A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

Abstract

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.

Figure 1

Construction outline of the MCPtacs promoter clusters. Fragment 5CPtacs with the flanking sequence was amplified by PCR with p5TG as the template. Fragment 1 was generated by digesting fragment 5CPtacs with BamHI. Fragment 2 was digested from fragment 5CPtacs with BamHI and HindIII. Fragment 3 was linearized from the plasmid p5TG with HindIII. Then, the three fragments were assembled together under the action of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase in the isothermal process.

Figure 2

Determination of the transcription strength of the MCPtacs promoter clusters by fluorescence analysis. (A) The time dependent analysis of the fluorescence/OD₆₀₀ ratio; (B) The OD₆₀₀ dependent analysis of the fluorescence/OD₆₀₀ ratio.

Figure 3

Determination of the GFP expression of the MCPtacs promoter clusters by SDS-PAGE. After 16 h cultivation, cells were harvested and lysed completely in isometric PBS. 15 μL supernatant containing GFP was subjected to SDS-PAGE. GFP bands were measured with ImageJ software, and the areas of their corresponding peaks were used to denote the relative GFP amounts. Lane1 Protein Marker, lane2 5874.43, lane3 12432.74, lane4 14813.91, lane5 17585.91, lane6 18039.91, lane7 18487.33, lane8 17334.79, lane9 16975.38, lane10 20819.62, lane11 18758.20, lane12 20396.74.

Figure 4

PHB production in the engineered E. coli. Glucose consumption and cell growth. Solid circles OD₆₀₀ of DH5α/ΔpoxB::1TPHB, solid squares OD₆₀₀ of DH5α/ΔpoxB::5TPHB, open circles glucose consumption of DH5α/ΔpoxB::1TPHB, open squares glucose consumption of DH5α/ΔpoxB::5TPHB.