Development of Rhodococcus promoter for utilization in actinomycetes

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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