In situ bioconversion of compactin to pravastatin by Actinomadura species in fermentation broth of Penicillium citrinum

Article abstract of DOI:10.2478/s11696-013-0323-y

The biocatalytic production of pravastatin from compactin by hydroxylation has found many applications in health care and pharmaceuticals. Actinomadura macra, Actinomadura madurae, and Actinomadura livida can efficiently bioconvert compactin to pravastatin. The fermentation broth (Penicillium citrinum fermented media) harvested on the eighth day contained 388.90 mg L^-1 of compactin and an undetectable level of mycotoxin (citrinin). Bioconversion by A. macra was highest (87 %) in the yeast extract-amended medium. The anti-actinomadura effects of citrinin reduce the bioconversion capacity of Actinomadura. The in situ hydroxylation of compactin produced by P. citrinum represents a preferable alternative for the use of purified compactin, as a way to reduce cost and time processing.

Full text of DOI:10.2478/s11696-013-0323-y

Chemical Papers 67 (6) 667–671 (2013)
DOI: 10.2478/s11696-013-0323-y
SHORT COMMUNICATION
In situ bioconversion of compactin to pravastatin by
species in fermentation broth of
b
b
b
aAjaz Ahmad, Mohd Mujeeb, Rohit Kapoor, Bibhu Prasad Panda*
aDepartment of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh-11451; Saudi Arabia
bMicrobial & Pharmaceutical Biotechnology Laboratory, Centre for Advanced Research in Pharmaceutical Science,
Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
Received 20 August 2012; Revised 28 October 2012; Accepted 9 November 2012
The biocatalytic production of pravastatin from compactin by hydroxylation has found many
applications in health care and pharmaceuticals. Actinomadura macra, Actinomadura madurae, and
Actinomadura livida can efficiently bioconvert compactin to pravastatin. The fermentation broth
(Penicillium citrinum fermented media) harvested on the eighth day contained 388.90 mg L−1 of
compactin and an undetectable level of mycotoxin (citrinin). Bioconversion by A. macra was highest
(87 %) in the yeast extract-amended medium. The anti-actinomadura effects of citrinin reduce
the bioconversion capacity of Actinomadura. The in situ hydroxylation of compactin produced by
P. citrinum represents a preferable alternative for the use of purified compactin, as a way to reduce
cost and time processing.
c
ꢀ 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Actinomadura sp., P. citrinum, compactin, pravasatin, in situ bioconversion
Pravastatin suppresses cholesterol biosynthesis by
inhibiting a 3-hydroxy-3-methyl glutaryl CoA reduc-
tase. Initially, pravastatin was isolated as a metabolite
product of compactin from canine urine. Subsequently,
pravastatin was developed as a new therapeutic agent
for the treatment of hypercholesterolemia (Yamashita
et al., 1985; Shepherd et al., 1995; Barrios-González &
Miranda, 2010). Pravastatin is produced by chemical
synthesis from compactin. However, due to the high
cost and the occurrence of stereoisomers, pravastatin
production by microbial hydroxylation is preferred.
Pravastatin can be produced by a two-step process,
in which the first step is the production of compactin
by an appropriate microbial strain such as Penicillium
citrinum; subsequently, hydroxylation of compactin at
the C-6 position can be performed either by a chemi-
cal method or by a fermentation process (Chen et al.,
2006).
SANK 62585, Streptomyces sp. Y-110, catalysed by
the cytochrome P450 monooxygenase system, or by
Actinomadura sp. ATCC 55678, catalysed by the hy-
droxylase enzyme (Hosobuchi et al., 1993; Peng & De-
main, 2000; Park et al., 2003). To reduce the cost
and time of processing, the present research proposes
use of the entire fermented broth containing com-
pactin rather than the purified compactin. To this end,
a fermented broth containing spent medium, com-
pactin, mycotoxin (citrinin), and Penicillium citrinum
cell fragments were used for testing the bioconversion
of compactin to pravastatin by three Actinomadura
species, namely Actinomadura livida, Actinomadura
macra, and Actinomadura madurae.
Cultures of Penicillium citrinum MTCC 1256,
Actinomadura madurae MTCC 1120, Actinomadura
macra MTCC 2559, and Actinomadura livida MTCC
1382 were obtained from MTCC, IMTECH, India.
P. citrinum was maintained on PDA slants. A. madu-
rae and A. livida were maintained in a growth
medium, A. macra was maintained in a growth
An extensive survey of the literature shows the use
of purified compactin for the production of pravastatin
by actinomycetes such as Streptomyces carbophillus
*Corresponding author, e-mail: bibhu panda31@rediffmail.com

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A. Ahmad et al./Chemical Papers 67 (6) 667–671 (2013)
medium supplemented with 2 g L−1 CaCO3. All mi-
crobial cultures were stored at 4◦C and sub-cultured
every 30 days (Peng & Demain, 2000; Chakravarti &
Sahai, 2002a).
days, and A. livida was incubated at 28◦C for seven
days in an orbital shaker at 110 min−1. Each acti-
nomycetes culture (5 mL) with 6 × 107 CFU (colony-
forming unit) per mL was treated for 12 h with 1 mL of
P. citrinum fermentation broth (14th day). The CFU
of A. madurae, A. macra, and A. livida, as developed
in the growth media, were measured after incubation
for 3 d for A. madurae and A. macra and 7 d for
A. livida.
Spores of P. citrinum (4 × 106 spores per mL) were
suspended in a glycerol–water solution (15 g L−1). The
seed culture was prepared according to the procedure
given by Chakravarti and Sahai (2002b). Fermenta-
tive production of compactin was carried out as per
the method detailed by Ahmad et al. (2011a). Cell sus-
pensions of A. madurae, A. livida, and A. macra were
prepared in a basal medium (glucose 4 g L−1, malt
extract 10 g L−1, and yeast extract 4 g L−1) from ac-
tively growing slants; the medium pH was adjusted to
7.2 with 0.1 M KOH, or 0.1 M HCl. A. macra and
A. livida were incubated at 28◦C for two days, and
A. madurae was incubated at 37◦C for seven days.
The fermented medium containing compactin and
fungal cells (8, 10, 12, and 14 day-old culture) was ul-
trasonicated by a Vibra Cell VCX 130 (Sonics, USA)
at 20 kHz for 5 min and autoclaved at 121◦C for
20 min. Subsequently, the fermentation broth con-
taining depleted medium, compactin, mycotoxin (cit-
rinin), and non-viable Penicillium citrinum cell frag-
ments was used for the bioconversion of compactin
to pravastatin by A. livida, A. macra, and A. madu-
rae. The spent medium containing different concen-
trations of compactin, citrinin and cell fragments was
supplemented with Actinomadura growth-promoting
medium components (dextrose 4 g L−1, malt extract
10 g L−1, CaCO3 2 g L−1, yeast extract 4 g L−1, and
urea 4 g L−1, at pH 7.2). Seed cultures of A. macra,
A. livida, and A. madurae were added (5 vol. %) into
the medium separately. All the bioconversion reactions
were carried out in 250 mL flasks by A. macra and
A. livida at 28◦C and by A. madurae at 37◦C for four
Compactin production in a previously optimised
medium started from the second day onwards with
an initial concentration of 67.59 mg L−1, reaching
a maximum level of 541.41 mg L−1 on the 15th
day and remaining almost constant thereafter. Cit-
rinin biosynthesis in the optimised production me-
dia started from the 9th day with a concentration
of 113.6 mg L−1. The fermentation media (8th day,
10th day, 12th day, 14th day) containing compac−ti1n
(388.90 mg L−1, 419.26 mg L−1, 458.75 mg L
and 522.50 mg L−1) and citrinin (0.00 mg L−1
,
,
126.03 mg L−1, 165.92 mg L−1, and 252.36 mg L−1
)
were used for bioconversion. The amount of pravas-
tatin produced after bioconversion by either A. livida,
A. macra, or A. madurae in a medium containing com-
pactin 388.90 mg L−1 and citrinin 0.00 mg L−1 sup-
plemented with yeast extract (4 g L−1) is represented
in (Fig. 1a) with bioconversion of 87 %, 85 %, and
86 % by A. macra, A. livida, and A. madurae, respec-
tively. The bioconversion percentages of compactin to
pravastatin by A. livida, A. macra, and A. madu-
rae in a medium containing 419.26 mg L−1 of com-
pactin, and 126.03 mg L−1 of citrinin were 78.9 %,
81.12 %, and 81.13 %, respectively (Fig. 1b). The bio-
conversion was highest at 96th h of fermentation in
the yeast extract-supplemented medium. A sharp de-
crease in the percentage of the bioconversion of com-
pactin to pravastatin was observed in the P. citrinum
fermentation medium containing a higher amount of
citrinin, 165.92 mg L−1 harvested on the 12th day of
fermentation with bioconversion of 74 %, 72 %, and
75 % by A. macra, A. livida, and A. madurae, re-
spectively (Fig. 1c). In the media containing citrinin,
252.36 mg L−1 (14th day of fermentation), the bio-
conversion was 67 %, 70 %, and 68 % by A. macra,
A. livida, and A. madurae, respectively (Fig. 1d).
The pravastatin produced was analysed by UPLC-
MS. The retention time (Rt) of the pravastatin pro-
duced and unused compactin exhibited a coincidence
with the Rt of standard pravastatin (0.71 min) and
standard compactin (2.65 min) in Fig. S1 (see sup-
plementary data) with the m/z value of compactin
and pravastatin is 391 and 424 respectively (Fig S2).
The chromatograms of components in the fermenta-
tion broths of A. madurae, A. macra, and A. livida
showed a very similar pattern, confirming that all the
Actinomadura sp. tested were capable of bioconverting
compactin to pravastatin. The percentage of pravas-
tatin production in the medium supplemented with
days, shaking at 210 min−1
.
Citrinin was extracted from the fermentation broth
following the procedure detailed by Jackson and
Ciegler (1978) and quantified by HPTLC (Ahmad et
al., 2010). Compactin and pravastatin were extracted
from the fermentation broth following the procedure
detailed by Chakarvarti and Sahai (2002b) and anal-
ysed by HPTLC (Ahmad et al., 2011b). Liquid chro-
matographic and mass spectrophotometric (LC-MS)
analysis of compactin and pravastatin was carried
out by using an ultra-pressure liquid chromatogra-
phy system (UPLC, Waters, USA) using an AQUITY
UPLCꢀ BEH, C-18, (2.1 mm × 100 mm, 1.7 µm) ana-
lytical column tempered maintained at temperature to
40◦C. Acetonitrile–water (ϕr = 65 : 35) acidified with
0.1 vol. % formic acid at a flow-rate of 0.75 mL min−1
was used as the mobile phase. Detection was carried
out using PDA photodiode array (235 nm) and mass
detectors.
The cultures of A. madurae, A. macra, and
A. livida were grown in a growth medium. A. madu-
rae and A. macra were incubated at 37◦C for three

