Previously, We constructed a hybrid strain which is capable of mineralizing each components of a benzene, toluene, and p-xylene mixture simultaneously. In hybrid Pseudomonas putida mt-2, dihydrodiols formed from benzene, toluene, p-xylene by toluene dioxygenase in tod pathway could be channeled into the tol pathway by the action of cis-p-toluate dihydrodiol dehydrogenase, consequently leading to complete mineralization of a benzene, toluene, and p-zylene mixture. We called this strain TB103. But this strain had a problem. In order to insert toluene dioxygenase gene in Pseudomonas putida mt-2, we used RSF1010 vector. Stability of this vector in hybrid strain was very low.
To overcome this problem, we used some promoters and some genetic engineering tools. To improve protein expression level, we exchanged promoter of TDO with Pm, Su, and tac promoter And to improve stability of TDO gene, we inserted parB region into RSF1010. With these two method, we experimented BTX cleavage test by hybrid strain. parB region improved the gene stability, but we were not satisfied with this result because in real environmental situation gene stability must be equal to chromosomal gene stability. So, we researched other method. If we could insert TDO gene into chromosome, TDO gene stability was to be equal to chromosomal gene stability. So we insert TDO gene using suicide delivery system. Consequently, we improved gene stability. Next we researched the effects of Promoter of TDO gene. We used pm, su, and tac promoter. When this gene was on the plasmid, the effect of a kind of promoters on BTX cleavage pattern was very low. But, when this gene was on the chromosome, that effect was very large. Maybe,the reasons are gene length, gene orientation etc.
