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Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system

Synthetic and Systems Biotechnology 2022年第3期7卷 1002-1011页

作者： Ke Liu Gui-Hong Lin Kun Liu Yong-Jun Liu Xin-Yi Tao Bei Gao Ming Zhao Dong-Zhi Wei Feng-Qing Wang State Key Laboratory of Bioreactor Engineering Newworld Institute of BiotechnologyEast China University of Science and TechnologyShanghai200237China

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell *** some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a ***,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell ***,a single-step multi-copy integration method was established in *** by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration ***,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration ***,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene *** a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR *** six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild *** improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

关键词： Site-specific recombination phage integrase Xer recombinases Mycolicibacterium Multi-copy integration

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