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The emerging role of recombineering in microbiology

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Engineering Microbiology 2023年第3期3卷 75-83页

作者： Ruijuan Li Aiying Li Youming Zhang Jun Fu State Key Laboratory of Microbial Technology Shandong UniversityQingdao 266237China Chinese Academy of Sciences(CAS)Key Laboratory of Quantitative Engineering Biology Shenzhen Institute of Synthetic BiologyShenzhen Institute of Advanced TechnologyChinese Academy of SciencesShenzhenChina

recombineering is a valuable technique for generating recombinant DNA in vivo,primarily in bacterial cells,and is based on homologous recombination using phage-encoded homologous recombinases,such as Red βγ from the lambda phage and RecET from the Rac *** recombineering technique can efficiently mediate homol-ogous recombination using short homologous arms(∼50 bp)and is unlimited by the size of the DNA molecules or positions of restriction *** this review,we summarize characteristics of recombinases,mechanism of recombineering,and advances in recombineering for DNA manipulation in Escherichia coli and other ***,the broad applications of recombineering for mining new bioactive microbial natural products,and for viral mutagenesis,phage genome engineering,and understanding bacterial metabolism are also reviewed.

关键词： recombineering Natural products Viral mutagenesis Phage genome engineering Bacterial metabolism

Half a century after their discovery:Structural insights into exonuclease and annealase proteins catalyzing recombineering

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Engineering Microbiology 2024年第1期4卷 39-54页

作者： Lucy J.Fitschen Timothy P.Newing Nikolas P.Johnston Charles E.Bell Gökhan Tolun School of Chemistry and Molecular Bioscience and Molecular HorizonsUniversity of WollongongWollongongNSWAustralia The ARC Training Centre for Cryo-electron Microscopy of Membrane Proteins University of WollongongWollongongNSWAustralia Faculty of Science University of Technology SydneyUltimoNSW 2007Australia Department of Biological Chemistry and Pharmacology The Ohio State University College of MedicineColumbusOH 43210

recombineering is an essential tool for molecular biologists,allowing for the facile and efficient manipulation of bacterial genomes directly in cells without the need for costly and laborious in vitro manipulations involving restriction *** main workhorses behind recombineering are bacteriophage proteins that promote the single-strand annealing(SSA)homologous recombination pathway to repair double-stranded DNA *** there have been several reviews examining recombineering methods and applications,comparatively few have focused on the mechanisms of the proteins that are the key players in the SSA pathway:a 5′→3′exonuclease and a single-strand annealing protein(SSAP or“annealase”).This review dives into the structures and functions of the two SSA recombination systems that were the first to be developed for recombineering in ***:the RecET system from *** Rac prophage and the𝜆Red system from bacteriophageλ.By comparing the structures of the RecT and Red𝛽annealases,and the RecE and𝜆Exo exonucleases,we provide new insights into how the structures of these proteins dictate their *** the sequence conservation of the𝜆λExo and RecE exonucleases gives more profound insights into their critical functional ***,as recombineering accelerates and evolves in the laboratory,a better understanding of the mechanisms of the proteins behind this powerful technique will drive the development of improved and expanded capabilities in the future.

关键词： recombineering Redβ λExo RecE RecT Protein structure Annealase Exonuclease

A recombineering system for Bacillus subtilis based on the native phage recombinase pair YqaJ/YqaK

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Engineering Microbiology 2023年第3期3卷 21-29页

作者： Qingshu Liu Ruijuan Li Hongbo Shi Runyu Yang Qiyao Shen Qingwen Cui Xiuling Wang Aiying Li Youming Zhang Jun Fu State Key Laboratory of Microbial Technology Shandong UniversityQingdao 266237China Hunan Institute of Microbiology Xinkaipu Lu 18Tianxin DistrictChangsha 410009China

Bacillus subtilis plays an important role in fundamental and applied research,and it has been widely used as a cell factory for the production of enzymes,antimicrobial materials,and chemicals for agriculture,medicine,and ***,genetic manipulation tools for *** have low *** this work,our goal was to develop a simple recombineering system for *** showed that genome editing can be achieved in *** 1A751 through co-expression of YqaJ/YqaK,a native phage recombinase pair found in ***-tilis 168,and the competence master regulator ComK using a double-stranded DNA substrate with short ho-mology arms(100 bp)and a phosphorothioate modification at the 5′-*** gene knockouts and large DNA insertions were achieved using this new recombineering system in *** *** far as we know,this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in *** conclusion,this new recombineering system provides a simple and fast tool for genetic manipulation of ***-tilis,and it will promote studies of genome function,construction of production strains,and genome mining in ***.

关键词： Bacillus subtilis recombineering YqaJK system Genome editing

Simple and efficient genome recombineering using kil counter-selection in Escherichia coli

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Simple and efficient genome recombineering using kil counter...

