title: A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies.
abstract: Direct optimization of the metabolic pathways on the chromosome requires tools that can fine tune the overexpression of a desired gene or optimize the combination of multiple genes. Although plasmid-dependent overexpression has been used for this task, fundamental issues concerning its genetic stability and operational repeatability have not been addressed. Here, we describe a rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies (CIGMC), which uses the flippase from the yeast 2-μm plasmid. Using green fluorescence protein as a model, we verified that the fluorescent intensity was in accordance with the integration copy number of the target gene. When a narrow-host-range replicon, R6K, was used in the integrative plasmid, the maximum integrated copy number of Escherichia coli reached 15. Applying the CIGMC method to optimize the overexpression of single or multiple genes in amino acid biosynthesis, we successfully improved the product yield and stability of the production. As a flexible strategy, CIGMC can be used in various microorganisms other than E. coli.
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Pengfei
authors: P
authors: Gu
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Fan
authors: F
authors: Yang
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Tianyuan
authors: T
authors: Su
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Qian
authors: Q
authors: Wang
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Quanfeng
authors: Q
authors: Liang
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors: Qingsheng
authors: Q
authors: Qi
mesh_terms: D002876:Chromosomes, Bacterial; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D017930:Genes, Reporter; D059765:Homologous Recombination; D053858:Metabolic Networks and Pathways; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase