AUTHOR=Askitosari Theresia D. , Boto Santiago T. , Blank Lars M. , Rosenbaum Miriam A. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01990

DOI=10.3389/fmicb.2019.01990

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Pseudomonas aeruginosa</italic> harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [<italic>phzA1-G1</italic> (operon 1), <italic>phzA2-G2</italic> (operon 2)]. To optimize heterologous phenazines production in the biotech host <italic>Pseudomonas putida</italic> KT2440, we compared PCA production from both operons originating from <italic>P. aeruginosa</italic> strain PAO1 (<italic>O1.phz1</italic> and <italic>O1.phz2</italic>) as well as from <italic>P. aeruginosa</italic> strain PA14 (<italic>14.phz1</italic> and <italic>14.phz2</italic>). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes <italic>phzM</italic> and <italic>phzS</italic> required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, <italic>P. putida</italic> harboring <italic>14.phz2</italic> produced 4-times higher PCA concentrations (80 μg/mL), which resulted in 3-times higher current densities (12 μA/cm<sup>2</sup>) compared to <italic>P. putida</italic> 14.phz1. The respective PCA/PYO producer containing the <italic>14.phz2</italic> operon was the best strain with 80 μg/mL PCA, 11 μg/mL PYO, and 22 μA/cm<sup>2</sup> 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 <italic>P. putida</italic> for oxygen-limited biocatalysis.</p>