Medium-scale preparation of useful metabolites of aromatic compounds via whole-cell fermentation with recombinant organisms

Article abstract of DOI:10.1021/op020013s

The whole-cell fermentation of aromatic coumpounds with Escherichia coli JM109 (pDTG601) on a medium scale (10-15L) produces enantiopure cyclohexadienediols. A detailed procedure for the fermentation is described, and yields for several metabolites are provided. A similar procedure using E. coli JM109 (pDTG602) affords catechols. The dienediols are useful for asymmetric synthesis, and several important targets originating from these metabolites are tabulated.

Full text of DOI:10.1021/op020013s

Organic Process Research & Development 2002, 6, 525−532
Medium-Scale Preparation of Useful Metabolites of Aromatic Compounds via
Whole-Cell Fermentation with Recombinant Organisms
Mary Ann Endoma, Vu P. Bui, Jeff Hansen, and Tomas Hudlicky*
Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611-7200, U.S.A.
Abstract:
The whole-cell fermentation of aromatic coumpounds with
Escherichia coli JM109 (pDTG601) on a medium scale (10-15
L) produces enantiopure cyclohexadienediols. A detailed pro-
cedure for the fermentation is described, and yields for several
metabolites are provided. A similar procedure using E. coli
JM109 (pDTG602) affords catechols. The dienediols are useful
for asymmetric synthesis, and several important targets orig-
Figure 2. Natural degradation pathway of aromatics by
eukaryotic organisms and mutation points for specific enzyme
expression.
inating from these metabolites are tabulated.
the accomplishments from our group as well as those from
several others have been amply reviewed as recently as
1999.5 Many of the metabolites as well as some of the
synthetically useful intermediates derived from them have
become catalog items (see Table 1).
Introduction
More than 35 years ago Gibson et al. described the
isolation and structure determination of an optically active
diol derived from the biooxidation of an aromatic compound
with a blocked mutant of Pseudomonas putida Pp F1.1
Despite this and other reports from his laboratory, the organic
synthesis community waited 20 years before the first
application of these compounds to synthesis appeared in the
literature in the form of the polyphenylene process by ICI2
and the synthesis of racemic pinitol by Ley.3 Our group
pioneered this field in the U.S. by the disclosure in 1988 of
the conversion of toluene to a prostaglandin synthon in just
three steps,4 Figure 1.
Because of the increased interest in these compounds, we
published an Organic Syntheses6 procedure for the small-
scale (shake-flask) preparation of diols by fermentation with
the blocked mutant P. putida 39D. The use of this organism
requires induction with a known substrate, usually toluene
or chlorobenzene. This is an obvious disadvantage when
different substrates are used as the resulting diols require
separation from the diol derived from the inducer. As Gibson
has subsequently developed organisms in which the enzyme
expression is initiated by a nonaromatic promoter on the
plasmid,7 it is more convenient to prepare the metabolites
by whole-cell fermentation with recombinant Escherichia coli
organisms in which the protein synthesis is induced by
isopropylthiogalactose (IPTG). These recombinant organisms
contain the plasmid for expression of either the aromatic
dioxygenases [toluene (TDO), naphthalene (NDO), or bi-
(5) (a) Hudlicky, T.; Gonzalez, D.; Gibson, D. T. Aldrichimica Acta 1999, 32,
35. (b) Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep. 1998, 15, 309. (c)
Hudlicky, T. In Green Chemistry: Frontiers in Benign Chemical Syntheses
and Process; Anastas, P. T., Williamson, T. C., Eds.; Oxford University
Press: Oxford, UK, 1998; Chapter 10, p 166. (d) Hudlicky, T. In Green
Chemistry: Designing Chemistry for the EnVironment; Anastas, P. T.,
Williamson, T. C., Eds.; ACS Symposium Series 626; American Chemical
Society: Washington, DC, 1996; p 180. (e) Hudlicky, T.; Entwistle, D. A.;
Pitzer, K. K.; Thorpe, A. J. Chem. ReV. 1996, 96, 1195. (f) Hudlicky, T.
Chem. ReV. 1996, 96, 3. (g) Hudlicky, T.; Thorpe, A. J. J. Chem. Soc.,
Chem. Commun. 1996, 1993. (h) Hudlicky, T.; Reed, J. W. In AdVances in
Asymmetric Synthesis; Hassner, A., Ed.; JAI Press: Greenwich, CT, 1995;
p 271. Grund, A. D. SIM News 1995, 45, 59. (i) Brown, S. M.; Hudlicky,
T. in Organic Synthesis: Theory and Applications; Hudlicky, T., Ed.; JAI
Press: Greenwich, CT, 1993; Vol. 2, p 113. (j) Carless, H. A. J.
Tetrahedron: Asymmetry, 1992, 3, 795. (k) Sheldrake, G. N. In Chirality
and Industry; Collins, A. N., Sheldrake, G. N., Crosby, J., Eds. John Wiley
and Sons: Ltd., Chichester, UK, 1992; p 127. (l) Widdowson, D. A.;
Ribbons, D. W.; Thomas, S. D. Janssen Chim. Acta 1990, 8, 3.
Figure 1. Historically important isolation and applications of
arene cis-diols in synthesis.
The past decade witnessed an almost explosive growth
of applications of these metabolites to asymmetric synthesiss
* To whom correspondence should be addressed. E-mail: hudlicky@
chem.ufl.edu.
(1) Gibson, D. T.; Koch, J. R.; Schuld, C. L.; Kallio, R. E. Biochemistry 1968,
7, 3795.
(2) (a) Ballard, D. G. H.; Courtis, A.; Shirley, I. M.; Taylor, S. C. J. Chem.
Soc., Chem. Commun. 1983, 954. (b) Ballard, D. G. H.; Courtis, A.; Shirley,
I. M.; Taylor, S. C. Macromolecules 1988, 21, 294.
(3) Ley, S. V.; Sternfeld, F.; Taylor, S. Tetrahedron Lett. 1987, 28, 225.
(4) (a) Hudlicky, T.; Luna, H.; Barbieri, G.; Kwart, L. D. J. Am. Chem. Soc.
1988, 110, 4735. (b) Johnson, C. R.; Penning, T. D. J. Am. Chem. Soc.
1986, 108, 5655. (c) Johnson, C. R.; Penning, T. D. J. Am. Chem. Soc.
1988, 110, 4726.
(6) Hudlicky, T.; Stabile, M. R.; Gibson, D. T.; Whited, G. M. Org. Synth.
1999, 76, 77.
(7) Zylstra, G. J.; Gibson, D. T. J. Biol. Chem. 1989, 264, 14940.
10.1021/op020013s CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/10/2002
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Table 1. List of diol metabolites and intermediates derived from them, available from Aldrich catalog (prices are from 2001
edition)
Aldrich
catalog
number
diol metabolites
price
48,949-2 (1S-cis)-3-bromo-3,5-cyclohexadiene-1,2-diol, 96%
48,950-6 (1S-cis)-3-chloro-3,5-cyclohexadiene-1,2-diol, 98%
1 g $35.00;
5 g $139.00
1 g $31.00;
5 g $130.00
1 g $76.00
1 g $76.00
48,963-- (1S-cis)-3-phenyl-3,5-cyclohexadiene-1,2-diol, 98%
49,032-6 (1R-cis)-1,2-dihydro-1,2-naphthalenediol, 98%
Synthetically useful intermediates prepared from diols
49,035-0 [3aS-(3aR,4R,5R,7aR]-7-bromo-3a,4,5,7a-tetrahydro-2,2-dimethyl-1,3-benzodioxole-4,5-diol, 99%
49,038-5 [3aS-(3aR,4R,5R,7aR]-]-3a,4,5,7a-tetrahydro-2,2-dimethyl-1,3-benzodioxole-4,5-diol, 98%
49,085-7 [3aS-(3aR,5aR,6aR,6bR]-4-bromo-3a,5a,6a,6b-tetrahydro-2,2-dimethyloxireno[e]-1,3-benzodioxole-4,5-diol, 98%
49,088-1 [3aR-(3aR,5aâ,6aâ,6bR]-3a,5a,6a,6b-tetrahydro-2,2-dimethyloxireno[e]-1,3-benzodioxole, 98%
49,340-6 [3aS-(3aR,4R,5â,7aR]-5-azido-7-bromo-3a,4,5,7a-tetrahydro-2,2-dimethyl-1,3-benzodioxol-4-ol, 99%
49,388-0 (3aS,7R,7aS)-7,7a-dihydro-7-hydroxy-2,2-dimethyl-1,3-dibenzodioxol-4(3aH)-one, 98%
500 mg $135.00
500 mg $180.00
500 mg $160.00
500 mg $185.00
500 mg $220.00
500 mg $130.00
500 mg $135.00
500 mg $130.00
49,389-9 (3aR,7R,7aS)-7-(carbobenzyloxyamino)-7,7a-dihydro-2,2-dimethyl-1,3-dibenzodioxol-4(3aH)-one, 98%
49,390-2 (3aR,4S,7R,7aS)-7-(carbobenzyloxyamino)-3a,4,7,7a-tetrahydo-2,2-dimethyl-1,3-benzodioxol-4-ol, 98%
49,391-0 (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-7-(methoxycarbonylamino)-2,2-dimethyl-1,3-benzodioxol-4-ol-4-acetate, 98% 500 mg $130.00
phenyl (BDO)] or the dioxygenases and the corresponding
catechol dehydrogenases that convert either the diols or
aromatics to the corresponding catechols. The mutations of
the natural degradation pathway are shown in Figure 2.
The availability of the organisms for the fermentation now
offers the opportunity to prepare both homochiral diols and
the corresponding catechols, some of which are very difficult
to prepare in traditional ways.8 This contribution reports the
details of medium-scale synthesis of some of the more
common metabolites in the aromatic series.
A detailed description of the 10- and 15-L fermentation
follows, including the handling of metabolites. For a
complete listing of all known metabolites consult ref 5a.
Conclusions
The medium-scale fermentation of aromatics with recom-
binant organisms expressing toluene dioxygenase has been
described in detail. These fermentations yield, in some cases,
100-g quantities of metabolites, enantiopure cis-cyclohexa-
diene diols. A listing of important applications of these
compounds is provided in Table 4 divided into asymmetric
and nonasymmetric synthetic applications. We hope that this
disclosure, together with a detailed procedure, will further
stimulate the growth of this fascinating area.
Results and Discussions
The Organic Syntheses procedure for the use of P. putida
39D is straightforward and can be adapted for practice in a
laboratory not equipped for microbiology. It is useful for
small (<3 g) amounts of self-inducing substrates (toluene,
benzene. and chloro- and bromobenzene). By contrast, the
use of recombinant organisms requires more sophistication
and a relatively costly fermentor. However, the cost and
sophistication of these requirements is offset by the yields
of metabolites and the ease of handling the E. coli strains.
Table 2 lists many of the diols and Table 3 lists many of the
catechols that have been prepared by this procedure with
yields and references to the isolation and characterization
of each. The direct preparation of catechols in one step
represents an important “green” alternative to the sometime
arduous and lengthy chemical synthesis.8,17
Experimental Section
General Overview of Fermentation. The whole-cell
dihydroxylation of aromatic compounds is a three-day
process. On day one the cell cultures and fermentor are
(17) (a) Bui, V. B.; Hansen, T. V.; Stenstrom, Y.; Hudlicky, T.; Ribbons, D. W.
New J. Chem. 2001, 25, 116. (b) Bui, V. B.; Hansen, T. V.; Stenstrom, Y.;
Ribbons, D. W.; Hudlicky, T. J. Chem. Soc., Perkin Trans 1 2000, 1669.
(18) (a) Gonzalez, D.; Martinot, T.; Hudlicky, T. Tetrahedron Lett. 1999, 40,
3077. (b) Hudlicky, T.; Schilling, S.; Rinner, U.; Gonzalez, D.; Martinot,
T. J. Org. Chem. 2002, submitted.
(19) Stabile, M. R.; Hudlicky, T.; Meisels, M. L.; Butora, G.; Gum, A. G.;
Fearnley, S. P.; Thorpe, A. J.; Ellis, M. R. Chirality 1995, 7, 556.
(20) Hudlicky, T.; Boros, E. E.; Boros, C. H. Tetrahedron: Asymmetry 1993, 4,
1365.
(8) Bui, V. P.; Hansen, T. V.; Stenstrom, Y.; Hudlicky, T. Green Chem. 2000,
2, 263.
(21) Jerina, D. M.; Daly, J. W.; Jeffrey, A. M.; Gibson, D. T. Arch. Biochem.
Biophys. 1971, 142, 394.
(9) Boyd, D. R.; Hand, M. V.; Sharma, N. D.; Chima, J.; Dalton, H.; Sheldrake,
G. N. J. Chem. Soc., Chem. Commun. 1991, 1630.
(10) Boyd, D. R.; Dorrity, M. R. J.; Hand, M. V.; Malone, J. F.; Sharma, N. D.;
Dalton, H.; Gray, D. J.; Sheldrake, G. N. J. Am. Chem. Soc. 1991, 113,
666.
(22) Hudlicky, T.; Endoma, M. A. A.; Butora, G. Tetrahedron: Asymmetry 1996,
7, 61.
(23) Gonzalez, D.; Schapiro, V.; Seoane, G.; Hudlicky, T. Tetrahedron:
Asymmetry 1997, 8, 975.
(24) Hudlicky, T.; Bui. V. P.; Hansen, J.; Nguyen, M. Unpublished observations.
(25) Hudlicky, T.; Rulin, F.; Tsunoda, T.; Luna, H.; Andersen, C.; Price, J. D.
Isr. J. Chem. 1991, 31, 229.
(11) Kobal, V. M.; Gibson, D. T.; Davis, R. E.; Garza, A. J. Am. Chem. Soc.
1973, 95, 4420.
(12) Gibson, D. T.; Roberts, R. L. Wells, M. C.; Kobal, V. M. Biochem. Biophys.
Res. Commun. 1973, 50, 211.
(13) Taylor, S. C.; Turnbull, M. D. Eur. Pat. 0253485, ICI plc, 1988.
(14) Novak, B. H.; Hudlicky, T. Tetrahedron: Asymmetry 1999, 10, 2067.
(15) Stabile, M. R.; Hudlicky, T.; Meisels, M. L. Tetrahedron: Asymmetry 1995,
6, 537.
(26) (a) Hudlicky, T.; Olivo, H. F. Tetrahedron Lett. 1991, 32, 6077. (b) Johnson,
C. R.; Ple, P. A.; Su, L.; Heeg, M. J.; Adams, J. P. Synlett 1992, 388.
(27) Mandel, M.; Hudlicky, T.; Kwart, L. D.; Whited, G. M. J. Org. Chem.
1993, 58, 2331.
(28) Hudlicky, T.; Price, J. D.; Rulin, F.; Tsunoda, T. J. Am. Chem. Soc. 1990,
112, 9439.
(16) Hudlicky, T. Endoma, M. A. A.; Butora, G. J. Chem. Soc., Perkin Trans.
1 1996, 2187.
(29) Hudlicky, T.; Pitzer, K. K.; Stabile, M. R.; Thorpe, A. J.; Whited, G. M. J.
Org. Chem. 1996, 61, 4151.
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Table 2. Survey of useful diol metabolites prepared by fermentation
prepared. Preparation of the cell cultures involves making
the necessary solutions and sterilizing all solutions, glass-
ware, and spatulas in an autoclave. Flasks containing
solutions are capped with gauze. All other glassware and
spatulas are capped or wrapped with aluminum foil in
preparation for autoclaving. After sterilization the flasks are
allowed to cool before being inoculated with the cells and
being placed on an orbital shaker. The fermentor is made
ready by adding water and the necessary materials for the
culture medium then sterilizing at 120 °C for 20 min.
On day two the pH of the medium in the fermentor is
adjusted to 6.8, and preculture solutions are transferred to
(36) (a) Hudlicky, T.; Olivo, H. F. J. Am. Chem. Soc. 1992, 114, 9694. (b)
Hudlicky, T.; Olivo, H. F.; McKibben, B. J. Am. Chem. Soc. 1994, 116,
5108.
(30) Banwell, M.; Blakey, S.; Harfoot, G.; Longmore, R. J. Chem. Soc., Perkin
Trans. 1 1998, 3141.
(37) Gonzalez, D.; Shapiro, V.; Seoane, G.; Hudlicky, T.; Abboud, K. J. Org.
Chem. 1997, 62, 1194.
(31) (a) Rouden, J.; Hudlicky, T. J. Chem. Soc., Perkin Trans. 1 1993, 1095.
(b) Hudlicky, T.; Rouden, J.; Luna, H.; Allen, S. J. Am. Chem. Soc. 1994,
116, 5099.
(32) Hudlicky, T.; Natchus, M. J. Org. Chem. 1992, 57, 4740.
(33) Banwell, M. G.; McRae, Kenneth J. Org. Lett. 2000, 2, 3583.
(34) (a) Tian, X.; Hudlicky, T.; Konigsberger, K. J. Am. Chem. Soc. 1995, 117,
3643. (b) Hudlicky, T.; Tian, X.; Konigsberger, K.; Maurya, R.; Rouden,
J.; Fan, B. J. Am. Chem. Soc. 1996, 118, 10752.
(38) Hudlicky, T.; Abboud, K. A.; Entwistle, D. A.; Fan, R.; Maurya, R.; Thorpe,
A. J.; Bolonick, J.; Myers, B. Synthesis 1996, 897.
(39) Banwell, M. G.; McRae, K. J. J. Org. Chem. 2001, 66, 6768.
(40) Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J. Am. Chem. Soc.
1997, 119, 4856.
(41) Banwell, M.; McLeod, M. Chem. Commun. 1998, 1851.
(42) Banwell, M. G.; Darmos, P.; McLeod, M. D.; Hockless, D. C. R. Synlett
1998, 897.
(35) Tian, X.; Maurya, R.; Konigsberger, K.; Hudlicky, T. Synlett 1995, 1125.
(43) Butora, G.; Gum, A. G.; Hudlicky, T.; Abboud, K. A. Synthesis 1998, 275.
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Table 3. Survey of useful catechol metabolites prepared by fermentation
the fermentor. The cells are allowed to grow while being
fed glucose. The amount of dissolved oxygen is monitored
to ensure proper conditions for growth of the cells. When
the amount of cells is sufficient, IPTG (isopropyl â-D-
thiogalactopyranoside) is added to induce the production of
toluene dioxygenase.
On day three the substrate is added, and the transformation
is monitored. When the reaction is complete, the fermentation
(44) Hudlicky, T.; Seoane, G.; Pettus, T. J. Org. Chem. 1989, 54, 4239.
(45) Hudlicky, T.; Boros, C. H. Tetrahedron Lett. 1993, 34, 2557.
(46) Frey, D. A.; Duan, C.; Hudlicky, T. Org. Lett. 1999, 1, 2085.
(47) (a) Butora, G.; Hudlicky, T.; Fearnley, S. P.; Gum, A. G.; Stabile, M. R.;
Abboud, K. Tetrahedron Lett. 1996, 37, 8155. (b) Butora, G.; Hudlicky,
T.; Fearnley, S. P.; Stabile, M. R.; Gum, A. G.; Gonzales, D. Synthesis
1998, 665.
(48) Tran, C. H.; Crout, D. H. G.; Errington, W.; Whited, G. M. Tetrahedron:
Asymmetry 1996, 7, 691.
(49) (a) Davies, I. W.; Senanayake, C. H.; Castonguay, L.; Larsen, R. D.;
Verhoeven, T. R.; Reider, P. J. Tetrahedron Lett. 1995, 36, 7619. (b) Davies,
I. W.; Reider P. J. Chem. Ind. (London) 1996, 412.
(51) Johnson, C. R.; Adams, J. P.; Collins, M. A. J. Chem. Soc., Perkin Trans.
1 1993, 1.
(52) (a) Mandel, M.; Hudlicky, T. J. Chem. Soc., Perkin Trans. 1 1993, 13,
1537. (b) Hudlicky, T.; Restrepo-Sanchez, N.; Kary, P. D.; Jaramillo-Gomez,
L. M. Carbohydr. Res. 2000, 324, 200.
(53) (a) Ensley, B. D.; Ratzkin, B. J.; Osslund, T. D.; Simon, M. J.; Wackett, L.