Recent advances in genome-scale engineering in Escherichia coli and their applications

中国生物工程学会第十二届学术年会暨2018年全国生物技术大会

作者： Peng Wang Jian Wang Fang Huang Shanhu Li Beijing Institute of Biotechnology

Background: The precise modification of genes and their regulatory elements, including insertions, deletions, and point mutations, is critical both to metabolic engineering and to functional genome analysis in Escherichia coli. A highly efficient phage-based homologous recombination method, recombineering, has been developed as a powerful tool for genome editing. Kil gene expression has been associated with cell filamentation, disruption of cytokinesis in E. coli and loss-of-viability phenotypes. It is driven by promoter p L, which is in turn under stringent control of the temperature-sensitive allele of the phage CI repressor(cI857). These features meet the requirements of a negative-selection marker gene. In this study, we found that E. coli cells carrying the kil gene are efficiently killed after continuous induction at 42 °C, and the kil gene from DY330(the defective lambda prophage that includes the kil gene inserted into the E. coli K-12 W3110 chromosome) can be successfully removed using counter-selection at 42 °C. Therefore, we want to explore and develop kil as a counter-selection gene. Using DY330 as a parent strain, the DNA fragment from CIII to N was deleted, and regions from rexB to rexA were replaced by a positive selection gene, neo. Thus a 2,484-bp-long selection/counter-selection system neo-kil was constructed. Results: Having tested the potential of kil as a counter-selection marker gene, we constructed a neo-kil double-selection cassette by deleting unnecessary fragments. Counter-selection efficiency was significantly high using overlapping oligos to delete DNA fragments from the E. coli genome: 23 tested colonies were all successfully recombined. The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, in the lacI, ack, and dbpa loci using different E. coli strains. In all these tested strains and tested loci, the selection stringency of kil significantly exceeds that of sacB by 2-to 28-fol

关键词： recombineering metabolic engineering counter-selection

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Engineering Microbiology 2024年第1期4卷 28-38页

作者： Hui Gao Zhichao Qiu Xuan Wang Xiyuan Zhang Yujia Zhang Junbiao Dai Zhuobin Liang Institute of Molecular Physiology Shenzhen Bay LaboratoryShenzhen 518132China CAS Key Laboratory of Quantitative Engineering Biology Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics.Shenzhen Institute of Synthetic BiologyShenzhen Institute of Advanced TechnologyChinese Academy of SciencesShenzhen 518055China Regenerative Medicine Program Bellvitge Institute for Biomedical Research(IDIBELL)and Program for Clinical Translation of Regenerative Medicine in Catalonia(P-CMRC)L’Hospitalet de LlobregatBarcelona 08908Spain Faculty of Pharmacy and Food Science Barcelona UniversityBarcelona 08028Spain College of Life Sciences Northwest A&F UniversityShaanxi 712100China Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureKey Laboratory of Synthetic BiologyMinistry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120China

Owing to the rapid advancement of genome engineering technologies,the scale of genome engineering has expanded *** editing has progressed from one genomic alteration at a time that could only be employed in few species,to the simultaneous generation of multiple modifications across many genomic loci in numerous *** development and recent advances in multiplex automated genome engineering(MAGE)-associated technologies and clustered regularly interspaced short palindromic repeats and their associated protein(CRISPR-Cas)-based approaches,together with genome-scale synthesis technologies offer unprecedented opportunities for advancing genome-scale engineering in a broader *** approaches provide new tools to generate strains with desired phenotypes,understand the complexity of biological systems,and directly evolve a genome with novel ***,we review the recent major advances in genome-scale engineering tools developed for Escherichia coli,focusing on their applications in identifying essential genes,genome reduction,recoding,and beyond.

关键词： Genome-scale engineering E.coli recombineering CRISPR-Cas

用基于galK的同源重组的方法构建arc基因表达增强的载体

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华西医学 2008年第3期23卷 433-435页

作者：邓蓉康杜晓霞陈米娜季一飞崔义元四川大学华西医院神经分子生物学实验室四川成都610041

目的:利用同源重组的方法,将arcBAC(bacteria artificial chromosome)启动子区域的SRE(serumre-sponse element)改造成Gal4结合序列,以此通过Gal4/UAS系统增强arc基因表达。方法:在大肠杆菌中利用依赖于噬菌体基因的同源重组,通过galK... 详细信息

目的:利用同源重组的方法,将arcBAC(bacteria artificial chromosome)启动子区域的SRE(serumre-sponse element)改造成Gal4结合序列,以此通过Gal4/UAS系统增强arc基因表达。方法:在大肠杆菌中利用依赖于噬菌体基因的同源重组,通过galK正负筛选的方法改造arcBAC。结果:成功将arc启动子区域的SRE改造成Gal4结合序列,并且没有影响arc基因的其他序列。结论:成功改造BAC,可以将此BAC用在Gal4/UAS系统增强arc基因表达的实验中。

关键词： recombineering galK正负筛选细菌人工染色体