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RESEARCH PAPER New Biotechnology � Volume 32, Number 1 � January 2015
Use of three-carbon chain compounds asbiosynthesis precursors to enhancetacrolimus production in StreptomycestsukubaensisWanda Gajzlerska1, Justyna Kurkowiak2 and Jadwiga Turło1
1Medical University of Warsaw, Department of Drug Technology & Pharmaceutical Biotechnology, Banacha 1a, Warszawa 02-097, Poland2Medical University of Warsaw, Department of Physical Chemistry, Banacha 1a, Warszawa 02-097, Poland
Tacrolimus, a 23-membered polyketide macrolide, is isolated as a metabolite from the whole
fermentation broth of Streptomyces species. This potent immunosuppressive calcineurin inhibitor has
been widely used in various fields of medicine, including transplantology, dermatology and
pharmacotherapy of autoimmune diseases. The current study was focused on optimisation of tacrolimus
biosynthesis by Streptomyces tsukubaensis through the use of culture media supplements. Enrichment of
the fermentation medium with propionic acid, propylene glycol or propanol resulted in a 5.5-, 3.5- and
1.8-fold, respectively, increase in tacrolimus production. The optimal concentration of the precursors
was 0.25% for both propanol and propionic acid and 0.75% for propylene glycol. The mode of action of
each media supplement tested was unique. For instance, propionic acid acted as a tacrolimus
biosynthesis precursor while propylene glycol induced mycelial growth of S. tsukubaensis. Results from
the current study clearly demonstrate that a set of novel culture medium supplements considerably
increased tacrolimus production. Application of such promoters of tacrolimus biosynthesis may lead to a
substantial improvement in the production of tacrolimus by S. tsukubaensis in industrial fermentation
processes.
IntroductionSince first being isolated from a soil sample taken near mountain
Tsukuba in Japan as a product of fermentation of Streptomyces
tsukubaensis in 1984 [1], tacrolimus has attracted the attention of
scientists and clinicians as a potent immunosuppressant with
multiple applications. Tacrolimus (also referred to as FK-506) is
a 23-membered macrocyclic polyketide from the ‘limus’ family of
immunosuppressive macrolides [2]. The term ‘limus family’ refers
to a group of biologically active molecules with characteristic
macrocyclic structure obtained as isolates from Streptomyces sp.
strains or by chemical modification of the leading structure. It
includes tacrolimus (FK-506), sirolimus (rapamycin), everolimus,
biolimus A9, zotarolimus, pimecrolimus, temsirolimus and
ridaforolimus.
Corresponding author: Gajzlerska, W. ([email protected])
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Specifically, FK-506 inhibits calcineurin – a calcium-dependent
serine–threonine phosphatase. In the process of T-cell activation
the level of intracellular calcium increases. Calmoduline binds
calcium ions for activation of calcineurin and this leads to de-
phosphorylation of transcription factor NF-AT (nuclear factor of
activated T-cells). This pathway results in activation of genes
responsible for T-cell activation and genes coding cytokines like
interleukin-2, interleukin 3, interleukin 4, interferon g and tu-
mour necrosis factor (TNF-a).
The complex of tacrolimus and FKBP (FK-506 binding protein)
binds with calcineurin and deactivates its function. Its major
therapeutic effects are exerted by the inhibition of T-cell prolifer-
ation and activation, as well as inhibition of cytokine production
and impairment of T cell-mediated cytotoxicity [3–5]. Due to its
powerful immunosuppressive activity, FK-506 has several applica-
tions in medicine. Research and clinical trials over the past two
decades have been promising for newer applications of tacrolimus,
http://dx.doi.org/10.1016/j.nbt.2014.07.006
1871-6784/� 2014 Elsevier B.V. All rights reserved.
New Biotechnology �Volume 32, Number 1 � January 2015 RESEARCH PAPER
FIGURE 1
The proposed utilisation of three-carbon chain compounds in the biosynthesis of FK-506 (tacrolimus). Methylmalonyl-CoA is derived from propionyl-CoA by
propionyl-CoA carboxylase (PCC). The macrolide ring of FK-506 is formed by cyclisation of a polyketide chain synthesised by PKS over ten elongation cycles using
two malonyl-CoAs, five methylmalonyl-CoAs, two methoxymalonyl-acyl carrier protein (-ACP) and allylmalonyl-CoA extender unit.Modified from Mo SJ and Goranovic D [15,19,24].
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resulting in many clinical indications for this drug. For instance, it
has been licensed as an immunosuppressive drug for use in trans-
plantology, primarily for subsequent immunosuppression and
rescue therapy following solid organ or bone marrow rejection,
but also for pharmacotherapy of numerous autoimmune diseases
[3,5,6]. Currently, for large-scale industrial production, tacrolimus
can only be synthesised and isolated as a metabolite from the
whole fermentation broth of various Streptomyces sp. cultivated in
submerged cultures [7–9]. Due to high production cost and low
yield, the cost of tacrolimus pharmacotherapy is very high [10–12].
Methods to increase the production of FK-506 in different
strains of Streptomyces have been of great interest to scientists
for many years. To date, efforts have included attempts to isolate
mutants that over-produce tacrolimus, as well as the use of meta-
bolic regulation through supplementation of cultivation medium
with various compounds [13–16]. The current study describes
optimisation of tacrolimus biosynthesis in S. tsukubaensis and
development of a novel fermentation process for more efficient
and economical industrial production of tacrolimus. Biosynthesis
of tacrolimus in Streptomyces sp. has been the subject of structural,
enzymological and genetic researches [17–19]. Production of sec-
ondary metabolites in Streptomyces is mainly determined by the
availability of biosynthetic precursors, such as acetyl-CoA and
malonyl-CoA [20]. In the case of tacrolimus, malonyl-CoA and
methylmalonyl-CoA are the main blocks (along with methoxy-
malonyl-acyl carrier protein (-ACP) and allylmalonyl-CoA which
are biosynthesised). Synthesis of tacrolimus in S. tsukubaensis
requires complex type I polyketide synthases (PKSS) and additional
modification enzymes [17,18]. According to literature describing
macrolide biosynthesis [21–23], the macrolide ring is a product of
enzymatic head-to-tail condensation of acetate and propionate,
resembling the fatty acid biosynthesis pathway. Previous studies
on macrolide biosynthesis have shown that shorter chain mole-
cules containing 2, 3 or 4 carbon units, such as acetate or propio-
nate, may be used as precursors for the macrolide ring.
Furthermore, for the biosynthesis of numerous macrolides, such
as candicin, nystatin, amphotericin B and lucensomycin, propio-
nate has been shown to be a more potent precursor [21]. According
to Mo et al. [15,24], extension of the polyketide chain requires
malonyl-CoAs, methylmalonyl-CoA and allylmalonyl-CoA (bio-
synthesised from propionylmalonyl-CoA). Propionate and acetate
play the main role in that process as substrates for the biosynthesis.
When propanol and propionic acid are used as precursors to enrich
fermentation medium, biosynthesis of many macrolides can be
enhanced. Three-carbon chain compounds are directly trans-
formed into malonyl-CoA and accumulate, whereas acetyl needs
to undergo a carboxylation reaction to be converted to propio-
nate. Thus, three-carbon chain compounds appear to be more
effective. Analysis of metabolic flux of tacrolimus biosynthesis
(Fig. 1) reveals great potential to enhance production of FK-506
biosynthesis by using three-carbon chain compounds as precur-
sors for polyketide chain extension units. Glycerol and three-
carbon molecules with free hydroxylic group have been reported
to induce stimulation of various polyene and non-polyene macro-
lides by various mechanisms [15,21,25]. However, to date there
has been limited research on the effect of propanol, propionic
acid and propylene glycol on the biosynthesis of FK-506 in S.
tsukubaensis.
