Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis

Abstract

The slow rate of extracellular electron transfer (EET) of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems. Intracellular NAD(H/+) (i.e., the total level of NADH and NAD+) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. However, how the total level of intracellular NAD(H/+) impacts the EET rate in Shewanella oneidensis has not been established. Here, we use a modular synthetic biology strategy to redirect metabolic flux towards NAD+ biosynthesis via three modules: de novo, salvage, and universal biosynthesis modules in S. oneidensis MR-1. The results demonstrate that an increase in intracellular NAD(H/+) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis, thereby enhancing intracellular electron flux and the EET rate.

Electron Communication of Bacillus subtilis in Harsh Environments

“…NAD, a key class of cofactors, serves as essential electron donor or acceptor in all biological organisms (Liu et al., 2018) and drives major catabolic and anabolic reactions to maintain cellular redox homeostasis and energy metabolism (Xiao et al., 2018). NAD(H/+) is a considerable source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways (Li et al., 2018).…”

“…Whether a microorganism is autotrophic or heterotrophic, free living or obligate parasite, energy generation is essential for the cell to survive (Hernandez and Newman, 2001). Energy metabolism is dominated by oxidation-reduction reactions, in which electron transfer plays a fundamental role, supplying the reducing power and maintaining the intracellular redox balance through the regeneration of the redox cofactor nicotinamide adenine dinucleotide (NAD) (Li et al., 2018, Nealson and Rowe, 2016). Extracellular electron transfer (EET) is the process by which electrons generated by microbial metabolism are transported to extracellular substrates that act as electron acceptors.…”

Microbial electro-fermentation for synthesis of chemicals and biofuels driven by bi-directional extracellular electron transfer

“…Intracellular NAD (H/ + ) and redox state (NADH/NAD + ratio) are the sources of EET process. Modular strategies to increase intracellular NAD (H/ + ) pool and the ratio of NADH/NAD + could significantly enhance the current production of exoelectrogens [ 68 , 69 ]. Other strategies including heterogeneous overexpression of porins to improve cell membrane permeability [ 70 , 71 ], increasing the synthesis of endogenous redox mediators [ 2 , 71 ], expanding the substrate utilization spectrum to accelerate substrate utilization [ 36 , 72 ], and modification of genes related to biofilm formation [ 73 ] could promote EET in electroactive microorganisms.…”

Construction of a third NAD+de novobiosynthesis pathway

“…The most frequently used strategy for increasing cellular NAD(H) levels is by reinforcing the NAD + salvage pathways instead of manipulating de novo biosynthesis. However, the few reported attempts to manipulate NAD + de novo pathways yielded only very limited 18 or nonexistent increases in cellular NAD(H) levels 19 , probably due to the stringent regulation of NAD + de novo biosynthesis at transcriptional 20 , translational 21 , and post-translational levels 22 . In addition, because both NAD + de novo biosynthesis pathways start from a proteinogenic amino acid, their activation may deplete amino acid pools required to efficiently overproduce proteins in engineered cell factories.…”