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

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 

机构地区：[1]State Key Laboratory of Bioreactor Engineering,Newworld Institute of Biotechnology,East China University of Science and Technology,Shanghai,200237,China

出　　处：《Synthetic and Systems Biotechnology》2022年第3期1002-1011,共10页合成和系统生物技术（英文）

基　　金：supported by the National Natural Science Foundation of China(No.21776075);the Natural Science Foundation of Shanghai(No.20ZR1415100);the National Key Research and Development Program of China(No.SQ2020YFC210061).

摘　　要：Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,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 factories.First,a single-step multi-copy integration method was established in M.neoaurum 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 events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As 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 system.With 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 strain.The 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 

分 类 号：Q78[生物学—分子生物学]