Biosynthesis of phloroglucinol

Article abstract of DOI:10.1021/ja042340g

Substantial concentrations of phloroglucinol were synthesized by Pseudomonas fluorescens Pf-5 expressing the plasmid-localized phlACBDE gene cluster responsible for biosynthesis of 2,4-diacetylphloroglucinol. Expression in Escherichia coli of a single gene in this cluster, P. fluorescens Pf-5 phlD, led to extracellular accumulation of phloroglucinol. Purification of PhlD to homogeneity afforded an enzyme that catalyzed the conversion of malonyl-CoA into phloroglucinol with K_m = 5.6 μM and k_cat = 10 min^-1. Acetylase and deacetylase activities were observed with the catalyzed interconversions of phloroglucinol, 2-acetylphloroglucinol, and 2,4-diacetylphloroglucinol when phlACB was expressed in E. coli. Beyond the mechanistic implications attendant with the identification of an enzyme that catalyzes the conversion of malonyl-CoA into phloroglucinol, PhlD provides the basis for environmentally benign syntheses of phloroglucinol and resorcinol from glucose. Copyright

Full text of DOI:10.1021/ja042340g

Published on Web 03/26/2005
Biosynthesis of Phloroglucinol
Jihane Achkar,† Mo Xian,† Huimin Zhao,‡ and J. W. Frost*,†
Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824, and Department of Chemical
and Biochemical Engineering, UniVersity of Illinois, 600 South Mathews AVenue, Urbana, Illinois 61801
Received December 20, 2004; E-mail: frostjw@chemistry.msu.edu
Scheme 1a
Phloroglucinol 1 (Scheme 1) is found as a substituent in a variety
of natural products. However, biosynthesis of phloroglucinol 1 as
a free-standing molecule has not been delineated. As part of a search
for such biosynthetic activity, biosynthesis of acetylphloroglucinols
encoded by the phlACBDE gene cluster found in Pseudomonas
fluorescens Pf-5 was examined.1 In addition to 2,4-diacetylphlo-
roglucinol 7 and 2-acetylphloroglucinol 6 (Scheme 1), formation
of phloroglucinol 1 was detected. Subsequent heterologous expres-
sion of phlD led to accumulation of phloroglucinol 1 in Escherichia
coli cultures. PhlD-catalyzed formation of phloroglucinol 1 suggests
an alternative to the previously proposed route for the biosynthesis
of acetylphloroglucinols.2 In addition, PhlD activity expressed by
intact microbes provides the basis for the formulation of new
syntheses (Scheme 2) of phloroglucinol 1 and resorcinol 11.
The condensation of three malonyl-CoA molecules required for
the biosyntheses of phloroglucinol 1 and triacetic acid lactone 3
(Scheme 1) may differ only in the timing of a single decarboxy-
lation. Decarboxylation of the priming malonyl-CoA may lead to
3,5-diketohexanoate 2b (Scheme 1), while retention of the car-
boxylate of the priming malonyl-CoA may lead to 3,5-diketohep-
tanedioate 2a (Scheme 1).11 A stabilized C-4,5 enolate in 2b may
cyclize to triacetic acid lactone 3, while decarboxylation of 2a and
cyclization of a C-6 carbanion may lead to phloroglucinol 1. Tri-
acetic acid lactone 3 has been synthesized by Gerbera hybrida 2-
pyrone synthase,3 mutated BreVibacterium ammoniagenes fatty acid
synthase B,4 and mutated Penicillium patulum 6-methylsalicylic
acid synthase.5 Phloroglucinol was not formed by any of these
enzymes.
a (a) Biosynthesis of acetylphloroglucinols 6 and 7 via phloroglucinol
1. (b) Previously proposed biosynthesis of acetylphloroglucinols 6 and 7.2
(c) Biosynthesis of triacetic acid lactone 3.
Scheme 2a
Prospecting for the biosynthesis of phloroglucinol 1 led to P.
fluorescens Pf-5 and the biosynthesis of 2,4-diacetylphloroglucinol
7 (Scheme 1).1 Acetylphloroglucinol biosynthesis is encoded by a
gene cluster consisting of phlACBD, a protein for product export
encoded by phlE, and a divergently transcribed phlF-encoded
regulator.2 PhlD has been suggested to be involved in the formation
and cyclization of an activated 3,5,7-triketooctanoate 5 (Scheme
1).2 The resulting intermediate 2-acetylphloroglucinol 6 is then
presumably acetylated to form 2,4-diacetylphloroglucinol 7 (Scheme
1).2 Biosynthesis of phloroglucinol 1 is not an activity that has been
assigned to PhlD.
