A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli.
BACKGROUND
For metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-tac-promoters (MCPtacs) in tandem.


RESULTS
Through a uniquely designed in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB) production proved that it was efficient. Integration of the phaCAB genes with the 5CPtacs promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation.


CONCLUSIONS
The transcription strength of the MCPtacs promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtacs promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Peoples Republic of China.
Mingji
M
Li
Junshu
J
Wang
Yanping
Y
Geng
Yikui
Y
Li
Qian
Q
Wang
Quanfeng
Q
Liang
Qingsheng
Q
Qi
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D006885:Hydroxybutyrates / Q000378:metabolism; D060847:Metabolic Engineering; D011091:Polyesters / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020080:Tandem Repeat Sequences; D014158:Transcription, Genetic; D000094122:Polyhydroxybutyrates
D029968:Escherichia coli Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates