RESUMEN
Sufficient supply of oxygen is a major bottleneck in industrial biotechnological synthesis. One example is the heterologous production of rhamnolipids using Pseudomonas putida KT2440. Typically, the synthesis is accompanied by strong foam formation in the reactor vessel hampering the process. It is caused by the extensive bubbling needed to sustain the high respirative oxygen demand in the presence of the produced surfactants. One way to reduce the oxygen requirement is to enable the cells to use the anode of a bioelectrochemical system (BES) as an alternative sink for their metabolically derived electrons. We here used a P. putida KT2440 strain that interacts with the anode using mediated extracellular electron transfer via intrinsically produced phenazines, to perform heterologous rhamnolipid production under oxygen limitation. The strain P. putida RL-PCA successfully produced 30.4 ± 4.7 mg/L mono-rhamnolipids together with 11.2 ± 0.8 mg/L of phenazine-1-carboxylic acid (PCA) in 500-mL benchtop BES reactors and 30.5 ± 0.5 mg/L rhamnolipids accompanied by 25.7 ± 8.0 mg/L PCA in electrode containing standard 1-L bioreactors. Hence, this study marks a first proof of concept to produce glycolipid surfactants in oxygen-limited BES with an industrially relevant strain.
RESUMEN
Bioelectrochemical systems (BESs) are ruled by a complex combination of biological and abiotic factors. The interplay of these factors determines the overall efficiency of BES in generating electricity and treating waste. The recent progress in bioelectrochemistry of BESs and electrobiotechnology exposed an important group of compounds, which have a significant contribution to operation and efficiency: surface-active agents, also termed surfactants. Implementation of the interfacial science led to determining several effects of synthetic and natural surfactants on BESs operation. In high pH, these amphiphilic compounds prevent the cathode electrodes from biodeterioration. Through solubilization, their presence leads to increased catabolism of hydrophobic compounds. They interfere with the surface of the electrodes leading to improved biofilm formation, while affecting its microarchitecture and composition. Furthermore, they may act as quorum sensing activators and induce the synthesis of electron shuttles produced by electroactive bacteria. On the other hand, the bioelectrochemical activity can be tailored for new, improved biosurfactant production processes. Herein, the most recent knowledge on the effects of these promising compounds in BESs is discussed.
RESUMEN
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.