P. fluorescens Pf-5/pME6031 was examined for products that
accumulated in its culture supernatants. In addition to accumulation
of 2,4-diacetylphloroglucinol 7 and 2-acetylphloroglucinol 6,
formation of phloroglucinol 1 was discovered (entry 1, Table 1).
To increase the concentration of biosynthesized phloroglucinols,
P. fluorescens Pf-5 was transformed with pJA2.232, a plasmid
derived from the insertion of the phlACBDE gene cluster into
pME6031. The goal was to evade regulation by genomically
encoded PhlF by presenting multiple copies of the biosynthetic gene
a (a) Na2Cr2O7, H2SO4; (b) Fe, HCl; (c) H2SO4, 108 °C; (d) see ref 4;
(e) Dowex 50 H+, MeOH; (f) Na, MeOH, 185 °C; (g) 12 N HCl; (h) phlD-
expressing microbe; (i) i. H2, Rh on Al2O3, ii. 0.5 M H2SO4, reflux; (j)
SO3, H2SO4; (k) NaOH, 350 °C; (l) HZSM-12, propene; (m) i. O2, ii. H2O2,
iii. H+.
cluster. This approach resulted in large increases in the concentra-
tions of synthesized phloroglucinols 1, 6, and 7 (entry 2 vs entry
1, Table 1).
Further analysis followed from heterologous expression from a
T7 promoter of phlACBDE genes in Escherichia coli (entry 3-7,
Table 1). All E. coli constructs also carried a chromosomal gene1
insert encoding the T7 RNA polymerase. E. coli BL21(DE3)/
pJA3.085, which carried a phlACBDE plasmid insert, synthesized
phloroglucinol 1 and 2-acetylphloroglucinol 6 but no 2,4-di-
acetylphloroglucinol 7 (entry 3, Table 1). The absence of the phlE-
encoded product exporter in E. coli BL21(DE3)/pJA3.156 had only
a modest impact on the concentrations of biosynthesized phloro-
glucinol 1 and 2-acetylphloroglucinol 6 (entry 4, Table 1). Product
formation attendant with heterologous expression of only phlD was
† Michigan State University.
‡ University of Illinois.
9
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J. AM. CHEM. SOC. 2005, 127, 5332-5333
10.1021/ja042340g CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Maximum Concentrations of Phloroglucinol 1,
2-Acetylphloroglucinol 6, and 2,4-Diacetylphloroglucinol 7
Biosynthesized by Constructs Expressing phlACBDE Genes
PhlD is of particular importance in establishing the outline of
new syntheses of phloroglucinol 1 and resorcinol 11 (Scheme 2).
Phloroglucinol is currently synthesized (Scheme 2) from 2,4,6-
trinitrotoluene 8 by a route involving an oxidation utilizing Na2-
Cr2O7.6 Beyond the explosion hazard, environmentally problematic
chromates are generated along with other salts as waste streams
during synthesis of phloroglucinol 1 from 2,4,6-trinitrotoluene 8.
Recently, an alternate route (Scheme 2) to phloroglucinol 1 has
been elaborated involving microbe-catalyzed synthesis of triacetic
acid lactone 3.4 Multiple chemical steps are needed to convert
triacetic acid lactone 3 into phloroglucinol 1 via intermediacy of
the methyl ethers 9 and 10 (Scheme 2).7 In contrast to these
chemical and chemoenzymatic routes to phloroglucinol, heterolo-
gous expression of PhlD in E. coli allows phloroglucinol 1 to be
made in a single microbe-catalyzed step from glucose (Scheme 2).
