ArticleNature communications2024
ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism.
Article in Nature communications, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed.
- High-throughput editing boosts the construction of next-generation microbial cell factories.Synthetic and systems biotechnology · 2026Review
- Development of efficient genetic toolkits and heterologous chassis for bioprospecting of myxobacteria.Nature communications · 2026Article
- rDNA-mediated multicopy integration and gene dosage quantification system for microbial zeaxanthin biosynthesis.World journal of microbiology & biotechnology · 2026Article
- Systematic engineering of Escherichia coli for biosynthesis of 3-hydroxypropionic acid from glucose and malonate.BMC biotechnology · 2026Article
- A recombineering-based platform for high-throughput genomic editing inApplied and environmental microbiology · 2025Article
- SELECT: high-precision genome editing strategy via integration of CRISPR-Cas and DNA damage response for cross-species applications.Nucleic acids research · 2025Article
- The Strategy and Application of Gene Attenuation in Metabolic Engineering.Microorganisms · 2025Review
Corrections and comments
- Erratum issued
Authors and funding
12 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering.
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