Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria

Research output: Contribution to journalResearch articleContributedpeer-review


  • Xue Wang - , Shandong University (Author)
  • Wentao Zheng - , Shandong University (Author)
  • Haibo Zhou - , Shandong University (Author)
  • Qiang Tu - , Shandong University (Author)
  • Ya Jie Tang - , Shandong University (Author)
  • A. Francis Stewart - , Chair of Applied Genomics (Author)
  • Youming Zhang - , Shandong University (Author)
  • Xiaoying Bian - , Shandong University (Author)


Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-Assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional mutagenesis at multiple sites on kilobase scales in diverse bacteria without the generation of double-strand breaks or disturbance of the mismatch repair system. dReaMGE can achieve combinatorial genome engineering works, for example, alterations to multiple biosynthetic pathways, multiple promoter or gene insertions, variations of transcriptional regulator combinations, within a few days. dReaMGE adds to the repertoire of bacterial genome engineering to facilitate discovery, functional genomics, strain optimization and directed evolution of microbial cell factories.


Original languageEnglish
Article numberE15
JournalNucleic acids research
Issue number3
Publication statusPublished - 22 Feb 2022

External IDs

PubMed 34792175
ORCID /0000-0002-4754-1707/work/142248121


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