Expression of a cloned cellulase/xylanase gene from Prevotella ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola.
A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.
Daniel
A S
AS
Rowett Research Institute, Bucksburn, Aberdeen, UK.
Martin
J
J
Vanat
I
I
Whitehead
T R
TR
Flint
H J
HJ
D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D018720:Prevotella / Q000201:enzymology* / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium