Benzene-Free Synthesis of Hydroquinone
J. Am. Chem. Soc., Vol. 123, No. 44, 2001 10929
Table 2. Concentrations and Yields of Quinic Acid and
3-Dehydroquinic Acid Biosynthesized under Glucose-Limited
Culture Conditions
The only metabolite other than quinic acid observed to
accumulate to significant concentrations in the culture super-
natants was 3-dehydroquinic acid. E. coli QP1.1/pKD12.112
synthesized (Table 2) 2.3 g/L of 3-dehydroquinic acid after 60
h while E. coli QP1.1/pKD12.138 synthesized (Table 2, Figure
1) 3.3 g/L of 3-dehydroquinic acid at 48 h. Concentrations of
3-dehydroquinic acid steadily increased in the culture superna-
tant of E. coli QP1.1/pKD12.138 reaching a maximum con-
centration of 7.1 g/L at 24 h (Figure 1). Concentrations of
3-dehydroquinic acid then steadily decreased between 24 and
48 h. This observation raises the intriguing possibility that some
portion of the quinic acid synthesized by E. coli QP1.1/
pKD12.138 was derived by transport back into the cytoplasm
and subsequent reduction of 3-dehydroquinic acid that had been
initially synthesized and exported into the culture supernatant.
Hydroquinone Toxicity. The toxicity of hydroquinone
toward ethanologenic E. coli cultured on xylose under fermenta-
tive conditions has been analyzed from the perspective of
hydroquinone’s inhibition of sugar catabolism and damage to
the plasma membrane.25 To gauge the toxicity of hydroquinone
toward E. coli cultured aerobically on glucose, E. coli QP1.1/
pKL4.33 was used. Because significant amounts of quinic acid
QAc
DHQc
QA yield, %d
total yield, %e
QP1.1
pKD12.112a
QP1.1
pKD12.138b
40
49
2.3
3.3
15
20
16
21
a Cultured for 60 h. b Cultured for 48 h. c Units: g/L of quinic acid
(QA), g/L of 3-dehydroquinic acid (DHQ). d (mol QA)/(mol glucose).
e (mol QA + DHQ)/(mol glucose).
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Figure 1. Biosynthesis of quinic acid under glucose-limited conditions
by E. coli QP1.1/pKD12.138. Legend: quinic acid (QA, 0), 3-dehy-
droquinic acid (DHQ, 9), dry cell weight (b).
E. coli QP1.1/pKD12.112 and E. coli QP1.1/pKD12.138 were
cultured under fermentor-controlled conditions at 33 °C, pH 7.0,
with dissolved oxygen maintained at a set point of 10% air
saturation. Glucose addition was controlled by dissolved O2
concentration with the rate of glucose addition dictated by a
proportional-integral-derivative (PID) control loop. When dis-
solved oxygen levels exceeded the set point value indicating
decreased microbial metabolism, the rate of glucose addition
was increased and conversely the rate of glucose addition was
decreased when dissolved oxygen levels declined below the set
point value indicating increased microbial metabolism. A
proportional gain (Kc) on the glucose PID control loop of 0.1
was used for culturing E. coli QP1.1/pKD12.112 and E. coli
QP1.1/pKD12.138. These conditions maintained a steady-state
concentration of glucose of approximately 0.2 mM.
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E. coli QP1.1/pKD12.112 synthesized (Table 2) 40 g/L of
quinic acid in 15% (mol/mol) yield in 60 h while E. coli QP1.1/
pKD12.138 synthesized (Table 2, Figure 1) 49 g/L of quinic
acid in 20% yield (mol/mol) in 48 h. The concentrations of
hydroaromatics synthesized by QP1.1/pKD12.112 that are listed
in Table 2 significantly differ from a previous report5 and are
the result of using crystalline quinic acid and 3-dehydroquinic
acid standards to determine response factors for 1H NMR
analyses of culture supernatants. Amplified expression of
transketolase clearly had an impact as reflected in the higher
concentrations and yield of quinic acid synthesized from glucose
by E. coli QP1.1/pKD12.138 relative to E. coli QP1.1/
pKD12.112. Overexpression of transketolase also had a pro-
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longer (60 h) periods of time. By contrast, quinic acid
biosynthesis in E. coli QP1.1/pKD12.112 required 60 h of
cultivation before quinic acid synthesis leveled off.
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