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

Synthetic and Systems Biotechnology 2023年第1期8卷 1-10页

作者： Yaru Chen Meijie Cheng Yan Li Lin Wang Lixia Fang Yingxiu Cao Hao Song Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering(Ministry of Education) School of Chemical Engineering and TechnologyTianjin UniversityTianjin300072China Key Laboratory of Systems Bioengineering(Ministry of Education) Tianjin UniversityTianjin300072China

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates.Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1.Since multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in S.oneidensis.Four approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster.Then,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,respectively.To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were screened.This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain.The maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type strain.This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.

关键词： Multiplex gene editing electroactive microorganisms Base editing Multiplexed engineering CRISPR Extracellular electron transfer

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Monitoring stratification of anode biofilms in bioelectrochemical laminar flow reactors using flow cytometry

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Environmental Science and Ecotechnology 2020年第4期4卷 18-25页

作者： Yuting Guo Luis FMRosa Susann Muller Falk Harnisch Department of Environmental Microbiology Helmholtz-Centre for Environmental Research e UFZPermoserstrasse 1504318LeipzigGermany

A laminar flow bioelectrochemical systems(BES)was designed and benchmarked using microbial anodes dominated with Geobacter spp.The reactor architecture was based on modeled flow fields,the resulting structure was 3D printed and used for BES manufacturing.Stratification of the substrate availability within the reactor channels led to heterogeneous biomass distribution,with the maximum biomass found mainly in the initial/middle channels.The anode performance was assessed for different hydraulic retention times while coulombic efficiencies of up to 100%(including also hydrogen recycling from the cathode)and current densities of up to 75 μA cm^(-2) at an anode surface to volume ratio of 1770 cm^(2) L^(-1) after 35 days were achieved.This low current density can be clearly attributed to the heterogeneous distributions of biomass and the stratification of the microbial community structure.Further,it was shown that time and space resolved analysis of the reactor microbiomes per channel is feasible using flow cytometry.

关键词： Microbial electrochemical technology Flow cytometry Laminar flow Microbial community electroactive microorganisms

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