Note: Descriptions are shown in the official language in which they were submitted.
2~
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OPTIMIZED NUTRIENT CONDITIONS FOR PRODUCING
MICROORGANISM; SECONDARY METABOLITES
_ . .
FIELD OF THE INVENTION
This invention relates to the culture of selected
fungi to enhance the production of secondary metabolites.
BACKGROUND OF THE INVENTION
In the field of biotechnology, a variety of fungi are
cultured in numerous types of media to produce secondary
metabolites having beneficial properties particularly in
the field of health care, for example, antibiotics.
Secondary metabolites are diverse natural products which
appear to be unnecessary for the growth of the organism
producing them. Metabolites have a restricted taxonomic
distribution with respect to microorganisms. They are
most prevalent among bacteria such as actinomycetes and
also among filamentous fungi.
A secondary metabolite, which has become very
important in the field of organ transplants, is
cyclosporin. This medicine is a secondary metabolite
derived from the aerobic cultures of the fungus
Tolypocladium inflatum Gams. Since the discovery of the
i~muno suppressi~e properties of this metabolite,
cyclosporin has become a very valuable medicine where its
use lies in the area of transplants, auto-immune disorders
and parasitology. The discovery of cyclosporin type A
antiparasitic activity has taken on worldwide significance
in view of the discovery of its effectiveness against
schistosomiasis and maleria which is thought to affect
over one billion people worldwide. A variety of types of
cyclosporins are produced by the culture of T. inflatum
under proper conditions. The cyclosporins are typified by
the initials CsA, CsB, CsC, CsD, CsE, CsF, CsG and CsH. A
summary of these cyclosporins are found in Kobel and
Traber (1982) Directed BiosY ~ losporins, Eur.
J. Appl. Microbiol. Biotechnol. 14, 237.
A number of publications disclose media for use in
culturing T. inflatum to optimize the production of the
cyclosporin metabolites. The disclosed media contain
sources of carbon, nitrogen, phosphate and trace elements
2 129Z9~2
to enhance culture. A typical media contains glucose,
sodium caseinate, ammonium phosphate, magnesium sulphate,
hydrate, potassium dihydrogenphosphate, sodium nitrate,
potassium chloride, iron sulphate hydrate in distilled
water. It has been discovered that to direct the
biosynthesis of various cyclosporins, for example A, B, C,
D and G, the media may be supplemented with the amino acid
which distinguishes cyclosporins B, C, D, and G from
cyclosporin A. It is generally understood that acceptable
production rates of cyclosporins by culturing T. inflatum
results in a volumetric production (maximum~ in the range
of 1.63 mg/L/h. The fungus is cultured in the media
normally for a period of approximately 12 to 14 days,
beyond which time production of the secondary metabolites
falls off considerably.
SUMMARY OF THE INVENTION
According to an aspect of the invention, a process
for increasing the production of secondary metabolites
during culture of a selected fungi comprises initiating
culture of the selected fungi in a media containing
assimilable sources of carbon, nitrogen and phospha~e.
The selected fungi is cultured in the media through its
growth phases to produce the secondary metabolites. The
culture is pulse fed during secondary metabolite
production from the selected fungi by periodically
supplementing the media with additional source of carbon
to enhance thereby continued production of the secondary
metabolites.
BRIEF DESCRIPTION OF THE DRAWINGS
.
Preferred embodiments of the invention are shown in
the drawings, wherein:
Figure 1 represents kinetic data from a typical
fermentation of T. inflatum;
Figure 2 represents production of CsA by T. inflatum
(wild type) cultured in sorbose medium supplemented with
maltose or citric acid;
Figure 3 represents kinetic data in production of CsA
and CsC in a maltose medium with supplementation of the
medium with additional maltose;
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Figure 4 represents kinetic data in the production of
CsA and CsC in an initial maltose medium with
supplementation with sorbose;
Figure 5 represents kinetic data for the production
of CsA and CsC with an initial sorbose medium supplemented
with sorbose; and
Figure 6 represents kinetic data for the production `
of CsA and CsC with an initial sorbose medium supplemented
with maltose.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Figure 1 is typical of ~he kinetic data derived from
the fermentation of T. inflatum (wild typel available from
the American Type Culture Collection under deposition
number 34921. The medium employed is that identified in
the above publication and reproduced in following Table l
except the suggested glucose was replaced by sorbose at 4%
weight per volume as the carbon source.
TABLE 1
~
Amount
Medium Component (g/L dist. water)
.
