|Whitehead, Terence - Terry|
Submitted to: Journal Of Applied Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/1995
Publication Date: N/A
Citation: Interpretive Summary: The breakdown and digestion of the fiber portion of feed material by farm animals such as cattle and sheep requires several different enzymes produced by bacteria that live in the rumen (stomach) of these animals. One approach to increase digestion of feed material by farm animals is to introduce genes into these bacteria so that the necessary enzymes are produced. We now report on introduction and expression of a gene in a bacterium from the rumen and the human intestinal tract that encodes an enzyme that is involved in the breakdown of fiber. This is the first report of expression of such a gene in a rumen bacterium. These modified bacteria will now be studied for increased fiber degradation.
Technical Abstract: A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRR12 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 othe 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 DH5a. Most of the xylanase activity 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.