P.; Gibson D. T. Science 1983, 222, 167. (b)Whited, G. Presented at Biotrans
’95, University of Warwick, 1995.
(50) Johnson, C. R.; Ple, P. A.; Adams, J. P. J. Chem. Soc., Chem. Commun.
1991, 1006.
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Table 4. Use of cis-dienediols in synthesis
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Table 4. (Continued)
a Syntheses in which either the asymmetry of the arene cis-diol has been destroyed or, if present in the product, was introduced by other means to a meso-diol.
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broth is harvested and centrifuged, and then the supernatant
is extracted with ethyl acetate. Removal of solvent from the
combined extracts provides relatively pure diol, which can
be further purified by recrystallization. The residue of cell
material is autoclaved before disposal.
level (optimum at 20%) by increasing the rate of stirring.
Hence, as the OD of the medium increases with time, which
means the density of bacteria increases, the rate of glucose
addition is also increased. Correspondingly, as glucose is
metabolized by the bacteria, the amount of dissolved oxygen
decreases, and to maintain a steady amount of dissolved
oxygen, stirring is increased. The rate of addition of glucose
and stirring rate must be balanced to maintain optimum
conditions for bacterial growth. If glucose is added too
rapidly oxygen consumption will be too large and no
reasonable amount of stirring could adequately compensate.
Also in this case, the addition of glucose is not optimum
because the cells are subjected to excess glucose. Likewise,
if glucose is added too slowly the bacteria will divide too
slowly. When the turbidity or OD of the medium has reached
15 times that of the blank as measured by UV absorbance,
IPTG (80 mg) is added to induce the production of toluene
dioxygenase.
Feeding of Substrate. On day three, the pH of the
fermentor medium is adjusted to 7.0 prior to the addition of
substrate. A sample is taken before the aromatic substrate is
introduced. This sample gives the value of the OD for this
particular fermentation batch. Also, this sample serves as
the blank for the measurement of absorbance of diol
chromophore in the UV-vis region (usually between 265
and 275 nm). The rate of feeding of the substrate mainly
depends on the type of substrate. In the case of bromoben-
zene the rate is 35 g h-1. If the substrate is added too rapidly
cells begin to die. When this occurs foam begins to form.
Samples are taken every 30 min to assess if there is an
increase in the amount of diol produced over time. When
the UV absorbance due to the diol levels off, substrate
addition is stopped, and the broth that contains the diol
metabolite is harvested. In the case of bromobenzene, the
amount of substrate added ranges from 90 to 120 g or about
3 h of biotransformation. The rates of addition for other
substrates are listed in Table 5.
Harvesting of Culture and Metabolite. The pH of the
medium in the fermentor is adjusted to 7.6 after the
biotransformation. The broth is centrifuged at 7000 rpm and
5 °C for 20 min. The supernatant liquid is decanted and saved
for extraction; the residue of cell material is collected and
autoclaved at 120 °C for 20 min prior to disposal.
Isolation of Metabolite. The supernatant liquid is ex-
tracted three times with one-third its volume of ethyl acetate.
The combined organic extracts are dried over Na2SO4,
filtered, and evaporated without heating to dryness to afford
the crude diol, which is essentially pure. Further purification
can be achieved by recrystallization from ethyl acetate (Na2-
CO3-washed)-hexanes mixtures. In the case of bromoben-
zene, typical yields are in the range of 90-120 g of diol for
8-10 L of culture medium.
Medium-Scale Fermentation with E. coli JM 109
(pDTG601)
Plate Preparation. Agar plates consist of bactotryptone
(10 g L-1), yeast extract (5 g L-1), NaCl (5 g L-1), agar (15
g L-1), and ampicillin (200 mg L-1). Previously thawed E.
coli JM 109 (pDTG601)8 cells in cryovials are streaked onto
a plate. The streaked plate is incubated at 35 °C for 2 d.
Single-cell colonies, which have a dark tinge, are chosen
for the preparation of preculture.
