Genetic Reconstruction Of Extracellular Electron Transfer Network In Shewanella Oneidensis MR-1

Dissimilatory metal reducing bacteria?DMRB?are a group of microorganisms capable of transferring the electrons generated from intracellular metabolism to extracellular metal oxides through electron transfer network,from which they can store energy for growth and metabolism.In addition to metal oxides,these microorganisms can also transfer electrons to metal ions,electrodes,and various pollutants.Thus,DMRB have a broad scope in application in the removal of heavy metals and radionuclides,bioremediation and energy generation.However,the practical application of DMRB has been somewhat hampered by its low extracellular electron transfer?EET?efficiency.This study,therefore used Shewanella oneidensis MR₁ as the chassis microorganism with the aim of enhancing its EET ability.For this purpose,we designed and developed various gene editing techniques,including the all-in-one CRISPR platform,gene network integration strategy,novel genome editing tool,and antibiotic-free gene carrier.Based on these techniques,this study further investigated the enhancement of pollutant biodegradation by refactoring and optimizing the EET pathway in genome,and explored the enhancement in energy generation by constructing the complex EET gene network.Moreover,this study also gave a scientific support to the application of S.oneidensis in practical environmental remediation.The main findings are summarized as follows:1.Gene editing of the electron transport chain in Shewanella oneidensis MR-1 based on CRISPR-Cas9.An all-in-one gene-editing platform was constructed with the CRISPR-Cas9 technology and the I-SceI plasmid-curing system,performing efficient multiple genomes editing in S.oneidensis with a maximum editing efficiency of 82.85%.Based on the CRISPR platform,this study developed a genome promoter-engineering strategy?GPS?for reconstituting the promoter of conductive Mtr complex production,and integrating additional expression cassettes of flavin biosynthesis and NADH generation into genome.The CRISPR-GPS significantly improved the production of the conductive c-type cytochrome?c-Cyts?complex,the electron shuttle of flavin,and the intracellular electron carrier of NADH.As a result,the current output capacity of S.oneidensis was enhanced by 2.79 times,and the U???reduction capacity was also promoted by 2.77 times.This work not only provides a new powerful and universal genome editing tool for DMRB,but also endows these microorganisms with an enhanced bioreduction ability to remediate the uranium-contaminated environments.2.Construction of the EET gene network platform in S.oneidensis MR-1.A plasmid shuttling engineering strategy?PSES?for DMRB was developed using the integration-recircularization system coupled with CRISPR-xCas9?3.7?.With PSES,the complex and variable DMRB gene network editing was transferred into the model strains of E.coli,in which the EET gene network platform of S.oneidensis was successfully constructed.Meanwhile,the constructed gene network platform could shuttle between the engineering strains and the model strains.Furthermore,the promoter of the metal reducing?MTR?gene network was optimized and reconstructed,and the flavin,GTP and purine gene network were quickly integrated with PSES,obtaining a 48-kb gene network platform containing 28 genes.The maximum current density of microbial fuel cell inoculated with engineered strains was increased by 7.61 times,the maximum power density was increased by 10.89 times,and the maximum output voltage was increased to 515.2 mV.These results indicate that the PSES could be used for the construction and optimization of large gene network,and its application in the engineering design of EET gene network of S.oneidensis achieved a highly efficient energy generation.3.Gene editing of the electron transport chain of S.oneidensis MR-1 based on I-SceI.A novel gene editing tool named as iEditing,was developed using the I-SceI homing endonuclease coupled with the hybrid RecET recombination system.The iEditing achieved highly efficient genome editing in S.oneidensis,with the highest editing efficiency up to 100%.With the iEditing tool,additional EET related pathways in S.oneidensis was successfully constructed by integrating the omB electron transfer pathway of G.sulfurreducens and the phenazine synthesis pathway of P.aeruginosa into the genome.The edited strains showed significant improvement of EET ability,which the maximum current density of microbial fuel cell inoculated with edited strains was increased by 3.14 times,and the maximum output voltage was increased by 1.70 times.The MO,Cr???,U???and ROX reduction abilities of the edited strains were also improved,for instance,the reduction of Cr???was enhanced by 4.1 times Moreover,The iEditing could also performed efficient genome editing in other Gram-negative bacteria,such as E.coli,P.putida and A.hydrophila.Therefore,the engineered iEditing toolkit provides both powerful gene editing tools for various Gram-negative bacteria,and new thought for environmental remediation.4.Construction of antibiotic-free genetic platform in S.oneidensis MR-1.The 161-kb endogenous large plasmid of S.oneidensis was successfully knocked out using I-SceI endonuclease,with no influence on the growth and energy generation of the strain.A new plasmid platform was developed based on the repA replication initiation factor of the endogenous large plasmid.An antibiotic-free plasmid system was constructed by introducing essential genes of infA or alr into the repA plasmid platform and deleting these essential genes from the genome.The antibiotic-free plasmid system could achieve multi-copy and stable gene expression without using antibiotics.Ultimately,the expression of the inner membrane menaquinone and outer membrane c-Cyts complex genes infA-dependent antibiotic-free plasmids significantly promoted the electrogenesis of S.oneidensis by 3.5 times,and accelerated the reduction of MO and Cr?VI?by 6.1 and 3.1 times,respectively.The antibiotic-free plasmids can not only relieve the close dependence of plasmid engineering on antibiotics,but also avoid the potential risk caused by antibiotics or resistance genes in practical environmental bioremediation using genetically engineered strains.