Electrofermentation As a Tool to Balance Redox in Engineered Pseudomonas Putida

Background: Pseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this work is to empower the anoxic metabolism of Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES).

Results: P. putida was able to survive and produce almost exclusively 2‑Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-KGA, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90% based on glucose. Seven different mediator compounds were tested and only those with redox potential above 0.207V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putidacells survived under anaerobic conditions and limited biofilm formation could be observed on the anode surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy by using the anode as terminal electron acceptor [1].

Having achieved a high 2-KGA yield of over 90 %, we then used metabolic engineering to accelerate sugar conversion rate by co-expressing the membrane-bound gluconate dehydrogenase (EC 1.1.5.2) and gluconate 2-dehydrogenase (EC 1.1.99.3) gene clusters in a duet expression vector. The expression level of all subunits of the above enzymes were controlled using similar strength synthetic ribosome binding sites and regulated by lacI / trc as well as by removing the RNase E binding site in the gluconate 2-dehydrogenase gene clusters to stabilise transcripts. The resulting strain exhibited an enhanced performance in the BES with fermentation time reduced by more than 55% while sugar uptake rate and peak current doubled in the BES compared to the base strain.

Conclusions: For the first time, this study proved the principle that a BES driven bioconversion of glucose can be achieved for an obligate aerobe. This non-growth bioconversion achieved high yield, high purity and also could deliver the necessary metabolic energy for cell maintenance. While metabolic engineering allowed to significantly increase the conversion rates. The presented approach is a powerful new way to produce bio-chemicals and fuels.

[1] Lai, B., Yu, S., Bernhardt, P.V., Rabaey, K., Virdis, B. and Krömer, J.O. (2016) Anoxic metabolism and biochemical production in Pseudomonas putida F1 driven by a bioelectrochemical system. Biotechnol Biofuels, 9:39 DOI: 10.1186/s13068-016-0452-y