U.S. patent application number 11/268649 was filed with the patent office on 2006-03-30 for biochemical synthesis apparatus.
This patent application is currently assigned to BIODIVERSITY LIMITED. Invention is credited to Frances Mary Giaquinto, Neil Porter.
Application Number | 20060068460 11/268649 |
Document ID | / |
Family ID | 26315293 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060068460 |
Kind Code |
A1 |
Porter; Neil ; et
al. |
March 30, 2006 |
Biochemical synthesis apparatus
Abstract
A biochemical synthesis apparatus comprises a receptacle for
containing a medium, and a support on which a microorganism can be
placed. The support can be placed in contact with the medium, so as
to allow access of the microorganism to the medium. The support
with the microorganism can be removed from the medium, allowing the
medium to be replaced. By this, the receptacle can firstly contain
a growth medium, and secondly a secondary medium capable of causing
the production by the microorganism of a potentially useful
biochemical.
Inventors: |
Porter; Neil; (Enfield,
GB) ; Giaquinto; Frances Mary; (Enfield, GB) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
BIODIVERSITY LIMITED
|
Family ID: |
26315293 |
Appl. No.: |
11/268649 |
Filed: |
November 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09936726 |
Jan 2, 2002 |
6991919 |
|
|
PCT/GB00/01000 |
Mar 17, 2000 |
|
|
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11268649 |
Nov 8, 2005 |
|
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Current U.S.
Class: |
435/41 |
Current CPC
Class: |
C12M 23/34 20130101;
C12P 1/02 20130101; C12P 1/04 20130101; C12M 37/00 20130101; C12M
23/08 20130101 |
Class at
Publication: |
435/041 |
International
Class: |
C12P 1/00 20060101
C12P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 1999 |
GB |
GB 9906206.9 |
Dec 10, 1999 |
GB |
GB 9929385.4 |
Claims
1-31. (canceled)
32. Apparatus for producing a biochemical, comprising: a container,
and microorganism support means, said support means forming a
dividing partition defining first and second volumes in said
container; wherein: said support means has a first face for
supporting a microorganism exposed to said first volume and a
second face exposed to said second volume; the first volume is in
communication with the ambient atmosphere via a gas permeable plug,
and a portion of the container which at least partially defines the
second volume is removable, whilst the first volume remains
substantially isolated from the ambient atmosphere.
33. Apparatus according to claim 32, including delivery means for
delivering medium supplied to said second volume to a microorganism
supported in use on said support means.
34. Apparatus according to claim 33, wherein said delivery means
defines a capillary pathway for delivery of medium.
35. Apparatus according to claim 32, wherein said portion of the
container which is removable comprises a first receptacle
exchangeable with a second receptacle and wherein, in use, said
first receptacle contains a first medium providing conditions for
growth of a microorganism supported on said support means and said
second receptacle contains a second medium providing conditions for
biosynthesis of said biochemical by said microorganism.
36. Apparatus according to claim 32, wherein said support means
comprises a wicking membrane.
37. Apparatus according to claim 32, wherein the support means
comprises hydrophilised polypropylene.
38. Apparatus according to claim 32, wherein said container
comprises: a fermentation receptacle of generally cylindrical shape
for storing medium for use by a microorganism; a lid threadingly
engageable to one end of said receptacle, said lid having a
throughbore defined by an annular flange extending into said
receptacle; a fermentation vessel having an end adapted for
taper-fitting to said flange, the opposite end of said vessel being
terminated at an acute angle to its longitudinal axis, and a
polystyrene foam filter for receipt in said throughbore.
39. Apparatus according to claim 32, which is sterilizable by steam
sterilization.
Description
[0001] The present invention is concerned with apparatus for use in
a biochemical reaction of a microorganism, and a process for the
synthesis of one or more biochemicals as a result of that
biochemical reaction.
[0002] Microorganisms such as fungi and bacteria produce a vast
diversity of chemical species through biochemical pathways which
constitute secondary metabolism. Secondary metabolism commences in
the absence of one or more nutrients essential to the performance
of primary metabolism. While primary metabolites and their
metabolism are essential for growth, secondary metabolites by
definition are not, but they are believed to contribute to the
survival of a microorganism in a number of ways, as set out in
"Diversity of Microbial Products--Discovery and Application" by N.
Porter and F. M. Fox (1993), Pesticide Science 39, pp 161-168.
Secondary metabolites, therefore, often exhibit diverse biological
properties and as such can provide the basis of new therapeutic
drugs.
[0003] As a consequence, microorganisms are constantly being
studied with a view to finding new and useful secondary
metabolites. However, commonly used processes for the fermentation
and production of samples containing secondary metabolites are
often not compatible with the requirements of modern drug screening
technologies. In small scale fermentations, secondary metabolism
cannot be controlled effectively and many different and often
randomly selected nutrient solutions must be used to achieve the
specific set of conditions required for secondary metabolism.
Additionally, secondary metabolites secreted by the microorganism
are diluted and contaminated with complex nutrients present in the
growth medium. This can lead to low quality samples for
screening.
[0004] In liquid fermentation, secondary metabolites are currently
produced by suspending a sample of the microorganism in a medium
consisting of an aqueous solution or suspension of a combination of
appropriate nutrients. The suspension is placed in a stoppered
flask which allows the ingress of oxygen and the flask is agitated
by shaking to mix and aerate the suspension. Growth and primary
metabolism of the microorganism occurs until one of the essential
nutrients in the medium is exhausted, at which point secondary
metabolism commences. Initially, after inoculating the nutrient
medium with microorganism there is often a variable delay or lag
period before growth commences. Then, in trophophase, the organism
grows in a linear or exponential fashion through primary metabolic
processes until the growth rate begins to decrease as an essential
nutrient, such as nitrogen or phosphate, becomes exhausted as the
organism enters idiophase. At that point, secondary metabolism is
induced as a result of a specific nutrient exhaustion and a
secondary metabolite is produced.
[0005] For an individual microorganism, the lag phase can vary due
to, amongst other things, the age and size of the culture inoculum.
Replicate cultures, while growing at the same rate, could have
different lag phases and therefore could finish growing and enter
idiophase at different times.
[0006] Moreover, different microorganisms could exhibit similar lag
phases but differ significantly in their growth rates so that they
consume essential nutrients at different rates, and they finish
growing at different times, consequently entering idiophase at
different times. The different growth rates could also be exhibited
by an individual microorganism growing on different nutrient
containing media.
[0007] For high throughput screening of secondary metabolites,
samples thereof need to be generated by cultivating microorganisms
in large batches. The inability to control secondary metabolism by
established processes means that the potential of each organism
within a batch to produce new secondary metabolites is not realised
because samples are prepared from fermentations after a fixed time
period at which it is expected that secondary metabolism will have
commenced. However, for the above reasons organisms may not have
begun secondary metabolism. Additionally, secreted secondary
metabolites will be mixed with complex nutrients from the growth
media. These can interfere with the drug screening procedures,
making screening less efficient and productive.
[0008] Therefore, it is an object of the present invention to
provide apparatus and a procedure which allows more predictable
production of secondary metabolite samples in a form compatible
with the operational requirements of high throughput screening
technologies.
[0009] A first aspect of the invention provides a biological
procedure including arranging biomass with access to a medium, said
medium being suitable to support biomass growth, and replacing said
medium with a replacement medium suitable to define conditions for
secondary metabolism in said biomass.
[0010] A second aspect of the invention provides a procedure for
generating a biochemical including the steps of causing an organism
to metabolise in the presence of a first medium for growth of
biomass and causing said organism to metabolise in the presence of
a second medium for generation of said biochemical.
[0011] A third aspect of the invention provides a procedure which
comprises the steps of growing an organism under conditions of
primary metabolism in the presence of excess essential nutrients
for growth, separating the organism from the essential nutrients
and allowing the organism to metabolise in the absence of essential
nutrients under conditions supporting secondary metabolism.
[0012] A fourth aspect of the invention provides a procedure which
comprises the steps of growing an organism under conditions of
primary metabolism in the presence of excess essential nutrients
for growth, separating the organism from the essential nutrients
and allowing the organism to metabolise in the presence of a
reduced concentration of one or more essential nutrients so as to
support secondary metabolism.
[0013] A fifth aspect of the invention provides a procedure which
comprises the steps of growing an organism under conditions of
primary metabolism in the presence of excess essential nutrients,
separating the organism from the essential nutrients, and placing
the organism in conditions supporting secondary metabolism thereby
to generate a secondary metabolite.
[0014] It is an advantage of the invention that secondary
metabolites generated in accordance therewith can be secreted into
a liquid medium containing no or limited amounts of defined
nutrients but substantially free from the complex mixture of
essential nutrients required for the growth of the organism.
[0015] It is a further advantage of the invention that defined
conditions can be selected to induce and support secondary
metabolism in a diverse range of microorganisms.
[0016] By providing a specific separation step, the exhaustion of
an essential nutrient can be carefully controlled, thereby inducing
secondary metabolism and controlling the production of secondary
metabolites.
[0017] A sixth aspect of the invention provides a biological
procedure including placing biomass with access to a medium
formulated for biomass growth, selectively removing said biomass
from said medium, and placing said biomass with access to a
secondary medium suitable to stimulate an alternative metabolic
pathway.
[0018] A seventh aspect of the invention provides apparatus for
arranging a microorganism for metabolism, the apparatus comprising
a receptacle for containing a nutrient medium, and a means for
supporting a microorganism which allows access to nutrient for
metabolism, wherein the means for supporting a microorganism can be
selectively separated from the nutrient in use.
[0019] An eighth aspect of the invention provides apparatus for
supporting biomass such that said biomass can be selectively
positioned for access to an environment for controlling a
biological process in said biomass in use.
[0020] A ninth aspect of the invention provides a procedure
including arranging biomass with access to a medium, said medium
being suitable to support biosynthesis with respect to said
biomass, and replacing said medium with a replacement medium from
which a product of said biosynthesis is distinguishable.
[0021] Further aspects and advantages of the present invention will
be appreciated from the following description of specific
embodiments and examples of the invention, with reference to the
accompanying drawings in which:
[0022] FIG. 1 is a schematic cross-sectional diagram of apparatus
in accordance with a first specific embodiment of the
invention;
[0023] FIG. 2 is a perspective view of a raft of the apparatus
illustrated in FIG. 1;
[0024] FIG. 3 is a perspective view of a fermentation vessel in
accordance with the first specific embodiment of the invention;
[0025] FIG. 4 is a cross-sectional view of the fermentation vessel
illustrated in FIG. 3;
[0026] FIG. 5 is a cross-sectional view of a fermentation vessel in
accordance with a second specific embodiment of the invention;
[0027] FIG. 6 is a schematic diagram of apparatus in accordance
with a third specific embodiment of the invention;
[0028] FIG. 7a is a chromatogram for a test sample prepared in
accordance with a first example of a specific method in accordance
with the present invention;
[0029] FIG. 7b is a chromatogram for a control sample illustrated
for comparison with the chromatogram of FIG. 7a;
[0030] FIG. 8a is a chromatogram for a first test sample prepared
in accordance with a second example of a specific method in
accordance with the present invention;
[0031] FIG. 8b is a chromatogram for a second test sample prepared
in accordance with a second example of a specific method in
accordance with the present invention;
[0032] FIG. 8c is a chromatogram for a reference sample illustrated
for comparison with the chromatograms of FIGS. 8a and 8b;
[0033] FIG. 9a is a chromatogram for a first test sample prepared
in accordance with a third example of a specific method in
accordance with the present invention;
[0034] FIG. 9b is a chromatogram for a second test sample prepared
in accordance with a third example of a specific method in
accordance with the present invention;
[0035] FIG. 9c is a chromatogram for a third test sample prepared
in accordance with a third example of a specific method in
accordance with the present invention;
[0036] FIG. 9d is a chromatogram for a control sample illustrated
for comparison with the chromatograms of FIGS. 9a, 9b, and 9c;
[0037] FIG. 10a is a chromatogram for a first test sample prepared
in accordance with a fourth example of a specific method in
accordance with the present invention;
[0038] FIG. 10b is a chromatogram for a second test sample prepared
in accordance with a fourth example of a specific method in
accordance with the present invention;
[0039] FIG. 10c is a chromatogram for a control sample illustrated
for comparison with the chromatograms of FIGS. 10a and 10b;
[0040] FIG. 11a is a chromatogram for a first test sample prepared
in accordance with a fifth example of a specific method in
accordance with the present invention;
[0041] FIG. 11b is a chromatogram for a second test sample prepared
in accordance with a fifth example of a specific method in
accordance with the present invention;
[0042] FIG. 11c is a chromatogram for a control sample illustrated
for comparison with the chromatograms of FIGS. 11a and 11b;
[0043] FIG. 12 is a schematic cross-sectional diagram of
fermentation apparatus in accordance with a fourth specific
embodiment of the invention;
[0044] FIG. 13 is a side elevation of a fermentation vessel of the
fermentation apparatus illustrated in FIG. 12;
[0045] FIG. 14 is a schematic cross-sectional diagram of the
fermentation apparatus illustrated in FIG. 12, in a mode of use
operative to generate secondary metabolites;
[0046] FIG. 15 is a schematic cross-sectional diagram of the
fermentation apparatus in accordance with a fifth specific
embodiment of the invention;
[0047] FIG. 16 is a side elevation of a fermentation vessel of the
fermentation apparatus illustrated in FIG. 15;
[0048] FIG. 17 is a schematic cross-sectional diagram of the
fermentation apparatus illustrated in FIG. 15 in a mode of use
operative to generate secondary metabolites;
[0049] FIG. 18 is a perspective view of a fermentation vessel of
fermentation apparatus in accordance with a sixth specific
embodiment of the invention;
[0050] FIG. 19a is a spectrum generated by mass spectrometry of a
sample generated in a sixth example in accordance with the
invention;
[0051] FIG. 19b is a view of an expanded portion of the spectrum
illustrated in FIG. 19a;
[0052] FIG. 20 is a spectrum generated by mass spectrometry of a
control sample corresponding with the sample generated in the sixth
example;
[0053] FIG. 21a is a spectrum generated by mass spectrometry of a
further sample generated in the sixth example;
[0054] FIG. 21b is a view of an exposed portion of the spectrum
illustrated in FIG. 21a;
[0055] FIG. 22 is a spectrum generated by mass spectrometry of a
control sample corresponding with the further sample of the sixth
example;
[0056] FIG. 23 is a spectrum generated by mass spectrometry of a
sample generated in a seventh example in accordance with the
invention; and
[0057] FIG. 24 is a spectrum generated by mass spectrometry of a
control sample corresponding with the sample whose spectrum is
illustrated in FIG. 23.
[0058] FIG. 1 shows a fermentation apparatus 2 comprising a
fermentation receptacle 10, which is generally cuboidal in shape.
The upper end of the receptacle 10 is open, and has a lid 12 fitted
thereon. The receptacle 10 and the lid 12 are made of a plastics
material capable of withstanding temperatures of up to 121.degree.
C. in order to allow for sterilisation thereof in the presence of
steam. However, it will be appreciated that other materials, such
as stainless steel or glass, would also be appropriate.
[0059] The lid 12 has a window 14 including a gas permeable foam
insert 16, which allows the transfer of oxygen and carbon dioxide
therethrough, as indicated by arrows in FIG. 1.
[0060] The receptacle 10 contains an aqueous solution/suspension 18
of a combination of nutrients appropriate to the metabolism of a
microorganism to be grown in the fermentation apparatus 2.
Particular examples of nutrients and microorganisms will be
described later.
[0061] Floating on the surface of the aqueous solution 18 is a raft
20. Accordingly, the volume of the aqueous solution/suspension 18
provided in the receptacle 10 is sufficient to allow flotation of
the raft 20. The construction of the raft 20 is best illustrated
with reference to FIG. 2. The raft 20 has a generally square
laminar body 22 with a square through aperture 24 located centrally
therein. A flange 26 extends downwardly as illustrated in FIG. 2
around the periphery of the square body 22.
[0062] As illustrated in FIG. 1, the raft 20 is constructed of a
material which renders it sufficiently buoyant as to float in the
aqueous solution 18, such that the surface of the aqueous solution
18 reaches the level of the square laminar body 22.
[0063] A fermentation vessel 28 is placed on the raft 20. The
vessel 28, illustrated in FIG. 3, consists of a generally square
frame 30 supporting a membrane 32.
[0064] FIGS. 4 and 5 illustrate two alternative embodiments of the
vessel 28 of different constructions. The first embodiment of the
vessel 28 is illustrated in FIG. 4. The membrane 32 of the vessel
28 is constructed of a polypropylene sheet 34 with a pore size of
0.3 micrometers, welded to the frame 30. The polypropylene sheet 34
is treated with a silicone-polyether copolymer to make it water
permeable. On the inside (upper) face of the polypropylene sheet 34
is placed a square melt cast polypropylene fibre hydrophilised
membrane 36, such as a polypropylene membrane sold as a pre-filter
by Millipore Corporation, 80 Ashby Road, Massachusetts, USA.
[0065] The solution/suspension held in the receptacle 10 soaks
through the polypropylene sheet 34 and is wicked by the membrane
36, so that any microorganism sample inoculated on to the membrane
36 has access to the solution/suspension 18. The soaking through of
the solution/suspension can be by means of a pressure gradient
derived from the weight of the raft 20 and fermentation vessel 28
in combination.
[0066] The second specific embodiment is illustrated in FIG. 5. The
vessel 28' is constructed in the same manner as the vessel 28 of
the first specific embodiment, except that the membrane 32' thereof
has a polypropylene-fibre hydrophilised membrane 34', welded to the
frame 30, in place of the polypropylene sheet 34.
[0067] In the case of the second specific embodiment, since both
membranes 34', 36 are hydrophilic, solution/suspension 18 can soak
into the membranes 34', 36 by wicking, brought about via capillary
action.
[0068] A third specific embodiment of the invention is illustrated
in FIG. 6. As far as the apparatus 2' of the third embodiment has
features corresponding to features in the first and second
embodiments, those features are provided with the same reference
numerals. The fermentation receptacle 10 of the apparatus 2'
includes a drain outlet 40 which is closeable by means of a drain
valve 42. In use, liquid contents of the fermentation receptacle 10
can be drained away through the drain outlet 40, which allows the
fermentation receptacle 10 to be emptied without lifting and
tipping thereof. Whereas the apparatus 2' of the third embodiment
of the invention has been provided with a vessel 28 corresponding
to the vessel 28 illustrated in FIG. 4, it will be appreciated that
the vessel could also take the form of the vessel 28' illustrated
in FIG. 5.
[0069] Application of the above described first, second and third
specific embodiments of the invention will now be described with
reference to the following specific examples. The examples involve
analysis of two fungi and three actinomycete bacteria.
[0070] The microorganisms need to be prepared in order to generate
sufficient mycelial growth for investigation. This requires the use
of formulated growth media. The present invention allows the use of
complex growth media.
[0071] Growth media suggested for promoting mycelial growth in
fungi include FS and HC4, whose formulations are set out in Tables
1 and 2 below. TABLE-US-00001 TABLE 1 FS g/l Sheftone -Z soy
peptone 10 Malt extract, Oxoid L39 21 Glycerol 40 Junlon 110
(Honeywell & Stein) 1 Adjust to pH 6.3
[0072] TABLE-US-00002 TABLE 2 HC4 g/l Beet molasses, British Sugar
20 Glycerol 25 Casein NZ-Amine AS 7.5 K.sub.2HPO.sub.4 (Anhydrous)
0.3 CaCO.sub.3 2.5 Tween 80 1 ml
[0073] Growth media suggested for promoting mycelial growth in
actinomycetes include SV2 and MPGS, whose formulations are set out
in Tables 3 and 4 below. TABLE-US-00003 TABLE 3 SV2 g/l D-Glucose
15 Glycerol 15 Sheftone -Z soy peptone 15 NaCl 3 CaCO.sub.3 1
Adjust to pH 7
[0074] TABLE-US-00004 TABLE 4 MPGS g/l Beet molasses, British Sugar
20 Sheftone -Z soy peptone 5 D-Glucose 10 Sucrose 20 CaCO.sub.3
2.5
[0075] In order to induce secondary metabolism in a microorganism,
a culture of the microorganism must be kept in an environment
lacking (or having a reduced concentration in) one or more of the
nutrients essential to primary metabolism and growth. Therefore,
the growth medium selected from the lists set out above must be
replaced by a nutrient deficient medium. Several different nutrient
deficient media require investigation for each new microorganism,
to ensure the identification of the most effective conditions for
efficient secondary metabolism. For fungi, the replacement media
listed in Table 5 are used in the following examples to investigate
secondary metabolism using the apparatus of the specific embodiment
of the invention. TABLE-US-00005 TABLE 5 Replacement media 1. Water
2. Glucidex (Roquette Freres), 10 g/l 3. Trehalose, 10 g/l 4.
Glycerol, 10 g/l 5. Mannitol, 10 g/l
[0076] Water is used as a control, and the other four media contain
a source of carbon. For actinomycetes, the replacement media set
out in Table 6 are used in the following examples to investigate
secondary metabolism using the apparatus of the specific embodiment
of the invention. TABLE-US-00006 TABLE 6 Replacement media 1. Water
2. Glucidex, 10 g/l 3. Glucidex, 10 g/l + Proline, 1.5 g/l (C:N is
approximately 30:1) 4. Glycerol, 10 g/l 5. Glycerol, 10 g/l +
Proline, 1.5 g/l (C:N is approximately 30:1)
[0077] Again, water is used as a control. The other four media
contain either a source of carbon or a source of carbon and
nitrogen. In the case of media 3 and 5 (Table 6), the
carbon:nitrogen ratio (C:N) is set at 30:1 to establish conditions
which particularly favour secondary metabolism.
[0078] Two specific procedures will now be described, for later use
in the following examples.
Procedure 1 (Layer Inoculation)
[0079] The fermentation apparatus 2 is employed in a first
procedure solely for secondary metabolism of a microorganism.
