Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

Abstract

Summary The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real‐world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the intron‐encoded homing‐endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR‐1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome‐editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow‐up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.

Type I-F CRISPR-PAIR platform for multi-mode regulation to boost extracellular electron transfer in Shewanella oneidensis

“…In this work, the transcriptional activation was achieved by CRISPRa in S. oneidensis . To our knowledge, the previous approaches to increase gene expression level in S. oneidensis MR-1 is overexpression based on plasmids and genomic knock-in ( Fan et al., 2021a , 2021b ; Yi and Ng, 2021 ). These methods are labor-intensive and time-consuming, owing to the difficulty of plasmid construction, inevitable steps of codons optimization and appropriate promoter selection to achieve specially adapted strength for the EET improvement ( Cao et al., 2019 ).…”

“…To date, there have been various tools available to artificially carry out gene regulation. For enhancing gene expression, the current approaches, plasmid-based overexpression and genomic knock-in, are utilized to increase the expression level of the specific genes in S. oneidensis MR-1 ( Fan et al., 2021a , 2021b ; Min et al., 2017 ). For repressing gene expression, CRISPRi system efficiently blocks RNA polymerase binding or elongation to achieve transcriptional interference ( Cao et al., 2017 ; Li et al., 2020 ).…”

Electrochemical Microwell Plate to Study Electroactive Microorganisms in Parallel and Real-Time

“…This will open the door to perform microbial resource mining in habitats that are already well known for harbouring EAM like wastewater and soil ( Koch and Harnisch 2016a ; Logan et al, 2019 ) but also recently discovered ones like the oral ( Naradasu et al, 2020 ) or gut ( Tahernia et al, 2020b ; Rago et al, 2021 ) microbiome and especially to explore new habitats as resources. Further, we foresee that already exploited EAM, for instance, for microbial electrosynthesis of chemical building blocks ( Mayr et al, 2019 ; Wu et al, 2019 ), can be further improved using concepts and tools that are well established (for non-electrochemical means) like site directed mutagenesis, CRISPR-CAS, and techniques beyond in high-throughput ( Alves et al, 2017 ; Fan et al, 2020 ). Even longer and more complex measurements will become a possibility by integrating microfluidics ( Yoon et al, 2018 ; Yates et al, 2021 ; Li et al, 2011 ), e.g., to replenish culture media or to enable complex co-cultivation experiments.…”

Control of synthetic microbial consortia in time, space, and composition

“…In any discussion of temporal control of microbes, it is also necessary to underscore the importance of advances in genome engineering [48][49][50][51]. While these advances are certainly vital to the advancement of nearly all of synthetic biology, they are of particular importance to consortial engineering.…”

“…There have already been significant efforts to expand synthetic biology beyond traditionally domesticated organisms, especially in applications that have proven challenging for E. coli and S. cerevisiae like extracellular electron transfer where Shewanella oneidensis already succeeds [50]. These efforts have recently developed beyond single-strain engineering but there is an enormous gap between the potential and current performance of such systems due to their infancy [75].…”