Abstract
Summary 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.
Highlights
Pseudomonas putida has previously demonstrated hallmark features of an interesting microbial chassis for industrial and environmental processes (Wackett, 2003; Poblete-Castro et al, 2012; Nikel et al, 2014; Loeschcke and Thies, 2015), due to its versatile metabolism towards a wide range of carbon sources (La Rosa et al, 2016; Molina et al, 2019) and high resistance to environmental stresses (Nikel et al, 2016)
In addition to D-glucose, D-fructose and D-ribose can be used for P. putida KT2440’s aerobic cultivation, a slower growth was observed for both substances (Fig. S2A)
When cultivated in a bioelectrochemical system (BES) in which the anode provided a surface to deposit electrons and where the electrode potential and current between the electrodes were continuously monitored, P. putida KT2440 cultures fed with D-glucose produced the highest peak current, followed by cultures with D-galactose, L-arabinose or Dribose (Fig. 1A), yet no cell growth was detected in any of the cultures (Fig. S3A)
Summary
Pseudomonas putida has previously demonstrated hallmark features of an interesting microbial chassis for industrial and environmental processes (Wackett, 2003; Poblete-Castro et al, 2012; Nikel et al, 2014; Loeschcke and Thies, 2015), due to its versatile metabolism towards a wide range of carbon sources (La Rosa et al, 2016; Molina et al, 2019) and high resistance to environmental stresses (Nikel et al, 2016). The published efforts to include selected fermentative or anaerobic respiratory pathways to establish anaerobic electron balancing in the cells (Sohn et al, 2010; Nikel and de Lorenzo, 2013; Steen et al, 2013) still relied on fermentation by-products or co-substrates as electron sinks. These strategies were able to improve the bacterium’s survival under anoxic conditions, no substantial anaerobic growth was observed, along with a persisting problem of energy shortage and redox imbalance
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