[0080] In this case, mycelial growth of the microorganism under
investigation is generated in a liquid culture, to serve as an
inoculum later referred to as a layer inoculation. This is achieved
in a plurality of 250 ml flasks each containing 50 ml growth
medium. Each flask is inoculated, in sterile conditions, from
microorganism grown on agar slopes, and incubated, with agitation,
at 25.degree. C. or 28.degree. C., for 3 to 5 days.
[0081] A one litre flask, provided with automatic temperature
regulation and stirring devices, is filled with 300 ml of the same
growth medium as used in the 250 ml flasks above. This is
inoculated with 5% cell culture (about 15 ml) taken from the 250 ml
flasks. The vessel is then stirred, using a 45 mm cross-shaped
magnetic follower, at 300 rpm and incubated at 25.degree. C. for
fungi and 28.degree. C. for actinomycetes. The culture is allowed
to grow for up to 5 days, depending on the nature of the
microorganism and its growth rate, in order to maintain the culture
in growth phase, known as trophophase.
[0082] A fermentation apparatus 2 as described above is provided
with a vessel 28' as illustrated in FIG. 5. In order to inoculate
the apparatus 2, the vessel 28' is temporarily removed from the
receptacle of the apparatus 2, and a 50 ml aliquot of the culture
contained in the one litre flask is transferred directly to the
membrane surface 36. The supernatant is allowed to drain away
before the vessel 28' is replaced in the receptacle 10, which
contains 60 ml of a replacement medium as described above.
Procedure 2 (Plug Inoculation)
[0083] The apparatus 2 is used in a second exemplary method both
for the preparation and growth of mycelium of a microorganism for
inoculation and for subsequent nutrient secondary metabolism of the
microorganism. Apparatus 2 in accordance with the first embodiment
is provided as described above with reference to FIGS. 1 to 4 of
the drawings. The receptacle 10 of the apparatus 2 is filled with a
nutrient solution to a level sufficient to support flotation of the
vessel (typically 60-70 ml).
[0084] For fungi, a plug of agar taken from the growing edge of a
stock Petri dish culture of the microorganism under investigation
is deposited on the centre of the membrane 34, 36 of the vessel 28
on the raft 20.
[0085] For actinomycetes, inoculation is carried out by placing a
spore/mycelial suspension onto the membrane of the vessel 30, the
suspension having been prepared from a stock culture of the
organism maintained, for instance, on a slope.
[0086] The inoculated vessel 30 is retained in the fermentation
receptacle 10 for fifteen days, before it is transferred
aseptically to a new fermentation receptacle 10 containing 60 ml of
a replacement medium as identified above.
Secondary Metabolism
[0087] After placement in contact with a replacement medium, fungal
cultures are incubated at 25.degree. C., and actinomycete cultures
at 28.degree. C., for up to 2 weeks to achieve maximum productivity
of secondary metabolites.
[0088] Notwithstanding the existence of water as a control
replacement medium, control samples are also advisedly established
in investigations, in which sample no transfer to a replacement
medium takes place. In the case of plug inoculation, a control is
established which comprises a fermentation apparatus 2 inoculated
with a plug of mycelial growth, which is then left in the same
growth medium for the duration of the trials. In the case of layer
inoculation, a control is established by transferring mycelial
biomass to a vessel 28 and allowing it to drain through. The vessel
28 is then placed in a fermentation receptacle 10 containing the
same growth medium as was used to generate the layer inoculation,
again for the duration of the trials.
Metabolite Isolation
[0089] Secondary metabolite can be produced in the cells of the
microorganism under test, in the fermentation broth in which the
microorganism resides, or in both. Therefore, samples of both
mycelium and filtrate are taken. The mycelium sample is extracted
with 10 ml methanol for a minimum of twelve hours, following which
the extract is subjected to chromatographic analysis. The broth
sample is diluted in suitable HPLC mobile phase, following which it
is also subjected to chromatographic analysis. Suitable HPLC
conditions will be described for each example outlined below.
[0090] Each example outlined below demonstrates the use of the
fermentation apparatus of the present invention in the execution of
a number of different tasks. The examples demonstrate
investigations into the effectiveness of the fermentation apparatus
illustrated in FIG. 1, and the method of transferring a
microorganism into conditions supporting secondary metabolism, to
generate secondary metabolite from five microorganisms treated in a
variety of different ways. The five microorganisms investigated in
the examples are Phoma sp. F16006 and Trichoderma longibraciatum
5602E, which are fungi, and Amycolatopsis orientalis C2726,
Nocardiopsis sp. 5997E, and Streptomyces citricolor C2778 which are
actinomycetes.
[0091] Each of the fungi are to be treated in the same manner,
likewise the actinomycetes. The microorganisms should be tested
under all combinations of available conditions. In respect of each
fungus, twenty fermentation apparata 2 need to be prepared. A first
group of five fermentation apparata 2 are prepared with a layer
inoculum from a liquid culture generated in FS growth medium and a
second group of five with a layer inoculum from liquid culture
prepared in HC4 growth medium, in accordance with procedure 1. A
third group of five apparata 2 are prepared with plug inoculated
cultures grown on FS medium and a fourth group of five apparata 2
with plug inoculated cultures grown on HC4 growth medium, in
accordance with procedure 2.
[0092] Each receptacle 10 of the five apparata 2 in each group is
filled with a respective one of the five replacement media set out
in Table 5. The twenty fermentation apparata 2 so inoculated are
maintained for ten days before harvest.
[0093] Four control apparata 2 are also arranged, two of which are
layer inoculated from four day old liquid cultures (one from each
of the two available growth media), and the other two of which are
inoculated using the plug inoculation technique (from the two
available growth media). The fermenting receptacles 10 are filled
with corresponding growth media, not replacement media. The
apparata are left for fifteen days before harvest for layer
inoculated cultures, and twenty five days before harvest for plug
inoculated cultures.
[0094] Each of the actinomycetes are to be treated in the same
general manner, but with some differences in the specific
procedures employed. Again, twenty test apparata 2 and four control
apparata 2 are assembled, since two growth media SV2, MPGS and five
replacement media (Table 6) are available. However, the duration of
each stage is in some cases different. In the case of Procedure 1
for actinomycetes, liquid culture for layer inoculation is grown
for five days rather than four as per fungi. Incubation after
transfer to replacement medium is conducted for ten days rather
than the eleven day period set down for fungi. Again, layer
inoculum control cultures are grown for 5 days before transfer to
apparata 2 containing growth media.
[0095] After completion of the relevant incubation period,
investigations are put in place to measure the production of
metabolite in cell extract and broth extract. In order to measure
concentrations of secondary metabolite, the extract under
investigation is subjected to HPLC under suitable conditions.
[0096] The operating parameters and mobile phase formulations for
all examples, except Example B, are set out in Table 7. Chemical
standards are used to identify chromatographic peaks corresponding
to the secondary metabolites produced by the test organisms.
TABLE-US-00007 TABLE 7 Time (Min) % Mobile Phase B Flow (ml/min) 0
0 1 20 100 1 30 100 1 32 0 1 35 0 1 Mobile Phase A: 5 g/litre
sodium lauryl sulphate + 10 ml/ litre 0.1M NH.sub.4H.sub.2PO.sub.4,
pH 2.5. Mobile Phase B: 75% CH.sub.3CN + 5 g/litre sodium lauryl
sulphate + 10 ml/litre 0.1M NH.sub.4H.sub.2PO.sub.4, pH 2.5.
Column: Spherisorb 15 cm C5 5 micron.
[0097] The conditions for Example B has formulation set out in
Table 8. TABLE-US-00008 TABLE 8 Time (Min) % Mobile Phase B Flow
(ml/min) 0 0 1 1 0 1 30 100 1 35 100 1 36 0 1 40 0 1 Mobile Phase
A: 0.1% TFA. Mobile Phase B: 75% CH.sub.3CN + 0.1% TFA. Column:
Hypersil 15 cm C18 3 micron.
[0098] Finally, standard shaken cultures in accordance with known
techniques are also carried out as a comparison of general
bioreactor performance. The growth media for these cultures are FS
(formulation previously described), SM37, BFMS and K252/P1. The
formulations for the latter three media are: TABLE-US-00009 SM37
g/l BFMS g/l K252/P1 g/l Lactose 25 Arkasoy 10 Glucose 5
KH.sub.2PO.sub.4 4 Glucose 18 Soluble starch 30 CaCO.sub.3 10
CaCO.sub.3 0.2 Arkasoy 20 Pharmamedia 20 CoCL.sub.2.6H.sub.2O 0.001
Yeast extract 5 pH to 6.5 Na.sub.2SO.sub.4 1 Corn steep liquor 5
Molasses 18 CaCO.sub.3 3 Sucrose 18 pH to 7.2
[0099] The results of the HPLC tests for selected samples produced
by the following examples are illustrated as chromatograms in FIGS.
7a and 7b, FIGS. 8a, 8b and 8c, FIGS. 9a, 9b, 9c and 9d, FIGS. 10a,
10b and 10c and FIGS. 11a, 11b and 11c. A chromatogram is a graph
of Absorbance (measured in milli Absorbance Units) against
retention time (measured in Minutes). Each chromatogram is marked
with an arrow pointing at a peak which represents the expected
secondary metabolite for that particular sample.
EXAMPLE A
Phoma sp. F16006
[0100] This fungus produces compound GR 195359. The results of the
procedures applied to the microorganism are set out in Table 9.
TABLE-US-00010 TABLE 9 Growth Replacement Extract Conc. Ref:
Organism Metabolite Inoculum Type Medium Medium Type (mg/l) TEST A1
Phoma sp F16006 GR 195359 Layer FS water cell 0 A2 Phoma sp F16006
GR 195359 Layer FS glucidex cell 0 A3 Phoma sp F16006 GR 195359
Layer FS trehalose cell 0 A4 Phoma sp F16006 GR 195359 Layer FS
glycerol cell 0 A5 Phoma sp F16006 GR 195359 Layer FS mannitol cell
0 A6 Phoma sp F16006 GR 195359 Layer FS water broth 0 A7 Phoma sp
F16006 GR 195359 Layer FS glucidex broth 0 A8 Phama sp F16006 GR
195359 Layer FS trehalose broth 0 A9 Phoma sp F16006 GR 195359
Layer FS glycerol broth 0 A10 Phoma sp F16006 GR 195359 Layer FS
mannitol broth 0 A11 Phoma sp F16006 GR 195359 Layer HC4 water cell
0 A12 Phoma sp F16006 GR 195359 Layer HC4 glucidex cell 0 A13 Phoma
sp F16006 GR 195359 Layer HC4 trehalose cell 0 A14 Phoma sp F16006
GR 195359 Layer HC4 glycerol cell 0 A15 Phoma sp F16006 GR 195359
