Functional large-scale production of a novel Jonesia sp. xyloglucanase by heterologous secretion from Streptomyces lividans.
The gene encoding a novel xyloglucanase (Xeg) belonging to family 74 glycoside hydrolases was isolated from a Jonesia sp. strain through functional screening in Escherichia coli. The encoded xyloglucanase is a protein of 972 aminoacyl residues with a 23 residue aminoterminal signal peptide. Over-expression of Xeg in B. subtilis or E. coli failed. In contrast, Xeg was successfully over-expressed and secreted in Streptomyces lividans TK24. To this end Xeg was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (vsi) from Streptomyces venezuelae. The native Xeg signal peptide derived from Jonesia sp. is only poorly functional in S. lividans. Under optimal growth conditions, significant amounts of mature Xeg (100-150 mg/l) are secreted in the spent growth media. A protocol to rapidly purify Xeg to homogeneity from culture supernatants was developed. Biophysical and biochemical analyses indicate that the enzyme is intact, stable and fully functional. Xeg is the longest heterologous polypeptide shown to be secreted from S. lividans. This study further validates use of S. lividans for production of active heterologous proteins and demonstrates that heterologous polypeptides of up to 100 kDa are also tractable by this system.
Sianidis
Giorgos
G
Institute of Molecular Biology and Biotechnology-FORTH and Department of Biology, University of Crete, P.O. Box 1527, Iraklio-Crete 71110, Greece.
Pozidis
Charalambos
C
Becker
Fiona
F
Vrancken
Kristof
K
Sjoeholm
Carsten
C
Karamanou
Spyridoula
S
Takamiya-Wik
Monika
M
van Mellaert
Lieve
L
Schaefer
Thomas
T
Anné
Jozef
J
Economou
Anastassios
A
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase