Expression and export of a Ruminococcus albus cellulase in Butyrivibrio fibrisolvens through the use of an alternative gene promoter and signal sequence.
Ruminococcal cellulase (Ruminococcus albus F-40 endoglucanase EgI) was successfully expressed in Butyrivibrio fibrisolvens OB156C, using the erm promoter from pAMbeta1. A newly identified signal peptide coding region of xynA from B. fibrisolvens 49 allowed efficient translocation of the foreign EgI into the extracellular fraction. First, B. fibrisolvens xynA with or without its own putative signal peptide (XynA SP) coding region was cloned into a shuttle vector to transform B. fibrisolvens OB156C. Both plasmids caused a 2- to 2.4-fold increase in xylanase activity. The transformant expressing XynA with the signal peptide showed a significantly higher proportion of activity in the extracellular fraction than the transformant with XynA lacking the signal peptide (75% vs. 19%), demonstrating the significance of XynA SP in the translocation of the expressed enzyme. Second, using the XynA SP coding region, secretion of EgI was attempted in B. fibrisolvens. Since the signal peptide of R. albus EgI did not function in B. fibrisolvens, it was replaced with the XynA SP. A high activity variant of EgI containing the XynA SP was transcribed using the erm promoter, resulting in a 27-fold increase in endoglucanase activity, most of which (>93%) was in the extracellular fraction of the B. fibrisolvens transformant. EgI without the XynA SP was scarcely detected in the extracellular fraction (<10%).
Kobayashi
Yasuo
Y
Graduate School of Agriculture, Hokkaido University, Sapporo, Japan. kyas@anim.agr.hokudai.ac.jp
Taguchi
Hidenori
H
Goto
Takashi N
TN
Koike
Satoshi
S
Ohmiya
Kunio
K
D001426:Bacterial Proteins / Q000096:biosynthesis; D015152:Blotting, Northern; D015153:Blotting, Western; D045856:Butyrivibrio / Q000235:genetics* / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D045857:Ruminococcus / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014990:Xylans; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium