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

采用新开发的转化系统进行高度纤维溶解瘤胃细菌的分子育种。

基本信息

批准号：
07660377
负责人：
KOBAYASHI Yasuo
金额：
$ 1.15万
依托单位：
MIE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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.

反刍动物粗饲料的高效利用是提高动物生产力的关键。我们尝试通过重组DNA技术培育出具有高纤维溶解活性的新瘤胃细菌。本研究的目的是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纤维素分解和木聚糖分解纤维溶解丁酸弧菌。结果如下： 1.用新开发的溶纤维芽孢杆菌和大肠杆菌之间的穿梭载体，通过电穿孔获得了含有R.albus纤维素酶或E.ruminantium木聚糖酶基因的阳性重组体。 2. 外源木聚糖酶基因重组后的木聚糖酶活性比亲本解纤维芽孢杆菌高9-11倍。然而，用外源纤维素酶基因重组并没有显示出纤维素酶活性的任何增加，这表明纤维素酶基因启动子在溶纤维芽孢杆菌中没有功能。 3.当该纤维素酶基因的启动子被作为筛选标记基因的红霉素抗性基因的启动子取代时，观察到该纤维素酶基因在溶纤维芽孢杆菌中成功表达。该重组体的纤维素酶活性是亲本菌株的2倍，并且在Western分析中也显示出阳性信号，证实了纤维素酶基因在溶纤维芽孢杆菌中表达。 4. 当从该纤维素酶基因中删除信号肽编码区时，纤维素酶活性进一步增加 (x3)，同时酶特性如最适温度和 pH 值也发生改变。我们成功地通过异源增强 B.fibrisolvens 的纤维溶解酶活性纤维素酶和木聚糖酶基因的表达按最初计划进行。此外，我们还证明，如果靶基因的转录出现问题，启动子替换对于成功表达是有效的。