Preculture Preparation and Inoculation. A mineral salts
broth (MSB, 600 mL) containing K2HPO4 (9.6 g), KH2PO4
(8.4 g), (NH4)2SO4 (3 g), yeast extract (9 g), glucose (18 g),
and MgSO4‚7 H2O (1.2 g) is divided into two 2.8-L Fernbach
flasks and sterilized. After the broth is cooled to room
temperature, ampicillin (60 mg in 6 mL of autoclaved water)
is added, and each flask is inoculated with a single colony
of E. coli JM 109 (pDTG601) from a fully grown plate.
These preculture flasks are placed in an orbital shaker (35
°C, 150 rpm) for 12 h.
Sterilization of Fermentor. The production medium in
the fermentor consists of an aqueous solution of KH2PO4
(60 g), citric acid (16 g), MgSO4‚7H2O (40 g), trace metal
solution [16 mL: Na2SO4 (1 g L-1), MnSO4 (2 g L-1), ZnCl2
(2 g L-1), CoCl2‚6H2O (2 g L-1), CuSO4‚5H2O (0.3 g L-1),
FeSO4‚7H2O (10 g L-1), pH 1.0], concentrated H2SO4 (9.6
mL), and ferric ammonium citrate (9.6 mL, 270 g L-1). The
fermentor containing these ingredients and approximately 8
L of water is sterilized at 120 °C for 20 min. The fermentor
is allowed to cool to 35 °C, and air is passed through the
fermentor at a flow rate of 3 L/min. The stirrer is set at 500
rpm while the pH is gradually adjusted to 6.8 by means of
automatic addition of ammonium hydroxide. Once the
desired pH is reached, thiamine hydrochloride (2.69 g in 8
mL of autoclaved water) and ampicillin (800 mg in 8 mL of
autoclaved water) are added to the fermentor.
Transfer of Precultures to Fermentor. On day two, the
pre-cultures are transferred to the fermentor, and stirring is
reduced to 300 rpm. A sample is taken from the fermentor
immediately after adding the precultures to serve as a blank
for monitoring the increase in optical density (OD) of the
fermentor medium. UV absorbance at 640 nm (1 to 100
dilution) is measured at 2 h intervals after transfer. During
this time, the dissolved oxygen content gradually decreases
until it reaches a minimum and then sharply increases, which
usually occurs 4 h after the addition of precultures. Glucose
(720 g L-1) is then introduced to the medium. The rate of
glucose addition is increased exponentially as the cells
multiply, and the rate is critical to the growth of the bacteria.
The optimum rate of glucose addition is determined by the
amount of dissolved oxygen in the medium (measured by
oxygen electrode) and rate of stirring. As glucose is added
and the cells grow and divide, more oxygen is consumed.
The amount of dissolved oxygen is maintained at a constant
SAFETY NOTE. The extraction of diol metabolites must
be done with base-washed solvents. Trace amounts of acid,
for example phenols, have been known to autocatalyze a
rapid and extremely exothermic dehydration and aromati-
zation with a serious potential for a flame-up or explosion,
especially on large scales. In one instance ∼80 g of a diol
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Table 5. Rate of substrate addition in g h-1
spontaneously dehydrated with complete obliteration of the
container. The diols are best stored in crystalline form
(extracted and crystallized from acid-free solvents), resus-
pended in the phosphate buffer, and frozen. Extraction and
immediate use is recommended on small- to medium-scale
(1-10 g). It is absolutley essential that all traces of acids
(including phenols) be removed from the diols when these
are in the crystalline and dry state and handled at room
temperature. The diols are stable indefinitely in crystalline
state when acid-free and stored at low temperature.
Medium-Scale Fermentation with E. coli JM109
(pDTG602). The process of generating catechols from the
corresponding arenes using E. coli JM109 (pDTG602)7 is
carried out in similar fashion as described for E. coli JM
109 (pDTG601). The metabolism of substrate is much more
limited in comparison to that of E. coli JM 109 (pDTG601).
In a typical experiment with bromobenzene, the yields are
in the range of 9-12 g of catechol for 8-10 L of culture
medium.
Acknowledgment
We are grateful to Professor David Gibson (U. Iowa) for
his continuing advice and gifts of the organisms. The
generous research support from the National Science Foun-
dation (CHE-9910412), TDC Research, Inc., and TDC
Research Foundation is acknowledged.
Received for review January 24, 2002.
OP020013S
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