Engineering Pseudomonas putida KT2440 for efficient bioelectrochemical production of glycolipids

  • Entwicklung von Pseudomonas putida KT2440 zur effizienten bioelektrochemischen Produktion von Glycolipiden

Askitosari, Theresia Desy; Agler-Rosenbaum, Miriam (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019


Sustainability of energy generation and careful use of environmental resources are two of our biggest challenges in the world today. Ecological and energy crises in every country enforce the need for development and exploration of sustainable bioenergy resources. One novel biotechnological approach to implement the means of converting and conserving resources are bioelectrochemical systems or BES. The main advantage of BES application is the generation of electric power or biochemical products from renewable materials and carbon-neutral waste materials. The recent exploration and study of natural microbial electron discharge to extracellular anodes might offer significant improvements strategies in bio electrochemical processes for the production of many valuable products, such as bio detergents. However, the natural activity of biocatalysts on electrodes is limited, and molecular engineering approaches are required to tailor new bioelectrochemical active production hosts. Schmitz et al has just reported a successful initial proof-of-principle study. propose a new concept of using an engineered strain of Pseudomonas putida to enable the utilization of an anode for electron discharge during oxygen-limited growth (Schmitz et al., 2015). Biotechnologically, this organism is already tailored to produce bio-detergents like rhamnolipids, one type of glycolipid surfactants, under aerobic conditions. But costly aeration and subsequent problems with vigorous reactor foaming, which is technically hard to handle with conventional antifoam technologies, are current drawbacks. This challenge might be overcome if the detergent production is combined with oxygen-limited growth in bioelectrochemical systems. In this study, following the work from Schmitz et al., we successfully expressed the other three phenazine synthesis gene originating from the phenazine synthesis operon two of P. aeruginosaPAO1 (PA1899-PA1905), operon one of P. aeruginosa PA14 (PA14_09410-PA14_09480), and operon two of P. aeruginosa PA14 (PA14_39880-PA14_39970). Notably, the phenazine-1-carboxylic acid (PCA) synthesis operon two from P. aeruginosa PA14 was found to be most active in the heterologous phenazine production within P. putida. This gene origin was chosen to be tailored further with rhamnolipid production. Hereinafter, the heterologous monorhamnolipid production in P. putida has been successfully coupled with phenazine production to generate the strains P. putida rhl-pca (produces PCA and mono-rhamnolipids) and P. putida rhl-Vpyo (produces PCA, pyocyanin (PYO), and mono-rhamnolipids). Based on the maximum titer of mono-rhamnolipids produced in aerobic shake flasks, P. putida rhl-pca was chosen for bioelectrochemical production experiments in BES. Oxygen-limited cultivations with redox balancing at an anode via phenazines can be coupled to rhamnolipid biosynthesis by employing plasmid-based genetic engineered P. putida. The result of our study showed that passive headspace aeration of BES was suitable to be applied for the bioelectrochemical production of foam-free rhamnolipids with P. putida rhl-pca. The increased carbon yield obtained by P. putida rhl-pca in passively aerated BES showed a potential economic advantage for glycolipid surfactant bioproduction. Overall, this work is an initial study showing that the bioelectrochemical production of foam-free glycolipid surfactants by utilizing phenazines as electron shuttles is possible.