Based on findings from previous studies, we hypothesised that
propanol, propionic acid and propylene glycol would be potent
precursors of tacrolimus biosynthesis. In the current study, we
evaluated FK-506 biosynthesis parameters in submerged cultures
of S. tsukubaensis using three different effectors with a three-carbon
chain as the leading structure: propanol, propylene glycol and
propionic acid.
Materials and methodsMicroorganism, media and inoculum preparationS. tsukubaensis (FERM BP-927) was obtained from the International
Patent Organism Depositary of National Institute of Advanced
Industrial Science and Technology (AIST) in Tsukuba, Ibaraki,
Japan. The S. tsukubaensis strain (FERM BP-927) used in this study
is a wild-type strain with relatively low FK-506 yields of FK-506.
The seed culture was grown in a 500 mL flask containing 200 mL
of basal medium (Malt Extract Broth; Biocorp Polska Co., Warsaw,
Poland) composed of 0.6% (w/v) malt extract, 0.6% glucose, 0.6%
maltose and 0.12% yeast extract. The pH of the medium was
adjusted to 7.3, the optimal value for mycelial growth observed
in previous studies. The pH of the culture broth was measured
using a digital pH meter (intoLab pH Level 1, WWT, Germany).
The fermentation was performed at 308C on a rotary shaker (New
Brunswick Scientific, Edison, NY, USA) at 110 rev/min for 10 days.
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RESEARCH PAPER New Biotechnology � Volume 32, Number 1 � January 2015
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The seed culture used was stored at 48C before the inoculation of
cultivation media.
Propanol, propylene glycol and propionic acid-enriched culturesThe fermentation medium chosen has been previously reported to
promote optimal mycelial growth and tacrolimus production
[26,27]. The cultures were grown in 500 mL flasks containing
200 mL of medium composed of 2% (w/v) maltose, 3% soy pep-
tone, 1% corn steep liquor, 0.05% MgSO4, 0.2% KH2PO4, 0.4%
Na2HPO4 and 0.3% CaCO3 at 308C on a rotary shaker at 110 rev/
min for 10 days.
Culture medium was enriched with multiple concentrations of
either propanol, propylene glycol or propionic acid. The tested
concentrations of each compound were 0, 0.25, 0.5, 0.75 or 1%.
The initial pH of the medium was adjusted to 7.3 with 0.1 N NaOH
solution. Mycelia were grown in 500 mL flasks containing 200 mL
of medium. Fermentation media were autoclaved at 1218C for
20 min, cooled to room temperature and inoculated with 10% (v/
v) of the seed culture. Cultures were incubated at 308C on a rotary
shaker at 110 rev/min for 10 days. Afterwards, the cultivation was
terminated, biomass was filtered and mycelial dry mass and tacro-
limus concentration were determined. The hypothesis regarding
the mechanisms of action of tested compounds was examined by
an alternative method. The amount of tacrolimus synthesised by
1 g of S. tsukubaensis biomass in each cultures was calculated and
expressed in mg/mycelial dry weight in g, isolated from 1 L of
cultivation broth, after 10 days of cultivation. All experiments
were conducted five times to ensure reproducibility.
The medium additives were tested independently. The experi-
ments were conducted at different times. To show the results
clearly and to be able to compare them, the control was cultivated
for each series of the experiments. The obtained results were
normalised – expressed in relation to the control which is assumed
as 100%.
Batch fermentations in a 5-L jar fermenterFermentation medium in a 5-L working volume bioreactor (Biotec,
UK) was inoculated with 10% (v/v) seed culture. Propylene glycol
at 0.25% (v/v) was then added to culture medium. The control
culture was also cultivated. At an initial pH of 7.3, fermentation
was performed at 308C, 2 vvm and 100 rev/min for 150 hours.
Eighty millilitre test samples were collected at regular intervals for
determination of FK-506 concentration and dry biomass weight.
Analytical methodsFor determination of mycelial growth, dry cell weight (DCW) was
monitored. Mycelia were harvested by filtration and dried at 608C.
