Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae.
To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.
Lee
J W
JW
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejin University, Yusong, Taejon, South Korea.
Kang
D O
DO
Kim
B Y
BY
Oh
W K
WK
Mheen
T I
TI
Pyun
Y R
YR
Ahn
J S
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012440:Saccharomyces / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D005087:Glucan 1,4-alpha-Glucosidase; D002480:Cellulase; C019826:carboxymethylcellulase