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most notable that the ED pathway flux was highly activated under
the PHB-producing condition. The fluxes of the pentose phosphate
(PP) pathway and cellular material synthesis were not greatly affected
when the PHB content was 49% but were significantly decreased by
more than 90% when the PHB content was 78%. It has been generally
accepted that the ED pathway is not functional in
E. coli
when glucose
is used as a carbon source. However, the in silico simulation studies
suggested that the ED pathway is important for PHB production from
glucose in recombinant
E. coli
. Since the in silico prediction cannot
be trusted 100%, the roles of the ED pathway on PHB biosynthesis
in recombinant
E. coli
were examined experimentally.
Step 4: Experimental validation.
To verify the importance of the ED
pathway on PHB production in
E. coli
as suggested by in silico sim-
ulation, actual experiments including gene manipulation, cultivation,
and proteome analysis were performed. The
eda
mutant strain KEDA
and its parent strain KS272 were transformed with pJC4, which harbors
the
A. latus
PHB biosynthesis operon, and were compared for their
ability to produce PHB in LB and MR media containing 20 g/l of glu-
cose at 30
C (table 7.2). The final PHB contents obtained with
the
eda
mutant strain KEDA (pJC4
°
pACYC184) were 61.9 wt% of dry
cell weight in LB medium and 44.0 wt% in MR medium, which
are lower than those (75.9% and 60%, respectively) obtained with
KS272 (pJC4
+
pACYC184). When the activity of Eda was restored by
the coexpression of the
eda
gene in KEDA (pJC4
+
pAC104Eda), the
PHB contents increased back to 72.5 wt% of DCW in LB medium and
64.5 wt% in MR medium.
+
Figure 7.11
Pictorial representation of the intracellular fluxes (mM/g dry
cell weight/h) in recombinant
E. coli
producing PHB versus wild-type
E. coli
not producing PHB; the flux values are given in parentheses for (a)
PHB content of 49%/non-PHB-producing condition, (b) PHB content of
78%/non-PHB-producing condition. Bold arrows indicate the fluxes that
are increased by more than 2-fold, while dotted arrows indicate the fluxes
that are decreased to less than a half. Abbreviations: 2K3D6PG, 2-keto-3-
deoxy-6-phosphogluconate; (R)-3HBCoA, (R)-3-hydroxybutyryl-CoA; 6PG,
6-phosphogluconate; AcetoCoA, acetoacetyl-CoA; AcCoA, acetyl-CoA; CIT,
citrate; T3P2, dihydroxyacetone-phosphate; E4P, erythrose-4-phosphate; F16P,
fructose-1,6-diphosphate; F6P, fructose-6-phosphate; FUM, fumarate; T3P1,
glyceraldehyde-3-phosphate; PHB, poly(3-hydroxybutyrate); ICT, isocitrate;
AKG, a-ketoglutarate; LAC, lactate; MAL, malate; OA, oxaloacetate; PEP,
phosphoenolpyruvate; PYR, pyruvate; R5P, ribose-5-phosphate; RL5P,
ribulose-5-phosphate; SUCC, succinic acid; Suc-CoA, succinyl-CoA; X5P,
xylulose-5-phosphate. Reproduced with permission from Hong et al. [95].
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