In this work we demonstrated the effectiveness of implanting a promoter to enhance degradation activity. The constructed strains expressed the bph operon at high levels not only in the absence of the pathway substrate but also in the presence of a repressive carbon source. We also demonstrated that a certain level of expression yields the highest degradation performance.
Transcription of the genes that encode a metabolic pathway is thought to be one of the bottlenecks which limit pollutant removal. In many cases, transcription of the metabolic genes is under the control of regulatory mechanisms and active transcription requires a sufficient concentration of cognate effector molecule(s). Moreover, transcription of the degradative genes is often under the control of catabolite repression that represses transcription in the presence of a favorable carbon source where the microorganism can grow rapidly (6
). The transcription of degradative genes is an important step in catabolism and hence a significant event if we are to use the catabolic potential. Thus, transcription is a good target in efforts to improve the efficiency of degradation.
Strains KH967, KH968, and KH981 showed different BphD activities (Fig. ), while they showed the same level of biphenyl-degrading activity (Fig. ). This indicates that transcription was no longer a rate-limiting step for biphenyl degradation in these strains. Presumably, other steps such as supplying the oxygen and/or NADH necessary for degradation, bioavailability of the substrates (28
), and translation efficiency could now be rate-limiting factors. It could be postulated that the ptac
promoter (KH968) was strong enough and that promoters with higher activity might not be suitable, because their use could lead to, for example, energy expenditure and instability of the genetic information. Although the reason was not clear, the presence of biphenyl at the onset of cultivation impaired the growth of strain KH967, which showed the highest BphD activity (data not shown). These observations suggest that the use of a strong promoter does not always give the desirable strain and that trials of different promoters are necessary to optimize degradation.
Implanting of a constitutive promoter was demonstrated to be effective for the creation of microorganisms with high degradation activity. This strategy is practical for a number of reasons. First, the strategy could be applicable to a wide variety of microorganisms relevant for biodegradation without needing to know how the transcription of the degradative operon is regulated. Second, the DNA element on a chromosome would be maintained more stably than on a plasmid and seems to be less likely to transfer horizontally (5
), reducing the risk of dispersal of human-derived genetic information into nature. Third, implanting leads to constitutive expression. Thus, we do not have to add inducing chemicals or take catabolite control into consideration to maintain the expression level (Fig. ) (6
). This advantage is pronounced in the degradation of PCBs that are made up of various isomers and not likely to induce bph
genes effectively. Fourth, overexpression leads to the degradation of some PCB isomers that are hardly degraded by the wild-type strain; i.e., overexpression has the potential to expand the range of substrates. Although the reasons for the apparent expansion of substrate specificity are unclear, it could be speculated that rapid conversion of PCBs or PCB degradation intermediates reduces their toxic effect on cell viability. Finally, constitutive expression of the entire operon would accelerate each step of mineralization; therefore, the target compound would be mineralized more completely, preventing the accumulation of intermediates. The accumulation of PCB degradation intermediates could cause undesirable effects. The 3-chlorinated, 4-chlorinated, and 4-hydroxy HOPDAs, which were formed from PCB degradation, inhibited BphD enzyme from Burkholderia cepacia
strain LB400 (25
). Recently, intermediate metabolites resulting from the degradation of PCBs were shown to inhibit cell separation of Comamonas testosteroni
strain TK102 (12
This approach is applicable, provided that the host strain is subject to homologous recombination, to any genes or operons to improve biological functions in the original strain. We expect that a combination of promoter implanting and the many genetic techniques developed thus far will contribute to the effective and rapid remediation of polluted environments.