The anoxic electrode‐driven fructose catabolism of Pseudomonas putida KT2440

Abstract

Pseudomonas putida (P. putida) is a microorganism of interest for various industrial processes, yet its strictly aerobic nature limits application. Despite previous attempts to adapt P. putida to anoxic conditions via genetic engineering or the use of a bioelectrochemical system (BES), the problem of energy shortage and internal redox imbalance persists. In this work, we aimed to provide the cytoplasmic metabolism with different monosaccharides, other than glucose, and explored the physiological response in P. putida KT2440 during bioelectrochemical cultivation. The periplasmic oxidation cascade was found to be able to oxidize a wide range of aldoses to their corresponding (keto-)aldonates. Unexpectedly, isomerization of the ketose fructose to mannose also enabled oxidation by glucose dehydrogenase, a new pathway uncovered for fructose metabolism in P. putida KT2440 in BES. Besides the isomerization, the remainder of fructose was imported into the cytoplasm and metabolized. This resulted in a higher NADPH/NADP + ratio, compared to glucose. Comparative proteomics further revealed the upregulation of proteins in the lower central carbon metabolism during the experiment. These findings highlight that the choice of a substrate in BES can target cytosolic and periplasmic oxidation pathways, and that electrode-driven redox balancing can drive these pathways in P. putida under anaerobic conditions.

Anaerobic glucose uptake in Pseudomonas putidaKT2440 in a bioelectrochemical system

“…The extremely low electrogenic activity of KT-G in BES was unexpected. Our former studies using P. putida F1 or using fructose as the substrate revealed the intracellular redox equivalents were largely shifted to the oxidized pools (Lai, Yu, et al, 2016;Nguyen et al, 2021), and thus we hypothesized that the increased NADPH supply via the glcT pathway might play an important role in providing redox equivalents under BES conditions. Unfortunately, it was difficult to obtain reproducible data for the intracellular redox pools in our experimental set-up, that would enable a direct assessment of the redox status of the cells.…”

“…Furthermore, it was shown in previous studies, that P. putida was able to metabolize a wide range of aldoses and fructose in BES, like. Therefore, the so far known possible product spectrum also includes (keto‐)aldonic acids, like (2‐keto) galactonic acid and arabinonic acid (Nguyen et al, 2021 ). This process might be also transferred to other compounds that could benefit from the anodic fermentation process (Kracke & Krömer, 2014 ) if the metabolic constrains of carbon turnover and uptake could be solved in the future.…”

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems

“…However, most of these studies only identify potential phenazine producers, but they do not evaluate or confirm the electroactivity of the identified strains. In addition to the search for new EAB, some success has been achieved by engineering industrially relevant microorganisms to generate synthetic electroactive biocatalysts (Kampers et al, 2019 , 2021 ; Nguyen et al, 2021 ; Philipp et al, 2020 ). For instance, phenazines heterologously expressed by the strict aerobe Pseudomonas putida allowed for a partial redox balancing in BES operated under oxygen‐limited conditions (Askitosari et al, 2019 , 2020 ; Schmitz et al, 2015 ).…”