A. Ahmad et al./Chemical Papers 67 (6) 667–671 (2013)
669
Fig. 1. Pravastatin production by A. madurae ( ), A. macra ( ), and A. livida ( ) in medium supplemented with yeast extract
containing fermentation broth obtained on 8th day (a), 10th day (b), 12th day (c), and 14th (d) day of P. citrinum
cultivation.
urea (4 g L−1) was lower than in the medium supple-
mented with yeast extract, with differences of 6 % for
A. macra, 7 % for A. madurae, and 5 % for A. livida,
respectively.
citrinin biosynthesis was found to be almost zero and
its production started during the second growth phase
of fungus until the idiophase was reached. The produc-
tion of compactin and citrinin is independent of each
other; this may be due to different biosynthetic gene
clusters and/or regulatory genes for compactin and
citrinin.
On the basis of the antibacterial effect of citrinin
(Chan, 2008), the anti-actinomadura activity of the
P. citrinum fermentation broth (14th day) containing
citrinin (252.36 mg L−1) was analysed in terms of the
number of colony-forming units (i.e. CFU) per mL.
There was a decrease from 6 × 107 CFU mL−1 to
7 × 102 CFU mL−1, 2 × 103 CFU mL−1, and 1 × 103
CFU mL−1 for A. madurae, A. livida, and A. macra,
respectively. The inhibitory effect of citrinin was great-
est against A. madurae. The percentage of bioconver-
sion of compactin to pravastatin was lowest in the cul-
ture broth containing maximum citrinin, i.e. in the
14th-day P. citrinum fermentation media.
Pravastatin is a natural HMG Co-A reductase in-
hibitor produced in two-step fermentation processes.
Initially, compactin (mevastatin) is produced by fer-
mentation using P. citrinum, then the purified com-
pactin is bioconverted to pravastatin by hydroxyla-
tion at the C-6 position. Biosynthesis of compactin by
P. citrinum and mycelium formation (glucosamine)
showed a parallel response which is revealed by the
fermentation kinetics. During the log phase growth,
In situ bioconversion of the compactin-containing
fermentation broth was carried out, instead of the use
of purified compactin, which would represent a possi-
bility to reduce cost and time required for processing.
Actinomadura species such as Actinomadura livida,
Actinomadura macra, and Actinomadura madurae
were selected for the bioconversion of compactin to
pravastatin. Bioconversion reaction was previously
achieved by a number of microorganisms, includ-
ing Amycolata autotrophica, Mucor hiemalis, Nor-
cardiaceae, Actinomycesa madurae, Streptomyces car-
bopilus, Streptomyces exfoliates, and Micromonospora.
However, these microbes proved to be highly sensi-
tive towards compactin concentration. The pro-drug
(compactin) of pravastatin is highly toxic to these mi-
croorganisms (Peng & Demain, 2000). However, bio-
conversion of pure compactin is 68 % after 6 days of
fermentation by Pseudonocardia carboxydivorans (Lin
et al., 2011).

670
A. Ahmad et al./Chemical Papers 67 (6) 667–671 (2013)
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Actinomadura growth-promoting medium components
(dextrose 4 g L−1, malt extract 10 g L−1, CaCO3
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Supplementary data
Supplementary data associated with this article
can be found in the online version of this paper (DOI:
10.2478/s11696-013-0323-y).
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