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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.

反刍动物对粗饲料的有效利用是提高动物生产力的关键。本研究尝试通过重组DNA技术培育一株具有高纤维分解活性的瘤胃细菌。本研究旨在从白色瘤胃球菌和反刍真杆菌中获得分别表达纤维素酶和木聚糖酶基因的解纤维丁酸弧菌。研究结果如下：1.通过电穿孔法将重组质粒转化到溶纤B菌和大肠杆菌之间，获得了含有白罗斯酵母纤维素酶基因和反刍兽疫杆菌木聚糖酶基因的重组子。2.外源木聚糖酶基因的重组子的木聚糖酶活性比亲本纤维布氏杆菌高9-11倍。然而，与外源纤维素酶基因重组没有表现出任何纤维素酶活性的增加，这表明纤维素酶基因启动子是没有功能的B.纤维溶剂。3.当该纤维素酶基因的启动子被用作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解纤B.该重组菌株的纤维素酶活性是亲本菌株的2倍，Western分析也显示阳性信号，证实纤维素酶基因在纤维溶剂芽孢杆菌中表达。4.当从该纤维素酶基因中删除信号肽编码区时，纤维素酶活性增加（x3），并且改变了酶的性质，如最适温度和pH。我们已经成功地通过异源表达纤维素酶和木聚糖酶基因来增强解纤B.fibrisolvens的纤维分解酶活性。此外，我们证明了启动子替换可以有效地成功表达，如果目标基因在其转录有问题。