Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440.

Simone Schmitz,Lars M Blank,Salome Nies,Miriam A Rosenbaum,Nick Wierckx

doi:10.3389/fmicb.2015.00284

Abstract

Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems (BES). We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 μg/mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 μA/cm2 anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains.

Highlights

Pseudomonas putida and closely related species such as Pseudomonas sp
P. putida pPhz Strain Construction and Evaluation The phenazine biosynthesis genes phzA1-G1, phzM, and phzS from P. aeruginosa were successfully cloned into the salicylateinducible vectors pBNT and pJNN under the control of the salicylate-inducible promoter NagR/pNagAa to result in pBNTphzA-G and pJNNphzMS (Figures S1A–C)
Heterologous Phenazine Synthesis enables Anodic Electron Discharge by P. putida KT2440 For the first time, we here show the molecular engineering of a non- or barely electroactive biotechnologically relevant microorganism—P. putida KT2440—to synthesize and utilize soluble redox mediators for metabolic electron discharge to an anode

Summary

Introduction

VLB120 are gaining increasing scientific and biotechnological interest because of their comparatively high tolerance toward amphiphilic and hydrophobic chemicals (Vickers et al, 2010; Lang et al, 2014; Nikel et al, 2014) This property allows these strains to grow in the presence of or even produce otherwise toxic chemicals like aromatic phenols and styrenes (Wierckx et al, 2005; Verhoef et al, 2009; Volmer et al, 2014). On the other hand, during the recombinant production of, e.g., valuable biodetergents like rhamnolipids (Wittgens et al, 2011), the required aeration leads to strong reactor foaming, which is technically hard to handle with conventional antifoam technologies (Küpper et al, 2013) In this case, the organism’s obligate aerobic nature poses a severe challenge for reactor and process engineering

Methods

Results

Conclusion