Molecular Breeding of Highly Fibrolytic Rumen Bacteria with newly developed transformation system.

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Efficient utilization of roughage in ruminants is a key to improve animal productivity. We have attempted to breed a new rumen bacterium having highly fibrolytic activity through recombinant DNA technique. This study was carried out to obtain more cellulolytic and xylanolytic Butyrivibrio fibrisolvens expressing cellulase and xylanase genes from Ruminococcus albus and Eubacterium ruminantium, respectively. Results are as follows : 1. Positive recombinant having gene of R.albus cellulase or E.ruminantium xylanase was obtained via electroporation with a newly developed shuttle vector between B.fibrisolvens and E.coli. 2. Recombinant with foreign xylanase gene showed 9-11 times higher xylanase activity than parental B.fibrisolvens. However, recombinant with foreign cellulase gene did not show any increase in cellulase activity, suggesting that cellulase gene promoter is not functional in B.fibrisolvens. 3. Successful expression of this cellulase gene in B.fibrisolvens was observed when its promoter was replaced by a promoter of erythromycin resistance gene functioning in screening as a marker gene. This recombinant showed 2 times of cellulase activity as compared with parental strain and also positive signal in Western analysis, confirming that cellulase gene is expressed in B.fibrisolvens. 4. When a signal peptide-coding region was deleted from this cellulase gene, cellulase activity was more incresed (x3) together with modified enzyme properties such as optimal temperature and pH.We have succeeded to enhance fibrolytic enzyme activity of B.fibrisolvens through heterologous expression of cellulase and xylanase genes as planned initially. Also, we demonstrated that promoter replacement could be effective for successful expression if target gene has a problem in its transcription.