Developing a base‐editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR‐1 for enhanced pollutant degradation

Abstract

Shewanella oneidensis MR‐1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR‐1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base‐editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR‐1. The C‐to‐T conversion of suitable C within the base‐editing window could be readily and efficiently achieved by the pCBEso system without requiring double‐strand break or repair templates. Moreover, double‐locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N‐acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR‐1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild‐type when glucose or GlcNAc was used as the sole carbon source. Such a base‐editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR‐1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

Complete genome sequence of Shewanella algae strain 2NE11, a decolorizing bacterium isolated from industrial effluent in Peru

“…Although the diversity of carbon source utilization in Shewanella is limited, the metabolic machinery for the catalysis of the other carbohydrates could not be fully elucidated in S. algae 2NE11. It has already been confirmed that several strains of Shewanella can improve the decolorization efficiency when grown under additives such as carbohydrates [10] . S. algae 2NE11 seems to have the complete genomic machinery necessary to metabolize lactate and N-acetylglucosamine, which has been related to the results obtained by physiological tests.…”

“…S. algae 2NE11 seems to have the complete genomic machinery necessary to metabolize lactate and N-acetylglucosamine, which has been related to the results obtained by physiological tests. Previous studies indicate that they are promoters of the decolorization efficiently, for which we highlight that these two sugars could be used to optimize the decolorization process on a larger scale with strain 2NE11 [ 10 , 55 , 58 ].…”

“…They have been found in almost all genomes of Shewanella genus and many isolated strains have been able to grown in N-acetylglucosamine as sole carbon source, ability also shared by 2NE11 [38] . Moreover, Nag genes has been recently related to glucose metabolism of Shewanella oneidensis MR-1 for enhanced pollutant degradation [10] . Likewise, it has been reported in multiple investigations that lactate can be used as electron acceptor in cellular respiration, allowing to improve the dye degradation process required [ 7 , 56 ].…”

Applications of Synthetic Biotechnology on Carbon Neutrality Research: A Review on Electrically Driven Microbial and Enzyme Engineering

“…By fusing Cas9 nickase (Cas9n (D10A)) with cytidine deaminase (rAPOBEC1), Cheng et al successfully developed the pCBEso system in S. oneidensis , whose double-locus simultaneous editing efficiency reached up to 87.5%. Compared with others, this system did not require double-strand break or repair templates and was successfully used for broadening carbon source utilization spectra for S. oneidensis ( Cheng L et al, 2020 ). The developed CRISPR-ddAsCpf1 system achieved 100% gene repression reported by green fluorescent protein (GFP).…”

Development of Whole Genome‐Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR‐1

“…[ 15b,17 ] Taking pCBEso system as an example, it requires that the Cs to be edited locate in the range of −18 to −13 upstream from PAM sequence. [ 12 ] These features of the base editing technologies confined the editable scope of genes in the genome, which further hindered their practical applications in editing EAMs and other microorganisms.…”