500
Chemistry Letters 2001
Synthesis of Citronellyl Acetate via a Transacetylation to Citronellol from Acetyl Coenzyme A
Produced from Glucose and Acetate in Growing Yeasts
Shinobu Oda,* Takeshi Sugai,† and Hiromichi Ohta†
Technical Research Laboratory, Kansai Paint Co. Ltd., 4-17-1 Higashi-Yawata, Hiratsuka, Kanagawa 254-8562
†Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
Yokohama, Kanagawa 223-8522
(Received Nobember 17, 2000; CL-001045)
A novel coupling system, which is an acetylation system of
primary alcohols with acetyl coenzyme A [acetyl-CoA] formed
via the metabolism of glucose and acetate, was developed with
Hansenula and Pichia. The supplementation of sodium acetate
to glucose as the source of acetyl-CoA was effective to enhance
the reaction rate and yield of acetylation of citronellol.
Three type culture yeasts, H. saturnus IFO 0809, Pichia
quercuum IFO 0949, and P. heedii IFO 10019 having the cou-
pling activity,2 were used. The basal medium (pH 6.0) consist-
ed of 5.0 g of peptone, 3.0 g of malt extract, 3.0 g of yeast
extract, 1.0 g of MgSO4·7H2O, 40.0 g of glucose, 0–50.0 g of
sodium acetate, and 1.0 L of distilled water. Agar powder
(15.0 g) was added to 1.0 L of the medium for the preparation
of agar plates. Three hundred µL of a cell suspension (1 loop-
ful/mL medium) of each strain was spread on the agar plate
prepared in a glass petri dish (surface area, 38.5 cm2; volume,
25 mL), and the plate was incubated at 30 °C for 1 day. Then,
8 mL of a 1% (w/v) solution of citronellol in decane was
added, and incubation was performed at 30 °C by allowing the
dish to stand. The concentrations of citronellyl acetate and cit-
ronellol in the decane layer were directly determined by gas
chromatography: the column (φ, 2.6 mm; length, 3 m) con-
tained Thermon-3000 /Chromosorb W (Chromato Packing
Center, Kyoto); the column temperature was raised from 100
to 240 °C at a rate of 7 °C/min; the carrier gas was N2 (flow
rate, 60 mL/min).
The coupling of transacetylation from acetyl-CoA to pri-
mary alcohols by the aid of alcohol acetyltransferase
[AATFase] and metabolism of glucose to acetyl-CoA is a
unique procedure for the production of various acetic esters
without any acetyl donor (we referred to this system as cou-
pling system).1–3 In this system, a high concentration of glu-
cose in a hydrophilic carrier of an interface bioreactor, which
is a nonaqueous bioreactor using a microorganism growing on
an interface between a hydrophilic carrier and a hydrophobic
organic solvent,4 contributes as the source of acetyl-CoA and
also depresses the unfavorable oxidation of the substrates, pri-
mary alcohols. However, the excess amount of glucose in the
carrier generally leads to decrease of growth rate of cells and
the coupling activity. For example, the activity of Hansenula
saturnus IFO 0809 decreases at over 5% (w/v) of glucose con-
tent.1 Moreover, the production rate of acetyl-CoA via the
metabolism of glucose is limited because of multiple steps of
its pathway. In this study, it is clarified that the supplementa-
tion of sodium acetate to glucose in the carrier is effective to
increase the reaction rate and the yield of the coupling system
(Figure 1).
In spite of strong biotoxicity of citronellol,5 three strains
tested could produce citronellyl acetate from 1% (w/v) of citro-
nellol due to toxicity alleviation effect of the incubation system
(growth on a solid–liquid interface).6 As shown in Figure 2,
adequate supplementation of sodium acetate was effective to
enhance the production rate of citronellyl acetate for all strains.
Especially, as for H. saturnus, supplementation of 3% (w/v) of
sodium acetate led to the most drastic increase. It is easily sup-
posed that the added acetate is readily converted to acetyl-CoA
and works as the acetyl donor in the transacetylation step. On
the other hand, oxidation of citronellol was not observed in any
strains because of repression of the production of citronellol-
oxidizing enzymes by the presence of high content of glucose
(4%, w/v).1–3,7 Thus, glucose must be used as a repressor of the
oxidation of primary alcohols in the coupling system with sodi-
um acetate.
Although the use of sodium acetate is effective as shown
above, the excess addition of sodium acetate inhibited the cou-
pling activity, i.e., the activities of P. quercuum and P. heedii
were reduced at over 4 and 3% (w/v) of sodium acetate, respec-
tively. It is well known that acetate exhibits strong biotoxicity
on many microorganisms. Indeed, while only 20 mM potassium
acetate strongly inhibits the uptake of L-serine into Bacillus
subtilis cells, growth of the cells is completely supressed by 100
mM of the salt, acetate.8 Furthermore, the uptake of phosphate
into Saccharomyces cerevisiae cells is completely inhibited by
the presence of 80 mM acetate because of destruction of a cell
membrane.9 In conclusion, the addition of sodium acetate is
Copyright © 2001 The Chemical Society of Japan
Oda, Shinobu