Standard
Glucose (C source) 50.0
3acto-peptone 10.0
KH2P4 5 . O
KCl 2.5
Cyclosporin A was produced by the third day of incubation
and was essentially completed by the tenth day of
incubation when 85~ of the initial carbon source sorbose
had been used. Beyond ten days incubation, there was a
considerable decrease in mycelial biomaqs and an apparent
; increase in pH.
An initial rapid growth phase associated with rapid
uptake of carbon source was followed by a ~lower growth
phase during which time most CsA production took place.
,.
- 4 lZ9Z962
The yields and production rates for this batch
fermentation are summarized in following Tables 2 and 3.
TABLE 2
5 Yieldsa Based on Batch Fermentation of T. Inflatum
Utilizing SS Medium Containing 4% (w/v) Sorbose as
Carbon Source
Yield - Units
10 Yx/s 0.39 g biomass/g sorbose
Yx/c 0.96 g bionass/g C
Yp/x 3.83 mg CsA/g biomass
Yp/s 1.48 mg CsA/g sorbose
Yp/c 3.70 mg CsA/g C
. _
a Yields are based on 10 day fermentations, as this is the
typical incubation period. Yields based on carbon neglect
the contribution of Bacto-peptone.
TABLE 3
Volumetric Production and Utilization Rates Based on
Batch aFermentation of T. Inflatum Utilizing SS
Medium Containing 4~ 7~/v) Sorbose as the Carbon
Source
_ _ _
Maximum-Rate Overall Rateb Units
0.26 0.19 mg CsA/L/h
30 0.12 0.05 g biomass/L/h
0.37 0.13 g sorbose/L/h
0.91 0.33 g C/L/h
: . . _ _ . . . _
a Standard medium with 4~ (w/v) Sorbose as C Source.
b Overall rates are based on 10 day fermentations, as this
the typical incubation period. Utilization rate of
carbon, neglects the contribution of Bacto-peptone.
Although the results of this culture of T. inflatum
provides production rates of CsA considerably lower than
1~9296Z
that in the published data, which is in the range of 1.63
mg CsA per liter/hour compared to the approxima~e 0.26 mg
CsA per liter/hour of the example of Figure 1, the medium
reported in published material may be different from media
used to obtain the reported higher production rates in the
range of 1.63 mg CsA per liter/hour. However, Figure 1 is
representative of CsA production whereby the tenth day of
culture and thereafter there is a decrease in biomass due
to fragmentation and lysis of the cells, secondary
metabolite production and carbon source utilization.
Hence, with the existing technology for culturing the T.
nflatum shown in Figure 1, the cyclosporin production is
at the optimum by the tenth day of incubation. Continued
incubation thereafter does not yield increased quantities
of cyclosporin and in fact, cyclosporin concentration may
fall off due to degradation of the metabolites.
It has been discovered that the approach to culturing
T. inflatum may be modified to realize significant
increase production of the cyclosporin secondary
metabolite well beyond the normal ten day cut-off period
and before harvesting of the secondary metabolite. As
noted with respect to Figure 1, the production of
cyclosporin CsA begins to level off after approximately
the eighth day. At this point approximately 80% of the
initial carbon source has been used. It has been
discovered that supplying additional carbon source just as
production of the metabolite commences to fall off
sustains the maximum production rate of the metabolite
well beyond the eighth day of fermentation. The amount of
additional carbon source added may be less than the
initial amount provided in the fermentation media. This
results in a more cost effective process. Tests using
excessive supplementations of the carbon source have
demonstrated that production of cyclosporin does not
exceed that experienced by using a lesser amount in the
supplementation phase. The specific examples discussed
with respect to Figures 2 through 6 demonstrate
supplementation on the eighth day of culture. However,
depending upon the characteristics of the selected fungus
6 1 Z 9 Z 9 ~ Z
and the medium used, pulse feeding by way of
supplementation of the carbon source may be adjusted and
may be done on a sequential basis, such as on the eighth
day, fourteenth day, twentieth day, etc.
It has been discovered that the preferred manner of
pulse feeding is to supplement the media with the same
carbon source as used in initiating the culture. However,
where improved biomass production is desired in the first
phase before supplementation, a carbon source can be
selected which optimizes biomass production. Thereafter,
a different carbon source, which favors production of the
metabolite, can be used in the supplementation phase.