Layer HC4 mannitol cell 246 A16 Phoma sp F16006 GR 195359 Layer HC4
water broth 0 A17 Phoma sp F16006 GR 195359 Layer HC4 glucidex
broth 0 A18 Phoma sp F16006 GR 195359 Layer HC4 trehalose broth 0
A19 Phoma sp F16006 GR 195359 Layer HC4 glycerol broth 0 A20 Phoma
sp F16006 GR 195359 Layer HC4 mannitol broth 0 A21 Phoma sp F16006
GR 195359 Plug FS water cell 134 A22 Phoma sp F16006 GR 195359 Plug
FS glucidex cell 529 A23 Phoma sp F16006 GR 195359 Plug FS
trehalose cell 525 A24 Phoma sp F16006 GR 195359 Plug FS glycerol
cell 519 A25 Phoma sp F16006 GR 195359 Plug FS mannitol cell 876
A26 Phoma sp F16006 GR 195359 Plug FS water broth 0 A27 Phoma sp
F16006 GR 195359 Plug FS glucidex broth 0 A28 Phoma sp F16006 GR
195359 Plug FS trehalose broth 0 A29 Phoma sp F16006 GR 195359 Plug
FS glycerol broth 0 A30 Phoma sp F16006 GR 195359 Plug FS mannitol
broth 0 A31 Phoma sp F16006 GR 195359 Plug HC4 water cell 0 A32
Phoma sp F16006 GR 195359 Plug HC4 glucidex cell 0 A33 Phoma sp
F16006 GR 195359 Plug HC4 trehalose cell 0 A34 Phoma sp F16006 GR
195359 Plug HC4 glycerol cell 0 A35 Phoma sp F16006 GR 195359 Plug
HC4 mannitol cell 85 A36 Phoma sp F16006 GR 195359 Plug HC4 water
broth 0 A37 Phoma sp F16006 GR 195359 Plug HC4 glucidex broth 0 A38
Phoma sp F16006 GR 195359 Plug HC4 trehalose broth 0 A39 Phoma sp
F16006 GR 195359 Plug HC4 glycerol broth 0 A40 Phoma sp F16006 GR
195359 Plug HC4 mannitol broth 0 CONTROL A41 Phoma sp F16006 GR
195359 Layer FS FS cell 0 A42 Phoma sp F16006 GR 195359 Layer FS FS
broth 0 A43 Phoma sp F16006 GR 195359 Layer HC4 HC4 cell 0 A44
Phoma sp F16006 GR 195359 Layer HC4 HC4 broth 0 A45 Phoma sp F16006
GR 195359 Plug FS FS cell 608 A46 Phoma sp F16006 GR 195359 Plug FS
FS broth 0 A47 Phoma sp F16006 GR 195359 Plug HC4 HC4 cell 0 A48
Phoma sp F16006 GR 195359 Plug HC4 HC4 broth 0 A49 Phoma sp F16006
GR 195359 Shaken SM37 culture 109
[0101] In the example, GR 195359 is produced, with two exceptions,
on FS medium in cultures inoculated by the plug method. GR 195359
is extracted only from the cell material. The nature of the
replacement medium affects the amount of GR 195359 produced by the
organism, as demonstrated by test samples A21-A25. In particular,
mannitol produces the highest titre of GR 195359 and is able to
stimulate production in layer and plug replacement cultures grown
on HC4 medium, as shown in samples A15 and A35 respectively.
Mannitol stimulates the production of GR 195359 significantly
beyond the level achievable in the corresponding control A45
arranged without transfer to replacement medium.
[0102] HPLC chromatograms reveal that in cell extracts A21-A25 in
respect of which the microorganism has been transferred to
replacement medium, the size of the GR 195359 peak relative to the
other component peaks is significantly greater than in control
samples. This indicates that there is a higher proportion of GR
195359 in cell extracts of replacement cultures. This is
illustrated in FIG. 7a, which illustrates sample A25, in comparison
with FIG. 7b, which shows its control A45.
[0103] Although the titres are not directly comparable, the
concentrations of GR 195359 in the described cell extracts are
superior to levels in whole culture extracts of Phoma sp. F16006
grown in traditional shake flasks on an optimised medium.
EXAMPLE B
Trichoderma longibraciatum 5602E
[0104] This fungus produces bisvertinolone. The results of the
procedures described above applied to the microorganism are set out
in Table 10. TABLE-US-00011 TABLE 10 Inoculum Growth Replacement
Extract Conc. Ref: Organism Metabolite Type Medium Medium Type
(mg/l) TEST B1 T. longibrachiatum 5602E bisvertinolone Layer FS
water cell 0 B2 T. longibrachiatum 5602E bisvertinolone Layer FS
glucidex cell 0 B3 T. longibrachiatum 5602E bisvertinolone Layer FS
trehalose cell 43.6 B4 T. longibrachiatum 5602E bisvertinolone
Layer FS glycerol cell 0 B5 T. longibrachiatum 5602E bisvertinolone
Layer FS mannitol cell 0 B6 T. longibrachiatum 5602E bisvertinolone
Layer FS water broth 406.3 B7 T. longibrachiatum 5602E
bisvertinolone Layer FS glucidex broth 115.6 B8 T. longibrachiatum
5602E bisvertinolone Layer FS trehalose broth 196.0 B9 T.
longibrachiatum 5602E bisvertinolone Layer FS glycerol broth 304.0
B10 T. longibrachiatum 5602E bisvertinolone Layer FS mannitol broth
168.3 B11 T. longibrachiatum 5602E bisvertinolone Layer HC4 water
cell 0 B12 T. longibrachiatum 5602E bisvertinolone Layer HC4
glucidex cell 0 B13 T. longibrachiatum 5602E bisvertinolone Layer
HC4 trehalose cell 322.6 B14 T. longibrachiatum 5602E
bisvertinolone Layer HC4 glycerol cell 456.1 B15 T. longibrachiatum
5602E bisvertinolone Layer HC4 mannitol cell 240.2 B16 T.
