A robust strategy for overexpression of DNA polymerase from Thermus aquaticus using an IPTG-independent autoinduction system in a benchtop bioreactor

Abstract

Abstract The DNA polymerase derived from Thermus aquaticus is the most widely utilized among various DNA polymerases, indicating its significant economic importance. Consequently, efforts to achieve a substantial yield of Taq DNA polymerase (Taq-pol) are ongoing. The expression of recombinant protein using T7-induced promoters presents challenges in cost-effectiveness, primarily due to the reliance on traditional induction method. Our study aims to enhance cost-efficiency, and scalability of our method for overproducing Taq-pol, particularly in comparison to traditional IPTG-induced techniques, which remain underreported in the current literature. To achieve those purposes, this work integrated the use of (1) a high copy number vector; (2) an optimized chemically defined medium; and (3) optimized fermentation conditions in a 5 L bioreactor. A total of 83.5 mg/L of pure Taq-pol was successfully synthesized in its active form, leading to a 9.7-fold enhancement in protein yield. This was achieved by incorporating glucose, glycerol, and lactose into a defined medium at concentrations of 0.1, 0.6, and 1%, respectively, under specific production conditions in a 5 L bioreactor: 300 rpm, 2 vvm, and 10% inoculant. The data collectively suggest that the strategy serves as a significant foundation for the future advancement of large-scale production of Taq-pol.

Keywords

• DNA polymerase
• Thermus aquaticus
• Autoinduction
• Chemically defined medium
• Escherichia coli