Environmental Engineering Reference
In-Depth Information
•
Completely deplete targeted congeners
•
Accumulate no intermediates of CB degradation
•
Not funnel produced chlorobenzoates into unproductive or toxic
pathways
Based on the results described above, we proposed that although none
of the investigated bacteria completely met all these criteria, the
ortho-directed strains RHA1 and LB400 catalyzed more efficient oxidation
of a wider range of CBs, with higher yield of chlorobenzoates, than did the
para-directed strains VP44 and NY05. Thus, the strains preferentially oxi-
dizing ortho-chlorinated CB rings are still more suitable for genetically engi-
neering bacteria capable of growth on Aroclor anaerobic dechlorination
products.
6.4.1.2 Conceptual proof of designing PCB growth pathway
In contrast to
in vivo
construction, the use of sequenced and well-charac-
terized genes permits targeting of specific compounds for degradation and
better prediction of expected intermediates and products with minimal
effects on existing cellular metabolism. In a two-phase anaerobic-aerobic
PCB bioremediation scheme, a limited number of ortho-, 2,6-, 2,4′-, and
2,4-chlorobiphenyls are major targets for the aerobic degradation of PCBs
(Quensen et al., 1990a, 1990b). These congeners, upon oxidation by BP
pathway enzymes, produce the respective ortho-, para-, and ortho- +
para-chlorobenzoates. Therefore, introduction of specific ortho- and
para-dechlorination genes into biphenyl-degrading bacteria should result
in growth on, and mineralization of, the targeted PCB congeners by the
recombinant organism. We have published (Hrywna et al., 1999) the first
results on construction of the recombinant variants using the PCB-come-
tabolizing
Comamonas testosteroni
strain VP44 as the host (Figure 6.5). The
dehalogenation genes used were the
fcb
operon for hydrolytic dechlorina-
tion of para-chlorobenzoate and ortho- + para-chlorinated biphenyls, such
as 2-, 2,2′-, 2,4,2′-, 2,4,4′-, which we previously isolated from
Arthrobacter
globiformis
strain KZT1 (Plotnikova et al., 1991; Tsoi et al., 1991), and the
ohb
operon for oxygenolytic dechlorination of ortho-halobenzoate, which
we isolated from
Pseudomonas aeruginosa
strain 142 under the related HSRC
project (Tsoi et al., 1999).
The
ohb
DNA region contains structural genes
ohbAB
encoding small
and large subunits, respectively, of the terminal oxygenase of the three-com-
ponent ortho-halobenzoate 1,2-dioxygenase, potential regulatory gene
ohbR
,
and
ohbC
, the gene of unknown function that overlaps the
ohbB
gene in an
opposite transcriptional direction. Prior results showed that expression of
the
ohb
genes in
Escherichia coli
and
Pseudomonas putida
cells results in dechlo-
rination and transformation of 2-CBA to catechol and, in the case of
P. putida
,
a recombinant pathway for growth on the chlorobenzoate. Plasmid pE43
(Tsoi et al., 1999), composed of the
ohb
operon cloned in a broad-host-range
vector pSP329, was introduced in strain VP44, and resulting transformants
