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

反刍动物对粗饲料的有效利用是提高动物生产能力的关键。我们试图通过重组DNA技术培育一种具有高溶纤维活性的瘤胃细菌。本研究旨在从白色瘤胃球菌和反刍真杆菌中分别获得更多表达纤维素酶和木聚糖酶基因的溶纤维丁酸弧菌和溶纤维丁酸弧菌。实验结果如下：1。利用新建立的溶纤维杆菌与大肠杆菌之间的穿梭载体，通过电穿孔法获得了含有白杆菌纤维素酶基因或反刍杆菌木聚糖酶基因的阳性重组体。2. 外源木聚糖酶基因重组菌的木聚糖酶活性比亲本溶纤维双歧杆菌高9-11倍。然而，外源纤维素酶基因的重组没有显示出纤维素酶活性的增加，这表明纤维素酶基因启动子在溶纤维双歧杆菌中不起作用。3. 当该纤维素酶基因的启动子被红霉素耐药基因的启动子取代时，该基因在溶纤维杆菌中成功表达。重组菌株的纤维素酶活性是亲本菌株的2倍，Western分析结果为阳性信号，证实纤维素酶基因在溶纤维杆菌中表达。4. 当该纤维素酶基因的一个信号肽编码区被删除后，纤维素酶的活性增加了3倍，同时酶的性质如最适温度和ph也得到了改变。我们已经成功地通过纤维素酶和木聚糖酶基因的异种表达，按照最初的计划增强了b.s isolfibr弧菌的纤溶酶活性。此外，我们还证明，如果靶基因在转录中有问题，启动子替换可以有效地成功表达。

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.