ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism

Wentao Zheng, Yuxuan Wang, Jie Cui, Guangyao Guo, Yufeng Li, Jin Hou, Qiang Tu, Yulong Yin, A. Francis Stewart (), Youming Zhang (), Xiaoying Bian () and Xue Wang ()
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Wentao Zheng: Shandong University
Yuxuan Wang: Shandong University
Jie Cui: Shandong University
Guangyao Guo: Tianjin University of Science and Technology
Yufeng Li: Shandong University
Jin Hou: Shandong University
Qiang Tu: Shandong University
Yulong Yin: Yuelushan Laboratory
A. Francis Stewart: Shandong University
Youming Zhang: Shandong University
Xiaoying Bian: Shandong University
Xue Wang: Shandong University

Nature Communications, 2024, vol. 15, issue 1, 1-19

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-54191-4

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