The methodology used in culturing the selected fungus
is principally the same for the prior art cultures and
those according to this invention, except for changes in
the sources of carbon and the pulse feeding in Figures 2
through 5. Each fermentation is conducted by the
submerged cultivation technique. The innoculum for the
media consists of mycelial suspensions of T. nflatum
obtained by addin~ sterile distilled water to cultures
(less than 4 months old) on sl~nts of malt-yeast-agar
medium in screw top tubes. The agar surfaces of the
slants are scraped with sterile spatulas to remove the
mycelial. The mycelial are suspended and introduced to
the flask containing malt yeast medium. Cultures are
incubated in these flasks for approximately 72 hours at
27C with rotational speeds of 200 rpm. From the prepared
innocula, 10 mls are introduced into larger flask
containing 100 mls of media. Incubations are maintained
at 27C and at 200 rpm for the shake flask system for 10
days or whatever other period indicated in the following
Examples.
With respect to the data reported in these examples,
the following determinations were made:
Biomass determination was made from the dry cell
weight of filtered cultures. Sartorius cellulose nitrate
filter papers, available from Sartorius GmbH, Goettingen,
Federal Republic of Germany. The filter papers are dried
for 4 hours at 105C and weighed for use in filtration.
7 12~29~2
After filtration, the residual fungal material was dried
at 105C to a constant weight which usually took about 16
hours.
Carbohydrate determinations were made to establish
consumption of the carbon source. The determinations were
by a color metric anthrone method adapted from the
procedures of Roe, J.H. (1955) The Determination of Suqar
in Blood and Spinal Fluid with Anthrone Reagent J. Biol.
Chem. 212, 355 and Weiner, J. (1978) Determination of
Total Carbohydrates in Beer J. Inst. Brew. 84, 222. The
amount of hexose units in a sample was determined by the
conversion of hexoses to furfural derivatives in strong
acid. These derivatives react with anthrone to form a
blue green colored compound which absor~s 625 nm light.
The reagent for use in this test was prepared by adding
850 mls of concentrated sulfuric acid to 220 mls of
distilled water. 1.00 g of anthrone was added to the
solution. To improve the stability of the reagent 10 g of
thiourea was added. The reagent was cooled and stored at
approximately 4C in accordance with known methodology.
Samples containing carbohydrates were treated with the
reagent and the absorbancy of each sample was determined
in a spectrometer at a wavelength of 625 nm. Curves for
standard concentrations of carbohydrates were prepared and
used as references for carbohydrate determination in the
culture sample.
Cyclosporin determination was accomplished by a high
performance liquid chromatographic system, such as that
available from Waters Scientific Ltd., Mississauga,
Ontario, Canada. Samples of the fermentation media were
extracted from time to time to determine the concentration
of cyclosporin produced. The isolated cultures were
macerated in accordance with standard techniques to
release the cyclosporin for analysis by HPLC technique.
Useful sources of carbon to initiate and supplement
the fermentation of the T. inflatum may be selected from
the group consisting of: sugar alcohols, ketoses, aldoses,
organic acids, disaccharides, polysaccharides and mixtures
thereof. The aldoses may include galactose,
8 1 ~ 9 Z ~ 6 Z
alpha-d-glucose, arabinose, xylose, beta-d-glucose, manose
and ribose. Ketoses may include fructose, sorbose;
disaccharides may include cellobiose, sucrose, maltose and
lactose. Typical polysaccharides includes dextrin. Sugar
alcohols include myo-inositol, sorbitol, manitol and
glycerol. Other acceptable carbohydrates include citric
acid, pyruvic acid, rhamnose and sodium acetate.
Examples of suitable sources of nitrogen for the
media include Bacto-peptone, Bacto-soytone and corn steep
liquor. The sources of these constituents for the
nitrogen component may be purchased from Bifco Labs.,
Detroit, Michigan. Bacto-peptone is the preferred source
of nitrogen.
A suitable source of phosphates for the medium is
potassium dihydrogenphosphate.
The preferred aspects of the process, when applied to
T. inflatum, demonstrates the preferred embodiments of the
invention. The fungus used is that of the wild type
available from American Type Culture Collection deposition
no. 34921. A mutant thereo which i~ particularly useful
in the production o cyclosporins i8 deposited at American
Type Culture Collection deposition no. 20798. The
improved aspects of this mutant are disclosed in
assignee's copending application S.N 540,705 filed June
26, 1987.
