Development of Rhodococcus promoter for utilization in actinomycetes

硝酸盐过程的微降解通过两种酶途径：硝酸盐水解酶（NHase）催化剂将硝酸盐转化为酰胺，其中硝酸盐催化剂将硝酸盐转化为酸和氨。硝酸酶是由ε-caprolactam或isovaleronitrile添加到培养介质的补充剂强有力地诱导的;形成的硝酸酶对应到35-Rhodococcus Rhodochrous J1中的aft soluble protein Io。在另一方面，NHS对培养介质的添加剂有很强的认识;形成的NHS对该菌株中所有可溶蛋白的50%的相应反应。首先，我们修剪了含有硝酸酶基因簇的DNA区域，并在基因簇上游或下游区域构建了几个等离子体，这些等离子体被删除。我们将每个质粒转化为大肠杆菌，并研究了最终转化体中的硝酸酶形成。SDS-PAGE和酶测定由HPLC揭示，红球菌硝酸酶表达系统不能在E中发挥作用。coli.下一步，我们试图调查硝酸酶调节系统是否在其他红球菌活性细胞中活跃或不活跃，并发现了一个功能良好的硝酸酶表达系统，以指示一个超表达载体的发展。我们也为NHase表达式系统提供了类似的结果。更进一步，我们试图构建一个红球菌宿主载体系统，并考虑到这种基因的有用特征。我们从微生物库存中心购买了超过100条红葡萄球菌菌株，并搜索了携带加密塑料的红葡萄球菌菌株。我们找到了三条直线。有一个加密的等离子体，并在使用其等离子体之一的Rhodococcus-E.大肠杆菌穿梭载体的构造中成功

The microbial degradation of nitrites proceeds through two enzymatic pathways : nitrite hydratase (NHase} catalyzes hydration of nitrite to amide, whereas nitrilase catalyzes hydrolysis of nitrite to acid and ammonium. Nitrilase is strongly induced by the addition of ε-caprolactam or isovaleronitrile to the culture medium ; the formed nitrilase corresponds to 35ーIo of aft soluble protein in Rhodococcus rhodochrous J1. On the other hand, NHase is strongly induced by the addition of urea to the culture medium ; the formed NHase corresponds to 50% of all soluble protein in this strain. At first, we trimmed the DNA region containing the nitrilase gene cluster, and constructed several plasmids where the upstream or downstream region of the gene cluster was deleted. We transformed each plasmid into Escherichia coli, and examined the nitrilase formation in the resultant transformants. SDS-PAGE and enzyme assay by HPLC revealed that the Rhodococcus nitrilase expression system was not able to function in E. coli. Next, we tried to investigate whether the nitrilase regulatory system is active or not in other Rhodococcus actinomycetes, and found that the nitrilase expression system functioned, indicating the development of a hyper-expression vector. We also obtained similar results for the NHase expression system. Furtheremore, we tried to construct a Rhodococcus host-vector system, considering the useful characteristics of this genus. We bought more than 100 Rhodococcus strains from microorganism stock centers, and searched for Rhodococcus strains carrying cryptic plasmids. We found three strains. with a cryptic plasmid, and succeeded in the construction of a Rhodococcus-E.coli shuttle-vector using one of their plasmids