Transcriptome Analysis to Identify Crucial Genes for Reinforcing Flavins-Mediated Extracellular Electron Transfer in Shewanella oneidensis

Abstract

Flavins serve as the electron mediators in Shewanella oneidensis, determining the extracellular electron transfer (EET) rate. Currently, metabolic engineering of flavins biosynthetic pathway has been studied for improving EET. However, the cellular response triggered by flavins that contribute to EET remains to be elucidated. In this study, the riboflavin-overproducing strain C5 (expressing the flavins synthetic genes in plasmid PYYDT) and the PYYDT strain (harboring the empty plasmid PYYDT) in the microbial fuel cells are applied for comparative transcriptomic analyses to investigate beneficial gene targets that could improve EET. From the differentially expressed genes, we select the significantly upregulated and downregulated genes for inverse engineering in S. oneidensis. The results show that overexpression of ahpC and ccpA, and inactivation of pubA, putB, and tonB are able to improve the EET capability. Combinatorial modulation of these five genes results in the recombinant strain CM4, achieving the maximum power density of 651.78 ± 124.60 mW/m2, 1.97 folds of the parental strain. These genes modulation is speculated to reduce the ROS damage and to promote cytochrome synthesis and heme accumulation, which coherently enhance EET. Our findings facilitate in-depth understanding of the mechanism of flavins-mediated EET and provide new insights in promoting EET of S. oneidensis for electricity generation.

Highly efficient multiplex base editing: One-shot deactivation of eight genes in Shewanella oneidensis MR-1

“…Considerable efforts have been made to modulate the flavin biosynthetic pathway in order to heighten RF-mediated EET [ 56 , 57 ]. Recently, a number of genes indirectly associated with RF-mediated EET have been identified by comparative transcriptome analyses, which were conducted between a RF-overproducing strain and a strain carrying empty plasmid [ 58 ]. Among the significantly downregulated genes candidates, the effectiveness of silence of each gene has been determined by the bio-electrochemical analysis [ 58 ].…”

“…Recently, a number of genes indirectly associated with RF-mediated EET have been identified by comparative transcriptome analyses, which were conducted between a RF-overproducing strain and a strain carrying empty plasmid [ 58 ]. Among the significantly downregulated genes candidates, the effectiveness of silence of each gene has been determined by the bio-electrochemical analysis [ 58 ]. As a next step, we here leveraged multiplexed base editing for combined reverse engineering, thus these genes were deactivated in combination for reinforcing RF-mediated EET.…”

“…Our previous work showed that the individual deactivation of gene exbB , hmuA , putA , pubC , and pubB had no significant helpful effect on the enhancement of EET [ 58 ]. Nonetheless, the maximum power density of HRF(8BE) was ∼1.22 folds higher than that of HRF(3BE), suggesting that combinatorial modification of genes that do not have positive effects alone might have unexpected outcome.…”

Efficient Enhancement of Extracellular Electron Transfer in Shewanella oneidensis MR-1 via CRISPR-Mediated Transposase Technology

“…Highly specific gene insertions at multiple sites are achieved by loading functional modules onto an eliminable plasmid vector. This approach overcomes limitations associated with exogenous plasmids and avoids common issues like antibiotic contamination and metabolic burden in practical applications. − This is particularly crucial for electroactive bacteria, which often are specialized strains designed for industrial or environmental applications. , …”

Modular Engineering Strategy to Redirect Electron Flux into the Electron-Transfer Chain for Enhancing Extracellular Electron Transfer in Shewanella oneidensis

“…Shewanella oneidensis MR-1, one of the most well-studied exoelectrogens, showed great promise in BESs as core biocatalysts. − The EET mechanism of S. oneidensis MR-1 has been extensively studied and elucidated, including contact-based direct electron transfer (DET) via a number of conductive outer-membrane c-cytochromes. − and indirect electron transport (IET) mediated by soluble electron shuttles. − Electrons generated by substrate oxidation were stored in the reduced form of nicotinamide adenine dinucleotide (NADH), which is the major releasable intracellular electron carrier; then, the electrons are transferred to extracellular electron acceptors via EET pathways. − Efforts toward synthetic biological strategies, , such as the de novo synthesis of intracellular NAD + and the acceleration of NADH regeneration, have been developed to enhance the intracellular releasable electron pool and the EET rate of S. oneidensis.…”