The dry mass percent of the filtered biomass was determined by the
use of moisture analyzer (Sartorius, Germany).
Preparation of a sample for FK-506 high-performance liquidchromatography (HPLC) detectionExtraction of tacrolimus from the fermentation broth was carried
out according to a modified method previously described by Kino
and Hatanaka [27,28]. FK-506 is produced intracellularly, but is also
excreted into the culture medium. As such, the whole fermentation
broth can be used for effective extraction. Eighty millilitre
test samples (whole fermentation broth) were homogenised in an
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ultrasonic bath for 10 min then extracted with 80 mL acetone while
being stirred for 1 hour. Extracts were filtered through Whatman
No. 5 filter paper, purified by chromatography on silica gel (0.2–
0.5 mm), evaporated to dryness and dissolved in 40 mL methanol.
HPLC tacrolimus determinationFK-506 concentration was determined by RP-HPLC (reversed phase
HPLC) using combined methods of Akashi et al. [29] and Nishi-
kawa et al. [30]. For preparation of external standards, FK-506
standard (Sigma, St. Louis, USA) was used. The calibration curve of
FK-506 was linear within the range of the concentrations mea-
sured. RP-HPLC was carried out using a Shimadzu LC-10AT gradi-
ent system (Shimadzu USA Manufacturing Inc., Calby, Oregon
City, USA) equipped with a UV–vis SPD-10A detector, a SCL-10-A
system controller and a CTO-10AC column oven. The column
used was a RP-18, 5 mm particle size, 25/4-mm Supelcosil DB
(Supelco, Sigma Aldrich Co., Bellefonte, USA). The methanol
extracts obtained as described above were injected into an HPLC
column. The eluent was acetonitrile/water 75:25 (v/v). The tem-
perature was 608C, the injection volume was 20 mL and the flow
rate was set to 1 mL/min. The wavelength of detection was
214 nm. All measurements were performed in triplicate. Tacroli-
mus retention time was 6.8 min. The content of FK-506 was
calculated based on the standard curve.
Statistical analysisA Dixon test was used to test for single outliers (at a 95% confi-
dence level) in the data. All data in groups were first tested for
normality using a Kolmogorov–Smirnov test and results were
confirmed by a Shapiro-Wilk test. To characterise parameters of
interest, the expected value for the mean, minimum value, maxi-
mum value, standard deviation (SD) and relative standard devia-
tion (RSD) were computed. All results graphically presented in this
paper are expressed as the mean of 5 replicates � SD. To compare
the means of two groups, a t-test was applied if the variances of the
two populations were equal. If not, a Cochran–Cox test was
applied. For comparison of the variance between two groups, an
F-test was employed. A P < 0.05 defined statistical significance. All
statistical analyses were conducted in Statistica 10, and graphical
representations were prepared in Microsoft Excel.
ResultsOptimisation of culture medium composition and cultivation
conditions is one of the main methods to enhance the productivi-
ty of a bacterial strain used for biosynthesis of a secondary metab-
olite, such as tacrolimus. Agents, such as precursors or macrolide
structure biosynthesis stimulators, that may significantly increase
productivity need to be identified. For example, propanol and
propionic acid are successfully used in industrial biosynthesis
processes for polyketide antibiotics (e.g., erythromycin).
The effect of three-carbon precursors on biosynthesis oftacrolimus and S. tsukubaensis growthAll tested compounds (propylene glycol, propionic acid and pro-
panol) enhanced strain productivity. Effectiveness and impact on
the mycelial growth differed for compound. The selected cultiva-
tion medium, determined in our previous research to be effective,
provided satisfactory S. tsukubaensis mycelial growth and facilitated
New Biotechnology �Volume 32, Number 1 � January 2015 RESEARCH PAPER
FIGURE 2
The influence of three-carbon chain substrates on mycelial growth andtacrolimus concentration in Streptomyces tsukubaensis cultures. The
relationship between the concentration of medium supplement (propylene
glycol, propionic acid or propanol) and the concentration of tacrolimus (a), S.
