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Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

ABSTRACT: Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes.

SUBMITTER: Lai B

PROVIDER: S-EPMC6632142 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

Lai Bin B Nguyen Anh Vu AV Krömer Jens O JO

Methods and protocols 20190330 2

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological descripti ...[more]

PMID: 31164607

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Project description:Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Project description:Acid mine drainage (AMD) is a typical source of environmental pollution ascribing to its characteristics of high acidity and heavy metal content. Currently, most strategies for AMD treatment merely focus on metal removal rather than metal recovery. However, bioelectrochemical system (BES) is a promising technology to simultaneously remove and recover metal ions from AMD. In this study, both cupric ion and cadmium ion in simulated AMD were effectively recovered by BES inoculated with a novel exoelectrogen, Pseudomonas sp. E8, that was first isolated from the anodic electroactive biofilm of a microbial fuel cell (MFC) in this study. Pseudomonas sp. E8 is a facultative anaerobic bacterium with a rod shape, 0.43-0.47 μm wide, and 1.10-1.30 μm long. Pseudomonas sp. E8 can agglomerate on the anode surface to form a biofilm in the single-chamber MFC using diluted Luria-Bertani (LB) medium as an energy substrate. A single-chamber MFC containing the electroactive Pseudomonas sp. E8 biofilms has a maximum output voltage of 191 mV and a maximum power density of 70.40 mW/m2, which is much higher than those obtained by most other exoelectrogenic strains in the genus of Pseudomonas. Almost all the Cu2+ (99.95% ± 0.09%) and Cd2+ (99.86% ± 0.04%) in simulated AMD were selectively recovered by a microbial fuel cell (MFC) and a microbial electrolysis cell (MEC). After the treatment with BES, the high concentrations of Cu2+(184.78 mg/L), Cd2+(132.25 mg/L), and total iron (49.87 mg/L) in simulated AMD were decreased to 0.02, 0.19, and 0 mg/L, respectively. Scanning electron micrograph (SEM), energy dispersive X-ray spectrometry (EDXS) and X-ray diffraction (XRD) analysis indicate that the Cu2+ and Cd2+ in simulated AMD were selectively recovered by microbial electrochemical reduction as Cu0 (together with trace amounts of Cu2O) or Cd0 on the cathode surface. Collectively, data suggest that Pseudomonas sp. E8 has great potential for AMD treatment and metal recovery.

Project description:UnlabelledTwo branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology.ImportanceThis study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

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Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

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Characterizing the Anoxic Phenotype of <i>Pseudomonas putida</i> Using a Bioelectrochemical System.

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