Signal peptide of Aureobasidiumpullulans xylanase: use for extracellular production of a fungal xylanase by Escherichia coli

• Published in: Journal of industrial microbiology & biotechnology Vol. 38; no. 8; pp. 967 - 973
• Main Authors: Ohta, Kazuyoshi, Tanaka, Hidenori, Yamakawa, Daisuke, Hamasuna, Hironori, Fujimoto, Hirohisa
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 2011
• Subjects: Biochemistry; Bioinformatics; Biomedical and Life Sciences; Biotechnology; Genetic Engineering; Inorganic Chemistry; Life Sciences; Microbiology; Original Paper; Xylanase; Signal peptide
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-010-0868-5

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli . The A . pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum . The gene fusion xynI :: A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β- d -thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.