tsukubaensis mycelial growth (b) and strain productivity expressed in mg oftacrolimus synthesised by 1 g of mycelial dry mass (c). Data are compared to
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good tacrolimus production. Biomass (DCW – dry cell weight)
accumulation for non-enriched medium was approximately 1.2 g
of mycelial dry weight per L of culture medium, while the concen-
tration of FK-506 in culture broth was approximately 4.7 mg/L. The
presented values are the mean of the results of the three series of
control experiments. The absolute values of FK-506 concentration
were 2.4, 5.0 and 6.7 mg/L. For the biomass accumulation they were
0.49, 1.7 and 1.3 g. The concentration of tacrolimus in the culture
broth was determined in each of the cultures grown in enriched
precursors medium. The results were compared with those obtained
from cultures grown in non-enriched medium (control) (Fig. 2).
Propylene glycolThe addition of propylene glycol to culture medium led to
increased biosynthesis of tacrolimus in the studied strain. It
was noted that the higher the concentration of propylene glycol,
the higher the biosynthesis rate of tacrolimus, reaching its
maximum at a concentration of propylene glycol equal to
0.75% (v/v). The concentration of tacrolimus increased from
2.4 mg/L in non-enriched medium to 9 mg/L in medium con-
taining 0.75% propylene glycol. However, when the concentra-
tion of propylene glycol exceeded 0.75%, FK-506 content in the
fermentation broth decreased (Fig. 2a). For strain growth, similar
trends were observed based on concentration of propylene gly-
col, but no growth inhibition was observed at higher concentra-
tions of propylene glycol. Optimal strain growth over 10 days
was observed in cultures containing an initial propylene glycol
concentration of 0.75% (equivalent to 1.04 g/L), which yielded
more than a twofold increase in tacrolimus production (Fig. 2b).
However, strain growth at 0.75% propylene glycol did not sta-
tistically differ from cultures containing 0.25%, 0.5% and 1%
propylene glycol.
Thus, the results indicate that both tacrolimus biosynthesis and
strain growth can be effectively stimulated by the use of propylene
glycol as a growth promoter. The evidence of strong correlation of
tacrolimus productivity and the strain growth for propylene glycol
is shown in Fig. 3a and is linear. In the following stage of the
studies the correlation of tacrolimus productivity and the strain
growth (productivity per 1 g of DCW) for all three compounds was
examined. The obtained results are presented in a bar graph
(Fig. 2c). There was no evidence that the productivity of 1 g of
S. tsukubaensis is related to the concentration of the supplementa-
tion with propylene glycol. For 0.75% propylene glycol in culture
medium, the observed productivity was 9.65 mg FK-506/g DW and
was significantly higher than in the culture medium containing
0% and 1%. For 0.25% and 0.5% propylene glycol, no statistically
significant difference was observed (P = 0.62 and P = 0.1, respec-
tively). When higher concentrations of propylene glycol were
used, tacrolimus productivity became lower than that of the
control, suggesting that propylene glycol enhances the productiv-
ity of the strain by acting only as a growth promoter. It is highly
probable that S. tsukubaensis uses propylene glycol as an additional
carbon source. Some tacrolimus producing strains of Streptomyces,
including S. tsukubaensis, Streptomyces antimycoticus and Streptomy-
ces hygroscopicus subsp. glebosus, utilise glycerine as a carbon source
[10]. Structural similarity of propylene glycol (1,2-propanediol)
and glycerine (1,2,3-propanetriol) may result in effective utilisa-
tion of propylene glycol by such strains.
