Environmental Engineering Reference
In-Depth Information
grown on Luria-broth (LB) medium containing tetracycline were then trans-
ferred onto solidified mineral medium K1 plates with 1.25 and 2.5 m
M
2-CBA
as a sole source of carbon. After an extended 8-week incubation, colonies for
containing plasmid pE43 were fully grown and confirmed for 2-CBA-specific
growth by transfer in liquid K1 + 2-CBA media. 4-CBA-growing recombinant
strain VP44(pPC3) was constructed by introduction of the recombinant plas-
mid pPC3 carrying a 4.36-Kb
fcb
DNA fragment from
A. globiformis
KZT1.
The
fcb
DNA fragment contains structural genes
fcbABC
encoding a denosine
triphosphate (ATP)-dependent coenzyme A (CoA) ligase, hydratase/deha-
logenase, and thioesterase, respectively. Prior results showed that expression
of the
fcb
operon in
E. coli
and
P. putida
results in dechlorination and con-
version of 4-CBA to 4-hydroxybenzoate (4-HBA) and, in the case of
P. putida
,
a recombinant pathway for mineralization of 4-CBA (Plotnikova et al., 1991;
Tsoi et al., 1991). Upon transformation of VP44 with plasmid pPC3, trans-
formants that grew on LB400 medium with tetracycline were transferred to
K1 plates with 4-CBA (5 m
M
) as a sole source of carbon. Transformant
VP44(pPC3) formed fully grown colonies on 4-CBA plates after less than 1
week of incubation.
Batches of recombinant VP44(pE43) and VP44(pPC3) grew efficiently on
respective chlorobenzoates and chlorobiphenyls as a sole source of carbon
and completely mineralized up to 10 m
M
of the chloroaromatics within 2
days, as confirmed optical density, chloride release, substrate disappearance,
and protein yield. Growth of VP44(pPC3) containing the
fcb
genes on 4-CBA
was similar to that of VP44(pE43::
ohb
) on 2-CBA, except for a shorter lag
period, in accordance with plate growth tendencies, perhaps due to higher
toxicity of 2-CBA. Protein yield was proportional to the concentration of
substrate and similar to that for benzoate-grown cultures.
When grown on VP44(pPC3) and VP44(pE43), recombinant strains 2-CB
and 4-CB, respectively, exhibited only transient accumulation of the corre-
sponding chlorobenzoates. Strain 4-CB exhibited rapid growth on 44(pPC3),
with only transient production of yellow color (HOPDA) and 4-CBA (max-
imal accumulation of the 4-CBA was about 50 μ
M
, or about 5% of the original
concentration) in the culture supernatant during log phase, with concomitant
release of inorganic chloride. Growth of strain 2-CB on 44(pE43) was slightly
slower, with a greater accumulation of 2-CBA, 125 μ
M
or 12.5% of original
substrate concentration, which persisted slightly longer.
The parent strain VP44 was capable of growth on low concentrations of
both 2- and 4-monochlorobiphenyls, with accumulation of stoichiometric
amounts of the corresponding chlorobenzoates in the culture supernatant.
Recombinant VP44(pE43) yielded approximately the same amount of protein
per mole of 2-CB, as compared to growth on biphenyl, and released stoichi-
ometric amounts of chloride. It also grew on 4-CB, however, releasing no
chloride and only about half as much protein compared to the growth on
2-CB and biphenyl, with a stoichiometric amount of 4-CBA accumulated.
While recombinant VP44(pPC3) growth on 4-CB was marked by stoichio-
metric chloride release and protein yield comparable to that observed on
