Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum.
The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.
Kikuchi
Yoshimi
Y
Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki, 210-8681, Japan. yoshimi_kikuchi@ajinomoto.com
Itaya
Hiroshi
H
Date
Masayo
M
Matsui
Kazuhiko
K
Wu
Long-Fei
LF
D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005972:Glutaminase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D020543:Proteome / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D020543:Proteome; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D005972:Glutaminase