An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Abstract

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

Effects of the immobilization of recombinant Escherichia coli on cyclodextrin glucanotransferase (CGTase) excretion and cell viability

“…Zhinan et al [37] showed that 2 × YT was the best medium for human epidermal growth factor (hEGF) production compared with 14 other media. In studies conducted by Low et al [38] and Kushoo et al [39], TB medium was chosen for further evaluation because the richness of this medium significantly enhances protein expression. However, in the present study, SOB medium was selected for the subsequent expression analysis because of the associated high CGTase excretion and low cell lysis in addition to the preservation of plasmid stability.…”

Cyclodextrin glycosyltransferase biosynthesis improvement by recombinantLactococcus lactisNZ:NSP:CGT: medium formulation and culture condition optimization

“…This is in good correspondence with the result obtained in this study. Normally, however, the optimum temperature for various producers of CGTase and for L. lactis producing different products is higher than 25 C. For example, Low et al [42] reported 34.7 C as the optimum temperature for CGTase biosynthesis by E. coli. Rosso et al [43], Mahat et al [31] and Higuti et al [44] concluded that 37 C was the optimum temperature for maximum CGTase production by various Bacillus strains.…”

Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

“…Since then, many CGTases from various bacteria have been expressed in E. coli, including an a-CGTase from a Paenibacillus macerans strain isolated in our previous studies [18]. To enhance the heterologous production in E. coli of an extracellular enzyme, several molecular approaches and culture strategies have been used, such as manipulation of the signal peptide [14], enhancement of the ABC transporter system [24], and site-directed mutagenesis of the a-CGTase gene [21,22]. In culture flask, changing the culture medium composition [18], concentration of the inducer [18], and medium supplements [7] (such as calcium [19] and glycine [17]) have also been studied.…”