A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies.

Pengfei Gu,Quanfeng Liang,Tianyuan Su,Qian Wang,Fan Yang,Qingsheng Qi

doi:10.1038/srep09684

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.

Highlights

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
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-mm plasmid
Development of the CIGMC system based on FLP/FLP recombination target (FRT) recombination

Summary

Introduction

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. Optimizing the overexpression of rate-limiting genes is a necessary step to direct more carbon flux into the biosynthesis pathway and to prevent the accumulation of toxic intermediate metabolites. To accomplish this task, plasmid-based overexpression has been extensively applied because of its easy manipulation and regulated expression[1,2,3]. In Escherichia coli, homologous recombination, site-specific recombination, and transposon-mediated gene transposition are often used to achieve chromosomal integration. Sabri et al.[15] constructed a series of knock-in/knock-out vectors to integrate large targeting DNA sequences into the chromosome Employing this strategy, a 7.3-kb DNA fragment at one locus and an 11.3-kb. To reduce the operation time and steps, St-Pierre et al.[21] recently developed a novel method named ‘‘clonetegration’’ to integrate DNA into prokaryotic chromosomes

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