Development of a fed‐batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat

To effectively achieve tight regulation and high‐level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30°→41°C stepwise upshift, the shake‐flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30°C. By exploiting the fed‐batch fermentation approach, the recombinant cell batch initially kept at 30°C in a lab‐scale fermentor was exposed to 41°C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37°C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880‐fold over that obtained at the shake‐flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale‐up of recombinant proteins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 459–466, 2003.