Developing a Base Editing System to Expand the Carbon Source Utilization
Spectra of Shewanella oneidensis MR-1 for Enhanced Pollutant Degradation
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 work, 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 roles of
the key genes involving in N-acetyl-glucosamine (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 work 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.