Signal peptide modification and signal peptide peptidase effect on cyclodextrin glucanotransferase excretion and cell lysis

Extracellular excretion of recombinant protein is beneficial as it can greatly reduce the cost of downstream processing and improve the product quality. However, the efforts in achieving high excretion level often leads to occurrence of cell lysis and low protein yield due to limited capacity of the transport machinery. The objectives of this study were to investigate the effect of amino acids and their locations in h-region signal peptide on cyclodextrin lucanotransferase (CGTase) excretion, and analyze the function of signal peptide peptidase (SPPase) in improving the membrane integrity of Escherichia coli (E. coli). Modification of the hydrophobic region of the N1R3 signal peptide (wild type) using site-saturation mutagenesis has improved the excretion of CGTase. The results indicated that the excretion of CGTase is highly dependent on properties of signal peptide which are hydrophobicity, secondary conformation and, the type and position of amino acids at the boundary and core segment of the h-region. Mutant signal peptides designated as M9F, V10L and A15Y enhanced the excretion of CGTase to three-fold and has demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate caused nine-fold increase in cell lysis as compared to the wild type. In dual-plasmid system for co-overexpression, gene expression of CGTase fused to A15Y signal peptide and SPPase, were regulated by T7lac and PBAD promoters, respectively, at induction temperature of 25ºC. It was shown that co-overexpression of SPPase and CGTase has reduced the occurrence of cell lysis that was reflected by ß-galactosidase activity from 128.6 U/ml to 0.12 U/ml, which equivalent to 99% decrease when compared to the E. coli that expressed CGTase alone. Further improvement of CGTase excretion was obtained by co-overexpression of CGTase and SPPase with addition of glycine which has successfully maintained the low ß- galactosidase level at 0.63 U/ml and increased 4.5 fold of CGTase excretion from 14.6 U/ml to 66.1 U/ml, as compared to the co-overexpression without glycine supplementation. The present results indicated that higher CGTase excretion with low cell lysis can be obtained by alteration of amino acids in the h-region signal peptide along with glycine supplementation and SPPase overexpression. This is the first report that highlights the combination of three approaches; site-saturation mutagenesis of signal peptide, SPPase overexpression and glycine supplementation in overcoming the problems of low secretion level of CGTase and high occurrences of cell lysis