Propionic acidThe addition of propionic acid to culture medium also resulted in
higher tacrolimus biosynthesis in S. tsukubaensis. The optimum
concentration of propionic acid was 0.25% (v/v), which led to
more than a 100% increase in tacrolimus concentration compared
to the control. Culture broth contained 12.7 mg/L of tacrolimus,
which was the highest concentration achieved in the entire ex-
periment (P < 0.05, with the exception of 0.75% propionic acid at
P = 0.06). With higher concentrations of propionic acid, FK-506
biosynthesis decreased. However, biosynthesis of tacrolimus in all
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RESEARCH PAPER New Biotechnology � Volume 32, Number 1 � January 2015
A
0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1
Strain Growth [g DW/L]
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10
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[mg/
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FIGURE 3
Correlation between strain growth and tacrolimus productivity. Results are
presented for (a) propylene glycol, (b) propionic acid and (c) propanol.
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propionic acid cultures was more effective than non-enriched
medium (Fig. 2a). Moreover, the presence of propionic acid in
the culture media caused a reduction in biomass (Fig. 2). Statistical
analysis showed a significant difference between 0.25% and 0.5%
propionic acid, but not between 0.25% and 0.75% or 1% propionic
36 www.elsevier.com/locate/nbt
acid (P = 0.09 and P = 0.06, respectively). There is no correlation
between the concentration of propionic acid and a decrease of S.
tsukubaensis mycelial growth, indicating that the mechanism of
strain growth inhibition by propionic acid was independent of
dose (Fig. 2b). The relationship between strain growth and FK-506
productivity was determined to be linear as shown in Fig. 3b,
which suggests that enhanced tacrolimus productivity by propio-
nic acid may be the effect of its function as a precursor for
biosynthesis. The amount of FK-506 synthesised by 1 g of myceli-
um was greatest when propionic acid concentration in the medi-
um was 0.25%, just as in the case of FK-506 biosynthesis and it was
15.9 mg FK-506/g DW, which is statistically significantly higher
than in the culture medium 0%, 0.75% and 1%. For 0.5% pro-
pionic acid, the difference was not statistically significant
(P = 0.088; Fig. 2c). Tacrolimus productivity per 1 g of DCW (cal-
culated in mg of tacrolimus per 1 g of DCW) is decreasing with
higher concentrations of propionic acid. This trend is similar to
that observed for tacrolimus biosynthesis (Fig. 2a). The mass of
tacrolimus synthesised by 1 g of mycelium increased 5.5 times
with 0.25% propionic acid, whereas productivity of the strain
(expressed in mg/L) increased by only 2.5 times, proving further
support that propionic acid is active as a precursor of tacrolimus
biosynthesis. The inhibition of the strain growth observed with
higher concentrations of propionic acid may be due to its antibi-
otic properties. Propionic acid is used, among others, in the food
industry as preservative E280 due to its antibacterial and antifun-
gal activity. The concentrations in which it is active have been
reported to range from 0.1 to 1% [31,32].
PropanolThe addition of propanol to culture medium did not have as great
an effect as the two previously discussed compounds. A consider-
able increase in FK-506 concentration was observed only for
0.25% propanol, while the tacrolimus concentration in culture
broth was determined to be 10.6 mg/L. With higher propanol
concentrations there was no significant difference compared to
the control (Fig. 2a). A similar trend was observed for biomass
production. Maximum strain growth was observed in cultures
enriched with 0.25% propanol (equal to 1.98 g/L), which was
significantly higher than in other concentrations (P < 0.05). The
addition of higher concentrations of propanol resulted in an
inhibitory effect on biomass production independent of dose
(Fig. 2b). The correlation between strain growth and tacrolimus
production in cultures containing propanol was non-linear
(Fig. 3c), suggesting that the influence of propanol on S. tsuku-
baensis and its production of tacrolimus is complex and requires
further studies. Tacrolimus productivity of 1 g of mycelium
reached its maximum with 1% propanol in culture medium,
yielding 20 mg/g DW. Differences in biomass production com-
pared with the control and other cultures were not statistically
significant (Fig. 2c).
As presented in Fig. 3c, there was no particular correlation
between strain growth and tacrolimus biosynthesis upon addition
of propanol. On the basis of these calculations we hypothesised
that the addition of low concentrations (0.25%) of propanol to the
cultivation medium may act as a growth promoter and with this
mechanism enhance tacrolimus biosynthesis. The linear portion
of the graph in Fig. 3c supports this hypothesis.
