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work, we tested whether it is able to grow on the predicted carbon source (D-fructose),
but the observed growth was very weak (only very small colonies grew on agar plates
after 3 days). This suggests that growth might be too inhibited by this strategy for it
to be of great use.
Table 5.
Summary of the characteristics of the
in silico
strains generated in the procedure of
optimization of the PHA production.
Blocked Enzymatic
Activity
Loci To Be
Blocked
AcCoA Production
[mmol gow
-1
.h
-1
]
Growth Yield
[gow·mol
c
-1
]
Strain
Carbon Source(s)
Min
Max
Limit
Sim
WT
WT
WT
L
-Serine
11.47
22.26
0.83
11.16
1
Triose-phosphate
isomerase
PP4715
D
-Fructose
7.7
29.74
0.83
3.5
6-Phosphoglucono
lactonase
PP1023
2
Glucose dehydroge-
nase (membrane)
PP1444
D
-Giucose
7.05
28.51
0.83
4.17
6-Phosphoglucono
lactonase
PP1023
3
lsocitrate dehydro-
genase
PP4011 or
PP4012
L
-Serine
22.41
23.01
6.66
10.67
Formate dehydro-
genase
PP0490 or
PP0491
PP2183 or
PP21 84 or
PP2185 or
PP2186
0.83
1.00
4
Citrate synthase
PP4194
L
-V aline
21.85
3.33
4.00
2-Methylcitrate
dehydratase
PP2338
5
Glycine hydroxym-
ethyl transferase
PP0322
L
-Leucine,
L
-
lysine,
L
-phenyl-
alanine
16.75
PP0671
Citrate synthase
PP4194
6
Glycine hydroxym-
ethyl transferase
PP0322
L
-Leucine,
L
-
isoleucine
9.35
6.66
9.33
PP0671
Citrate synthase
PP4194
One strategy suggested by the AcCoA pooling method (strategy 4) called for
knocking out 2-methylcitrate dehydratase (prpD/PP2338) and citrate synthase (gltA/
PP4194), and supplying
P. putida
with valine. Using this strategy, AcCoA pooling
could theoretically reach 21.9 mmolg
DW
−1
h
−1
, but at a severe expense in bacterial
growth (Figure 6B). The other strategies suggested by the AcCoA pooling method
highlight a somewhat linear tradeoff between growth and AcCoA pooling, which
could be investigated experimentally to determine how much growth disruption is ac-
ceptable in a bioengineered production strain of
P. putida
(Figure 6B).
