Accelerated Electro-Fermentation of Acetoin in Escherichia coli by Identifying Physiological Limitations of the Electron Transfer Kinetics and the Central Metabolism

Abstract

Anode-assisted fermentations offer the benefit of an anoxic fermentation routine that can be applied to produce end-products with an oxidation state independent from the substrate. The whole cell biocatalyst transfers the surplus of electrons to an electrode that can be used as a non-depletable electron acceptor. So far, anode-assisted fermentations were shown to provide high carbon efficiencies but low space-time yields. This study aimed at increasing space-time yields of an Escherichia coli-based anode-assisted fermentation of glucose to acetoin. The experiments build on an obligate respiratory strain, that was advanced using selective adaptation and targeted strain development. Several transfers under respiratory conditions led to point mutations in the pfl, aceF and rpoC gene. These mutations increased anoxic growth by three-fold. Furthermore, overexpression of genes encoding a synthetic electron transport chain to methylene blue increased the electron transfer rate by 2.45-fold. Overall, these measures and a medium optimization increased the space-time yield in an electrode-assisted fermentation by 3.6-fold.

Microbe–Anode Interactions: Comparing the impact of genetic and material engineering approaches to improve the performance of microbial electrochemical systems (MES)

“…So far, this approach was only pursued to transplant the electron transfer chain from S. oneidensis into E. coli . Although this approach was to some extent successful, it was not possible to reach EET rates in E. coli that would be similar to what is reached by Shewanella cells (Beblawy et al, 2020 ; Jensen et al, 2010 ; Sturm‐Richter et al, 2015 ; TerAvest, Zajdel, & Ajo‐Franklin, 2014 ). Hence, we have apparently not understood the biochemistry sufficiently to transplant it in way reaching its full capacity in other organisms yet.…”

“…Further applications of anodic BESs include the use for (I) microbial electrolysis for carbon capture (MECC; Lu, Huang, Rau, & Ren, 2015 ), (II) anode‐assisted fermentation of value‐added substances (Beblawy, Philipp, & Gescher, 2020 ; Härrer, Elreedy, Ali, Hille‐Reichel, & Gescher, 2023 ; Palma‐Delgado, Paquete, Sturm, & Gescher, 2019 ), and (III) biosensors (Golitsch, Bücking, & Gescher, 2013 ; Ivars‐Barceló et al, 2018 ; Modin, Wang, Wu, Rauch, & Fedje, 2012 ; Pasternak, Greenman, & Ieropoulos, 2017 ) among others.…”

Implementation of a flavin biosynthesis operon improves extracellular electron transfer in bioengineered Escherichia coli

“…Over the past two decades, different [15] , overexpression of the full MtrCAB complex in the outer membrane [16] and co-expression of the inner membrane cytochrome CymA [18] , to the expression of both membrane-associated and periplasmic cytochromes [22] . These advances in implementing direct EET mechanisms in E. coli are promising and have led to applications in electrode-assisted biosynthesis of chemicals and biosensing [4,6,17,20,23] . Beyond direct electron transfer through cytochromes, EET in engineered E. coli can be further improved using exogenous mediators [6,[17][18]21,23] .…”

“…Beyond direct electron transfer through cytochromes, EET in engineered E. coli can be further improved using exogenous mediators [6,17–18,21,23] . Indeed, commonly available lab strains of E. coli are limited in their ability to form biofilms, making it challenging to rely on direct electron transfer in BES with no specific immobilization on the electrode [24] .…”

“…These advances in implementing direct EET mechanisms in E. coli are promising and have led to applications in electrode-assisted biosynthesis of chemicals and biosensing [4,6,17,20,23] . Beyond direct electron transfer through cytochromes, EET in engineered E. coli can be further improved using exogenous mediators [6,[17][18]21,23] . Indeed, commonly available lab strains of E. coli are limited in their ability to form biofilms, making it challenging to rely on direct electron transfer in BES with no specific immobilization on the electrode [24] .…”