Thymidine-producing B. helvolum mutant strains were developed by chemical mutagenesis. In addition to chemical mutagenesis, the transformation of a foreign gene was needed in order to develop the higher thymidine-producing strain. In this study, the following experiments were carried out in order to solve few bottlenecks in developing a efficient thymidine-producing strain.
First, it was very difficult to isolate the transformed colonies because of the restriction system of the host cells. This bottleneck was solved by construction of B. helvolum R66 $(R^-M^+)$. B. helvolum R66 was the useful strain that foreign DNA could not be degraded by host restriction system but be methylated at the same pattern as the that of B. helvolum spp. The transformation system with the efficiency, was set up by using B. helvolum R66 as a mediator of gene transfer.
Second, analogue resistant mutants of B. helvolum constructed by chemical mutagenesis, showed the resistance to kanamycin that was used as selectable marker for gene transfer. To solve this problem, modified expression vector, pECEx1, was constructed. pECEx1 has $Cm^r$ gene under the control of innate promoter from B. helvolum, instead of $Kn^r$ gene. The transformants harboring pECEx1 vector showed to be resistant for about $30\mu g/ml$ of chloramphenicol.
Thus, Brevibacterium helvolum $R66(R^-M^+)$ and pECEx1 which were constructed in this study could be useful for the genetic manipulation of Brevibacterium spp.