In accordance with the prescribed methodology, T.
inflatum (wild type~ is cultured in the medium of Table 1
containing sorbose 5% weight per volume as the initiating
source of carbon. After 8 days of fermentation, the media
is supplemented with additional carbon source in the form
of maltose at 2% wlv or citric acid at 2% w/v. The
addition of the supplemental source of the carbon
component, noting that neither7additional sources of trace
elements nitrogen or phosphates are added, as shown in
Figure 2, results in an increased production of
cyclosporin beyond that achieved under normal culture
conditions with the same fungus. As shown in Figure 1,
the maximum production of cyclosporin is approximately 50
mg per liter. Whereas Figure 2 shows that by the feed
"
9 1;~9Z9~iZ
pulsing of the carbon source, the concentration of
cyclosporin at the end o~ the sixteen day fermentation
period is increased to approximately 95 mg/l. There is a
lag period after the addition of the supplemental carbon
source which may range from 2 to 4 days. The lag period
results from the supplemental carbon source being
different from the initiating carbon source. As a result,
it is necessary for the T. inflatum to adjust its
metabolism to the new carbon source before resuming
increased production of the secondary metabolites.
Further investigations were conducted on the mutant
of T. inflatum ATCC 20798 to optimize the conditions for
pulse fermentation involving the use of various carbon
sources. Several twenty-one day fermentations were
conducted to determine the effect of carbon source on-the
production of CsA and CsC. The medium used is the
standard SS medium of Table 1 with variations on the
initiating carbon source and the carbon source
supplemented on the eighth day of fermentation. A summary
of the yields based on twenty-one day fermentations of the
mutant ATCC 20798 are summarized in the following Table 5.
TABLE 5
Yields Based on 21 Day ~ermentations of M6 Mutant
Incubation
Conditions Yx/c ypa/x ypc/c Ypa/c YpC/c
M 0~9313.706.9012.68 6.20
MM 0.6339.1918.0024.60 11.30
MS 0.069.454.27 5.78 2.55
S 1.237.581.69 9.23 2.06
SS 0.826.253.42 5.16 2.82
SM 0.6315.096.759,45 4,23
. _
Key:
M-Standard SS Medium (Table 1) with 3% (w/v) Maltose*as C
Source.
MM-Same as M but supplemented on day 8 of incubation with
an additional 2% (w/v) Maltose.
MS-Same as M but supplemented on day 8 of incubation with
2% ~w/v) Sorbose.*
~, .
~! *trade marks
1292~2
S-Standard SS Medium (Table 1) with 3~ (w/v) Sorbose as C
Source.
SS-Same as S but supplemented on day 8 of incubation with
an additional 2~ (w/v) Sorbose.
SM-Same as S but supplemented on day 8 OL incubation with
2% (w/v) Maltose.
Yx/c - g biomass/g C
Yp/x - mg cyclosporin/g biomass
Yp/c - mg cyclosporin/g C
a - CsA
c -- CsC
The volumetric production rates based on the 21 day
fermentation of the mutant is summarized in the following
Table 6.
TABLE 6
Volumetric Production Rates* Based on 21 Day
Fermentation of M6 Mutant
Incubation Cyclosporin A Cyclosporin C
Conditions
Overa~rl MaximumOveraL1 ~-~h~
-- ---
M 0.27 0.63 0.14 0.83
MM 0.92 5.10 0.45 2.76
MS 0,21 0.63 0.09 0.83
2S -S 0.19 0.39 0,08 0.24
SS 0.19 0.39 0.11 0.24
SM 0.34 1.06 0.15 0.50
Key - See Key, Table 5
*mg cyclosporin/L/h
Figures 3, 4, 5 and 6 show in graph form the effect
of the supplementation with various carbon sources. In
addition to CsA and CsC production, pH, biomass and carbon
utilization are also recorded. Only four of the selected
carbon sources are shown in the attached drawings. Figure
3 represents incubation conditions MM. Figure 4
represents incubation conditions MS. Figure 5 represents
incubation conditions SS and Figure 6 represents
11 ~292~62
.
incubation conditions SM. From the results shown in
Figure 3, it is apparent that the incubation conditions
which most suit the mutant are an initiating source of
carbon at 3% w/v of maltose with a supplementation on day
8 of fermentation with an additional 2% w/v maltose. The
overall rates of production of mg of cyclosporin/L/h
reaches a maximum of 5.10 for the MM media as shown in
Table 6 which well exceeds the maximum reported production
rates of (1.63 mg/CsA per L/h) CsA from T. inflatum wild
type. As shown in the Tables and Figures, there is,
however, a marked improvement in the production of CsA by
the use of other types of carbon sources such as sorbose,
as the initiating and supplementary source of carbon, or,
the combined use of maltose with sorbose as the initiating
source of carbon. In the medium used in these conditions,
the source of phosphates is potassium dihydrogen
orthophosphate phosphate. The medium contains
approximately 0.5~ by weight of the source of phosphorous.
