural Sequence Space

Abstract

Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pseudomonas putida KT2440, we compared PCA production from both operons originating from P. aeruginosa strain PAO1 (O1.phz1 and O1.phz2) as well as from P. aeruginosa strain PA14 (14.phz1 and 14.phz2). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes phzM and phzS required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, P. putida harboring 14.phz2 produced 4-times higher PCA concentrations (80 μg/mL), which resulted in 3-times higher current densities (12 μA/cm²) compared to P. putida 14.phz1. The respective PCA/PYO producer containing the 14.phz2 operon was the best strain with 80 μg/mL PCA, 11 μg/mL PYO, and 22 μA/cm² current density. Tailoring phenazine production also resulted in improved oxygen-limited metabolic activity of the bacterium through enhanced anodic electron discharge. To elucidate the reason for this superior performance, a detailed structure comparison of the PCA-synthesizing proteins has been performed. The here presented characterization and optimization of these new strains will be useful to improve electroactivity in P. putida for oxygen-limited biocatalysis.

Keywords: PCA; Pseudomonas putida; bioelectrochemical systems; heterologous production; phenazine; pyocyanin.

FIGURE 1

Heterologous expression of phz genes in P. putida KT2440. (A) P. putida cultures (from left to right) expressing O1.phz1, O1.phz2, 14.phz1, and 14.phz2 gene for PCA synthesis (top) and with combination of phzM+S genes for PCA and PYO synthesis PCA and PYO (bottom). (B) Phenazine production of engineered P. putida strains in triplicate shake flask experiments (fully aerobic), whereby the “+” in the strain designation indicates presence of phzM+S to convert PCA to PYO.

FIGURE 2

Heterologous phenazine production with P. putida under oxygen-limited conditions in a BES. Current densities generation and phenazine production of P. putida KT2440 harboring either phz operon 1 or operon 2 originating from P. aeruginosa strains PAO1 (A,E,B,F) or PA14 (C,G,D,H) under active (A–D) and passive (E–H) aeration. (B,F,D,H) indicated as PAO1+ and PA14+ are in combination with the phzM+S genes to synthesize both PCA and PYO. In the active aeration experiments, liquid phase aeration for the first 48 h was followed by passive headspace aeration. The highest currents generation and phenazine production were obtained from the engineered P. putida carrying operon 14.phz2+ under active aeration BES (Figure 2D). The BES data are means from biological duplicates.

FIGURE 3

Nucleotide-based analysis of the phenazine clusters. (A) First intergenic regulatory element alignment; between phzA and phzB. Deleted nucleotides are marked as hyphen (-). Identical bases are marked as asterisk (^∗). (B) Second intergenic regulatory element alignment; between phzB and phzC. Deleted nucleotides are marked as hyphen (-). Identical bases are marked as asterisk (^∗). (C) Prediction of the expression tendency. Bars represent the number of genes in each operon detected with the highest or the lowest codon adaptation indexes, CAI, as detailed in Supplementary Table S2. Genes with equally high or low values are counted double.

FIGURE 4

Structural analysis of PhzE. (A) PA14-PhzE2 is used as the reference for the alignment (blue). RMSD for PA14-PhzE1 (yellow): 1.056 Å. RMSD for PAO1-PhzE1 (green): 0.836 Å. PAO1-PhzE2 is identical to PAO1-PhzE1. Unique amino acid changes regarding in PA14-PhzE2 [p.(F231L;I266L)] are marked in purple. Substrate (chorismic acid) molecules are marked in yellow. (B) Detail of the substrate binding site vicinity. (C) Detail of the unique amino acid changes in PA14-PhzE2 and location inside the structure showing the phenylalanine in positon 231 and the isoleucine in position 266 for PA14-PhzE2 (both blue) compared to the leucine substitutions for both positions in the other PhzE proteins (green and yellow).