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0 50 100 150 200Time [h]
FK-5
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L]
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ass p
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[g D
W/L
]
FK-50 6Biomass
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]
FK-50 6
Biomass
FIGURE 4
The time-course fermentation graphs for cell growth and tacrolimus production in medium containing propylene glycol. The graph illustrates tacrolimus
concentration in the culture broth and biomass accumulation upon addition of (a) 0%, (b) 0.25% propylene glycol.
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The effect of propylene glycol on S. tsukubaensis submergedculture: analysis of strain growth and kinetics of tacrolimusbiosynthesisBio-fermenter cultivationPropylene glycol was chosen for use as a precursor in further studies
since it previously demonstrated effective stimulation of tacrolimus
biosynthesis process and the most substantial effect on S. tsuku-
baensis biomass production. The culture medium was enriched with
0.25% (v/v) propylene glycol and compared with the control culti-
vation (Fig. 4a,b). Figure 4 summarises the changes in mycelial
growth and tacrolimus production during the cultivation of S.
tsukubaensis in a 5-L bioreactor. The graphs present a time-course
of tacrolimus concentration in the culture broth and mycelial
biomass. The results indicate that for the culture grown in medium
with 0.25% propylene glycol, the most effective tacrolimus biosyn-
thesis occurred during the logarithmic phase of growth. In the
control culture, the maximum level of FK-506 biosynthesis occurred
during the exponential (logarithmic) phase of growth, approxi-
mately between 10 and 40 hours into the cultivation (Fig. 4a),
whereas this growth phase was extended by an additional 50–
90 hours by supplementation of culture medium with propylene
glycol (Fig. 4b). A significant increase in the concentration of
tacrolimus was observed during the stationary phase of growth
(90–130 hours into the cultivation), indicating that the biosynthesis
process was extended. Tacrolimus concentration increased consid-
erably at the end of the stationary phase and during the lethal phase
of mycelial growth. Cell lysis is probably responsible for the ob-
served decrease in mycelial mass and the increase in tacrolimus
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concentration in the medium as a result of the cell disintegration
and liberation of intracellularly accumulated FK-506.
The addition of propylene glycol to the culture medium accel-
erated strain growth of S. tsukubaensis but also led the culture to
reach its idiophase earlier. Biomass yield was significantly higher
in the culture enriched with propylene glycol compared to the
control. Figure 4a,b illustrates that the addition of propylene
glycol has a specific effect on biomass accumulation where, at
the end of the stationary phase of the strain growth, there is a
significant increase in biomass similar to the end of the logarith-
mic phase. Such observation is probably connected to the uptake
of propylene glycol by S. tsukubaensis cells and a ‘switch’ in carbon
source from maltose to propylene glycol.
The biosynthesis of secondary metabolites (e.g., tacrolimus) in
Streptomyces is dependent on the availability of biosynthetic pre-
cursors which, in addition to co-factors, can be rate-limiting for
secondary metabolism. In a reaction similar to that of fatty acid
condensation, early stages of macrolide biosynthesis require short
chain (2–4 carbon units) compounds for the chain extension reac-
tionofanacylstarterunitbyPKSIenzymes.Assuch,wehypothesised
that exogenous three-carbon chain compounds may enhance tacro-
limus production by acting as a direct precursor. According to
observed results, supplementation of culture medium with each of
the tested as precursors of FK-506 compounds increased the yield of
tacrolimus in the culture broth. All three compounds also had an
effect on strain growth, though the effect was unique for each
compounds. Thus, the mechanism for increased FK-506 biosynthesis
and the mode of action of each of the tested compounds must be
different. Propionic acid and propylene glycol increased the yield of
FK-506. In propylene glycol-enriched cultures, tacrolimus produc-
tivity linearly correlated with strain growth, indicating that propyl-
ene glycol was probably used as an additional carbon source to
promote growth of S. tsukubaensis. Propionic acid effectively stimu-
lated tacrolimus biosynthesis and seemed to be a potent precursor of
the tacrolimus macrolide ring, which is consistent with the mecha-
nism that has been observed in the biosynthesis of other macrolides
[21]. Propionate is directly incorporated as three-carbon units. Pro-
pionyl-CoA (probably as methylmalonyl-CoA) is used together with
acetyl-CoA in the biosynthesis pathway. The influence of propanol
on S. tsukubaensis cultures seemed to be more complex. The enhanc-
ing effect of propanol on FK-506 biosynthesis was the weakest of all
tested media supplements. It is difficult to explain the metabolic
modeofactionatthispoint and moreexperiments arerequired. Also,
the optimal concentration of each media supplement differed. It was
0.75% (v/v) for propylene glycol and propanol, but only 0.25% for
propionic acid to provide a 1.8–5.5-fold increase in tacrolimus
concentration.
