Site-saturation mutagenesis of mutant l-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase

• Published in: Journal of industrial microbiology & biotechnology Vol. 44; no. 12; pp. 1627 - 1641
• Main Authors: Ismail, Abbas, Illias, Rosli Md
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.12.2017; Oxford University Press
• Subjects: Amino acids; Asparaginase; Asparaginase - chemistry; Asparaginase - genetics; Asparaginase - metabolism; Biochemistry; Bioinformatics; Biomedical and Life Sciences; Biotechnology; Cell culture; Cell death; Cyclodextrin; Cytoplasm; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Excretion; Genetic Engineering; Genetic recombination; Genetics and Molecular Biology of Industrial Organisms - Original Paper; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - secretion; Hydrophobic and Hydrophilic Interactions; Hydrophobicity; Inorganic Chemistry; L-asparaginase; Life Sciences; Microbiology; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Peptides; Protein folding; Protein Sorting Signals - genetics; Protein Sorting Signals - physiology; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - secretion; Saturation; Saturation mutagenesis; Secretion; Recombinant protein; Mutation; Secretory production; Asparaginase II signal peptide; L-Asparaginase II signal peptide; Escherichia coli
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-017-1980-6

The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.