, 2001, 11(2), 72–73
Microbial transformations of diterpene acids
Aleksei V. Vorob’ev,a Victoria V. Grishko,*a Irina B. Ivshina,b Emma N. Shmidt,a Leonid M. Pokrovskii,a
Maria S. Kuyukinab and Genrikh A. Tolstikova
a N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
630090 Novosibirsk, Russian Federation. Fax: +7 3832 34 4752; e-mail: benzol@nioch.nsc.ru
b Institute of Ecology and Genetics of Microorganisms, Urals Branch of the Russian Academy of Sciences, 614081 Perm,
Russian Federation. Fax: +7 3422 64 6711; e-mail: ivshina@ecology.psu.ru
10.1070/MC2001v011n02ABEH001414
The ability of Rhodococcus bacteria to transform dehydroabietic and isopimaric acids with a high degree of conversion (up to
95%) was detected.
The microbial oxidation of various compounds, including diter-
penoids, is of considerable interest as a key stage in the syn-
thesis of practically valuable compounds because of the high
stereoselectivity of biotransformations and the ability of micro-
organisms to modify chemically non-activated molecular frag-
H
ments.1–3
Dehydroabietic acid 1 and isopimaric acid 2 are naturally
available compounds, and they were selected as the test com-
pounds for two reasons. First, the hydroxylated derivatives of
these acids are interesting as intermediates in the synthesis of
antiviral agents.4–6 Second, the microbial degradation of resin
acids is an essential stage in waste treatment at pulp mills.7 We
decided on nocardiaform actinomycetes, in particular, Rhodo-
coccus bacteria, as potential biocatalysts for the hydroxylation
of terpenoids.
In this study, we tested 276 rhodococci strains [R. erythro-
polis (121†), R. fascians (2), R. “longus” (9), R. opacus (6), R.
rhodochrous (15) and R. ruber (123) maintained in the Region-
al Specialised Collection of Alkanotrophic Microorganisms at
IEGM8] and 39 freshly isolated strains of Rhodococcus sp. for
the ability to transform diterpene acids. Microorganisms were
grown in a mineral medium (pH 6.8–7.0) of the following
composition (g dm–3): KNO3, 1.0; KH2PO4, 1.0; K2HPO4, 1.0;
NaCl, 1.0; MgSO4, 0.2; CaCl2, 0.02; FeCl3,8 0.001; yeast extract,
0.1; and dehydroabietic or isopimaric acid as a carbon source
(dissolved in ethanol), 0.5. In some cases, n-hexadecane (0.5–
1.0 vol.%) was used as a co-substrate. Cultivation was carried
out at 28 °C in a rotary shaker (150 rpm) for 6 to 7 days.
The majority of the tested strains exhibited insignificant trans-
forming activities (no higher than 5%), whereas some rhodococci
were able to transform from 35.3 to 95.4% initial diterpenoids.
Thus, R. erythropolis IEGM 267, R. ruber IEGM 472 and IEGM
474 selectively oxidised dehydroabietic acid, and R. erythropolis
IEGM 192, R. ruber IEGM 457, IEGM 467 and IEGM 468
oxidised isopimaric acid.
It should be noted that the representatives of R. ruber and R.
rhodochrous having an orange-red non-diffusing pigment were
found most active when diterpenoids were used as sole growth
substrates. R. erythropolis strains exhibited the highest diter-
penoid-transforming activity when n-hexadecane was used as a
growth substrate.
The biotransformations of dehydroabietic acid 1 by R. ruber
IEGM 472 and IEGM 474 resulted in the appearance of a
compound with m/z = 328 (12.4 and 14.6%, respectively) in
the reaction mixture (samples were analysed by GC/MS). The
conversion of dehydroabietic acid was 75.6–82.3% (in terms of
a decrease in the concentration of the parent compound). The
1H NMR and mass spectra of the methylated reaction product
were consistent with the published data9 for 7-oxomethyldehydro-
abietate 3a.‡
H
COOH
H
COOH
1
2
R3
H
H
R1
R2
O
H
COOMe
COOMe
R1 + R2 = O, R3 = H
4
3a
3b R1 = R2 = H, R3 = OH
3c R1 = OH, R2 = R3 = H
3d R1 + R2 = O, R3 = OH
R1 = R3 = H, R2 = OH
3e
H
O
H
H
O
COOMe
MeOOC
5
6
7β-hydroxymethyldehydroabietate 3c‡ and 7-oxo-15-hydroxy-
methyldehydroabietate 3d.‡ It should be noted that the auto-
oxidation of methyldehydroabietate resulted in the formation of
7α-hydroxymethyldehydroabietate 3e.12
Rhodococci were found to exhibit high stereoselectivity in
the enzymatic oxidation of isopimaric acid 2. In the transfor-
mation of isopimaric acid by R. ruber IEGM 457, a compound
with m/z = 330 (14%) dominated in the reaction mixture; this
compound was identified as methyl 7-oxoisopimara-8(14),15-
dien-18-oate 4‡ by IR, UV and 1H NMR spectroscopy.13 At
the same time, R. ruber IEGM 467 and IEGM 468 converted
Selected data for 3a: mp 64–66 °C, [a]2D3 +15.1° (c 3.3, CHCl3); lit.,9
‡
mp 65–67 °C, [a]2D3 +7.8° (c 5.2, EtOH).
For 3b: mp 79–81 °C, [a]2D2 +44.7° (c 2.3, CHCl3); lit.,10 mp 82–83 °C,
[a]2D4 +54° (CHCl3).
For 3c: mp 95–96 °C, [a]2D1 +56° (c 1.5, CHCl3); lit.,11 mp 92–93 °C,
[a]2D4 +56° (1% EtOH).
According to our data, at 70% conversion of dehydroabietic
acid by R. erythropolis IEGM 267, the methylated reaction
products contained 15-hydroxymethyldehydroabietate 3b‡ (23.6%)
as the major constituent in a mixture with previously described
minor compounds,9–11 namely, 7-oxomethyldehydroabietate 3a,
For 4: mp 89–90 °C, [a]2D0 –11° (c 0.3, CHCl3); lit.,13 mp 90–92 °C,
[a]2D0 –10° (c 0.2, CHCl3).
For 5: mp 60–61 °C, [a]2D0 +88° (c 3.4, CHCl3); lit.,13 mp 58–59 °C,
[a]2D4 +88° (c 0.34, CHCl3).
IR, UV and 1H NMR spectra of 3a–d, 4 and 5 were consistent with the
corresponding published data.
†
Number of strains is indicated in the brackets.
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