Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch  

作　　者：Haoyu Guo Rongzhen Tian Yaokang Wu Xueqin Lv Jianghua Li Long Liu Guocheng Du Jian Chen Yanfeng Liu 

机构地区：[1]Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi,214122,China [2]Science Center for Future Foods,Jiangnan University,Wuxi,214122,China [3]Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology,Jiangnan University,Wuxi,214122,China [4]Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology,Jiangnan University,Wuxi,214122,China

出　　处：《Synthetic and Systems Biotechnology》2024年第3期577-585,共9页合成和系统生物技术（英文）

基　　金：supported by the National Key Research and Development Program of China(2020YFA0908300);the National Natural Science Foundation of China(32222069,32172349);the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005);the Natural Science Foundation of Jiangsu Province(BK20202002).

摘　　要：Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology.In comparison to plasmid-based multi-copy expression,the utilization of chromosomal multi-copy genes offers increased stability of expression level,diminishes the metabolic burden on host cells,and enhances overall genetic stability.In this study,we developed the“BacAmp”,a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis,which was achieved by employing a combination of repressor and non-natural amino acids(ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination.When the reversible switch is turned on,genome editing and gene amplification can be achieved.Subsequently,the reversible switch was turned off therefore stabilizing the gene copy number.The stabilized gene amplification system marked by green fluorescent protein,achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations.When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid(NeuAc)synthesis,the copy number of the critical gene increased to an average of 7.7,which yielded a 1.3-fold NeuAc titer.Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B.subtilis.

关 键 词：Bacillus subtilis Stabilized gene amplification system Homologous recombination RECA 

分 类 号：Q78[生物学—分子生物学]