The source of nitrogen is Bacto-peptone where the medium
contain~ approximately 1% by weight per volume thereof.
With respect to Figures 2 through 6, it is noted that the
carbon source supplement is introduced to the media on the
eighth day which also corresponds to an update of
approximately 80% of the initiating source of carbon. In
using the process of this invention, with other types of
fungus, similar guidelines based on uptake of carbon can
be used to determine the times when supplementation of
carbon source is desirable. In accordance with this
method, only the carbon source is supplemented without any
requirement to add any other nutrients of nitrogen,
phosphorous or trace elements.
Accordingly, this invention requires the use of low
concentration additions of carbon source to the fungus
over an extended fermentation period. Such "pulse"
fermentation circumvents the possible carbon catabolite
repressive effects of high carbon concentration. The
overall efficiency of carbon to cyclosporin conversion is
greatly increased and higher yields of cyclosporin are
attained by adopting this feeding strategy with a simple
12 1~92~3~2
batch fermentation. Cyclosporin yields achieved by pulse
fermentation are more than ten times the levels officially
produced by T. inflatum (wild type) cultivation. The
present production level of in excess of 500 ml CsA/L is
significant commercially since the CsA titre isolated from
the culture media are relatively pure which results in
reduced associated recovery and purification of the CsA.
The highest volumetric production of cyclosporin
produced by T. inflatum (wild type) at a level of lOS ml/L
of broth was obtained on a semi-synthetic medium
containing:
1) 3% w/v sorbose:
2) 1~ w/v Bacto-peptone;
3) 0.5% w/v potassium dihydrogen ortho phosphate;
and
4) 0.25% w/v potassium chloride
The media was adjusted to a pH of 5.3. Cultures were
incubated for 10 days at 27C with agitation.
By using a media containing an initial concentration
%o of maltose equal to 3% w/v supplemented on the eighth day
o incubation with an additionàl 2% w/v maltose and using
the T. inflatum mutant ATCC 20798, the highest production
of CsA and C5C was obtained at 537 mg CsA per liter of
broth and 250 mg of CsC per liter of broth respectively.
By using the pulse feeding mode, a more efficient approach
to process development than simple batch fermentation is
realized. The overall efficiency of carbon to cyclosporin
conversion is increased by more than twofold for pulse
fermentation compared to simple batch fermentation.
With reference to Figure 3, supplementing the
fermentation with additional maltose at 2~ w/v on the
eighth day of fermentation stimulated cyclosporin
productivity to a maximum of 5.10 mg CsA per L/h and 2.76
mg CsC per L/h. The maximum titre of Cs~ and CsC occurred
on the sixteenth day of fermentation at 537.5 mg per liter
and 250 mg per liter respectively. A slight decrease in
cyclosporin titre occurred after the sixteenth day of
fermentation. There was no lag in cyclosporin production
when the maltose medium was supplemented with additional
13 12929~2
maltose. However, when the medium was supplemented with
sorbose, there was a four day lag in production as shown
in Figure 4. This lag is probably due to the fungus not
having the metabolite machinery in place to utilize
sorbose when initiated in the presence of maltose.
However, the fungus recovers and the overall yields are
far beyond those obtained under standard batch
fermentation conditions.
The highest yield of biomass at 1.23 g biomass per
gram of carbon was attained in cultures grown in 3% w/v
sorbose medium as shown in Table 5. The maximum titre of
cyclosporin A produced in this medium was only 93 mg per
liter of broth after 21 days incubation. This indicates
that the T. _nflatum mutant does not utilize sorbose as
effectively as the wild type, because 10 day fermentations
of the wild type on the same medium produced greater than
100 mg CsA per liter. Therefore, it appears that the
mutant utilizes sorbose for growth and thus biomass
formation, whereas the ~ild type produces considerably
less biomass but increased output of cyclosporin. This is
conirmed as shown in Figure 5 where supplementing the
sorbose medium with the addition of 2% w/v sorbose was
directed towards the produc*ion of more biomass instead of
increased production of cyclosporin.
As shown in Table 6 and Figure 6, the addition of
maltose to the sorbose medium on the eighth day of
fermentation resulted in biomass productivity approaching
zero and after a short lag, cyclosporin productivity
increased to a maximum of 1.06 mg CsA per liter/h.
Although preferred embodiments of the invention have
been described herein in detail, it will be understood by
those skilled in the art that variations may be made
~- ~ - thereto without departing from the spirit of the invention
or the scope of the appended claims.