Resorcinol 11 is currently manufactured (Scheme 2) by alkali
fusion of 1,3-benzenedisulfonic acid 12 or hydroperoxidation of
1,3-diisopropylbenzene 13.8 Alkali fusion requires high temperatures
and generates large salt waste streams.8 Acetone hydroperoxide
formed during hydroperoxidation is an explosion hazard.8 In
addition, both 1,3-benzenedisulfonic acid 12 and 1,3-diisopropyl-
benzene 13 are produced from petroleum-derived, carcinogenic
benzene (Scheme 2). The new route to resorcinol 11 is based on
the Rh-catalyzed hydrogenation7 (Scheme 2) of microbe-synthesized
phloroglucinol 1. Acid-catalyzed dehydration of the resulting
dihydroresorcinol intermediate leads to resorcinol 11. Since phlo-
roglucinol 1 can now be synthesized from glucose, resorcinol joins
catechol9 and hydroquinone10 as a dihydroxy aromatic that is
amenable to synthesis from nontoxic, plant-derived glucose (Scheme
2).
phloroglucinols
(mg/L)
host/
plasmid
inserts
entry
plasmid
1
6
7
1
P. fluorescens Pf-5/
pME6031a
none
10
23
35
2
3
4
5
6
P. fluorescens Pf-5/
phlACBDE
phlACBDE
phlACBD
phlD
470
32
500
14
13
0
790
0
pJA2.232a
E. coli BL21(DE3)/
pJA3.085b
E. coli BL21(DE3)/
pJA3.156b
E. coli BL21(DE3)/
pJA2.042b
E. coli JWF1(DE3)/
pJA3.131Ac
22
0
720
780
0
phlD
0
0
7a
7b
7c
7d
0b
0b
0b
E. coli BL21(DE3)/
pJA3.169
37d
29e
22f
28d
16e
9f
3d
1e
0f
phlACB
a Cells were cultured in YM medium under shake-flask conditions. b Cells
were cultured under shake-flask conditions in TB medium and harvested.
Following resuspension in M9 minimal salts medium, cells were cultured
under shake-flask conditions. c Cells were cultured in M9 minimal salts
medium under fermentor-controlled conditions. Concentrations of phloro-
d
e
f
glucinols 48 h after addition of 1 (100 mg/L), 6 (100 mg/L), or 7 (100
mg/L) to cells cultured in M9 medium under shake-flask conditions.
then evaluated using E. coli BL21(DE3)/pJA2.042 (entry 5, Table
1). Only phloroglucinol 1 formation was observed. Synthesis of
phloroglucinol 1 from glucose in minimal salts medium under
fermentor-controlled conditions was examined using E. coli JWF1-
(DE3)/pJA3.131A (entry 6, Table 1). Under these culture conditions,
synthesis of phloroglucinol 1 occurred only during the log phase
and not during the stationary phase of growth. Triacetic acid lactone
3 (Scheme 1) was not observed in the culture supernatants in any
of the experiments summarized in Table 1.
PhlD was purified to homogeneity, and its in vitro enzymology
was examined. No activity was observed when acetyl-CoA alone
was employed as a substrate. Approximately equal specific activities
were observed when malonyl-CoA and acetyl-CoA were incubated
with PhlD relative to incubation of PhlD with only malonyl-CoA.
A Km ) 5.6 µM for malonyl-CoA and a kcat ) 10 min-1 were
determined for PhlD. No triacetic acid lactone 3 or 2-acetylphlo-
roglucinol 6 was observed when purified PhlD was incubated with
malonyl-CoA.
The products formed by microbes expressing phlD and during
incubation of purified PhlD with malonyl-CoA suggest that
cyclization of an activated 3,5-diketoheptanedioate 2a (Scheme 1)
leads to phloroglucinol 1. Stepwise acetylation of 1 might then lead
to acetylphloroglucinols 6 and 7 (Scheme 1). No phloroglucinols
were synthesized (entry 7a, Table 8) by E. coli BL21(DE3)/
pJA3.169, which carried plasmid-localized phlACB. However,
addition of phloroglucinol 1 to the culture medium of E. coli BL21-
(DE3)/pJA3.169 led to formation of acetylphloroglucinols 6 and 7
(entry 7b, Table 1). Deacetylase activity was also observed with
the conversion of 2-acetylphloroglucinol 6 into phloroglucinol 1
(entry 7c, Table 1) and the conversion of 2,4-diacetylphloroglucinol
7 into both phloroglucinol 1 and 2-acetylphloroglucinol 6 (entry
7d, Table 1).
Acknowledgment. Research was supported by Grant N00014-
02-1-0725 from the Office of Naval Research, and expedited by
conversations with Professor Joseph P. Noel.
Supporting Information Available: Plasmid maps; strain construc-
tion; culture conditions; enzyme assays; pH optimum for PhlD activity
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
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