In summary, all three-carbon skeleton compounds tested en-
hanced tacrolimus production. Their mechanisms of action
seemed to be unique, with propionic acid acting as a tacrolimus
biosynthesis precursor and propylene glycol specifically promot-
ing mycelial growth of S. tsukubaensis. The obtained results
38 www.elsevier.com/locate/nbt
revealed that for optimisation of tacrolimus biosynthesis the
tested compounds can be used separately. It is highly probable
that because of the different mechanism of action of the tested
compounds they can be mixed and added to the cultivation
medium for potentially even higher productivity of tacrolimus
but this problem will be undertaken in our future studies.
Upon examination of growth kinetics in relation to biosynthesis
of tacrolimus during batch fermentation in bioreactor, the control
culture produced the most tacrolimus during the exponential (log)
phase, whereas biosynthesis was extended into the stationary
phase in cultures supplemented with propylene glycol. Specifical-
ly, the addition of propylene glycol to the culture medium accel-
erated strain growth but also caused that the culture reached its
idiophase earlier. The bioreactor fermentation kinetics indicated
that production of tacrolimus was highest during the logarithmic
phase. Thus, cultivation time of S. tsukubaensis, cultured under the
condition presented in this work, should be no longer than
150 hours since, after this period, FK-506 biosynthesis decreases.
The observed results are promising and indicate that the pro-
posed media supplementation described in this paper and our
previous study [26] may significantly increase tacrolimus biotech-
nological production in submerged cultures of S. tsukubaensis.
Results from the current study can be widely applied to the
biosynthesis process of tacrolimus and may help to optimise the
biotechnological production process. Searching for biosynthesis
precursors as well as optimisation of the biosynthesis process is
important research that, when taken together with research on
regulation of FK-506 biosynthesis by molecular mechanisms, may
contribute to the development of an improved and efficient
industrial fermentation process.
ConclusionsThe addition of 3C precursors, such as propylene glycol, propanol
and propionic acid, to culture medium induces tacrolimus bio-
synthesis in S. tsukubaensis and exerts a specific effect on mycelial
growth of the strain. The proposed precursors of the FK-506
biosynthesis pathway and the compositions of culture medium
may hold promise for use in hyper-production of FK-506 on a large
bioreactor scale. Propanol, propylene glycol and propionic acid
increased the production of tacrolimus by S. tsukubaensis by 1.8–
5.5-fold. The activity of tested compounds, defined as the influ-
ence of the tested medium supplement on S. tsukubaensis produc-
tivity of FK-506 per 1 g of DCW, decreased in the following order:
propionic acid > propylene glycol > propanol. The current study
also demonstrated that the biosynthesis-stimulating mechanism
of 3C agents was not related to stimulation of strain growth,
proving that 3C agents specifically enhance tacrolimus biosynthe-
sis in S. tsukubaensis submerged cultures. Despite structural simi-
larities, the mechanisms of action of the tested compounds are
diverse. Application of findings may lead to improvement of
tacrolimus yield in industrial fermentation processes and reduce
the cost of this clinically important immunosuppressive agent.
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