longibrachiatum 5602E bisvertinolone Layer HC4 water broth 798.3
B17 T. longibrachiatum 5602E bisvertinolone Layer HC4 glucidex
broth 1448.0 B18 T. longibrachiatum 5602E bisvertinolone Layer HC4
trehalose broth 2505.0 B19 T. longibrachiatum 5602E bisvertinolone
Layer HC4 glycerol broth 3407.9 B20 T. longibrachiatum 5602E
bisvertinolone Layer HC4 mannitol broth 2328.1 B21 T.
longibrachiatum 5602E bisvertinolone Plug FS water cell 0 B22 T.
longibrachiatum 5602E bisvertinolone Plug FS glucidex cell 1669.9
B23 T. longibrachiatum 5602E bisvertinolone Plug FS trehalose cell
1325.2 B24 T. longibrachiatum 5602E bisvertinolone Plug FS glycerol
cell 901.7 B25 T. longibrachiatum 5602E bisvertinolone Plug FS
mannitol cell 1333.6 B26 T. longibrachiatum 5602E bisvertinolone
Plug FS water broth 1214.6 B27 T. longibrachiatum 5602E
bisvertinolone Plug FS glucidex broth 1439.8 B28 T. longibrachiatum
5602E bisvertinolone Plug FS trehalose broth 617.6 B29 T.
longibrachiatum 5602E bisvertinolone Plug FS glycerol broth 802.2
B30 T. longibrachiatum 5602E bisvertinolone Plug FS mannitol broth
1227.8 B31 T. longibrachiatum 5602E bisvertinolone Plug HC4 water
cell 432.9 B32 T. longibrachiatum 5602E bisvertinolone Plug HC4
glucidex cell 1046.2 B33 T. longibrachiatum 5602E bisvertinolone
Plug HC4 trehalose cell 219.6 B34 T. longibrachiatum 5602E
bisvertinolone Plug HC4 glycerol cell 276.6 B35 T. longibrachiatum
5602E bisvertinolone Plug HC4 mannitol cell 378.0 B36 T.
longibrachiatum 5602E bisvertinolone Plug HC4 water broth 798.4 B37
T. longibrachiatum 5602E bisvertinolone Plug HC4 glucidex broth
2821.6 B38 T. longibrachiatum 5602E bisvertinolone Plug HC4
trehalose broth 1510.8 B39 T. longibrachiatum 5602E bisvertinolone
Plug HC4 glycerol broth 3263.7 B40 T. longibrachiatum 5602E
bisvertinolone Plug HC4 mannitol broth 2078.7 CONTROL B41 T.
longibrachiatum 5602E bisvertinolone Layer FS FS cell 892.9 B42 T.
longibrachiatum 5602E bisvertinolone Layer FS FS broth 344.7 B43 T.
longibrachiatum 5602E bisvertinolone Layer HC4 HC4 cell 5256.5 B44
T. longibrachiatum 5602E bisvertinolone Layer HC4 HC4 broth 2451.2
B45 T. longibrachiatum 5602E bisvertinolone Plug FS FS cell 659.5
B46 T. longibrachiatum 5602E bisvertinolone Plug FS FS broth 660.5
B47 T. longibrachiatum 5602E bisvertinolone Plug HC4 HC4 cell
1470.4 B48 T. longibrachiatum 5602E bisvertinolone Plug HC4 HC4
broth 2186.5 B49 T. longibrachiatum 5602E bisvertinolone Shaken FS
culture 6400
[0105] From the results, it can be observed that the fungus
produces its secondary metabolite under most circumstances,
generally as effectively in the apparatus of the present invention
as in traditional shaken cultures.
[0106] The apparatus allows for secretion of secondary metabolites
into the highly defined replacement medium and the generation of
less complex mixtures of wholly fungal origin. This is exemplified
in FIG. 8a by the HPLC chromatogram for broth sample B19 which has
a flatter baseline and shows better peak separation than the
corresponding cell extract B14 illustrated in FIG. 8c. Where the
replacement medium is water as in sample B16, the chromatogram is
simplified even further (FIG. 8b).
EXAMPLE C
Amycolatopsis orientalis C2726
[0107] This actinomycete bacterium produces vancomycin. The results
of the procedures applied to the microorganism are set out in Table
11. TABLE-US-00012 TABLE 11 Inoculum Growth Replacement Conc. Ref:
Organism Metabolite Type Medium Medium Extract Type (mg/l) TEST C1
A orientalis C2726 vancomycin Layer SV2 water cell 0 C2 A
orientalis C2726 vancomycin Layer SV2 glucidex cell 0 C3 A
orientalis C2726 vancomycin Layer SV2 glucidex + proline cell 0 C4
A orientalis C2726 vancomycin Layer SV2 glycerol cell 0 C5 A
orientalis C2726 vancomycin Layer SV2 glycerol + proline cell 0 C6
A orientalis C2726 vancomycin Layer SV2 water broth 52.1 C7 A
orientalis C2726 vancomycin Layer SV2 glucidex broth 79.3 C8 A
orientalis C2726 vancomycin Layer SV2 glucidex + proline broth 49.8
C9 A orientalis C2726 vancomycin Layer SV2 glycerol broth 76.9 C10
A orientalis C2726 vancomycin Layer SV2 glycerol + proline broth
58.6 C11 A orientalis C2726 vancomycin Layer MPGS water cell 0 C12
A orientalis C2726 vancomycin Layer MPGS glucidex cell 0 C13 A
orientalis C2726 vancomycin Layer MPGS glucidex + proline cell 0
C14 A orientalis C2726 vancomycin Layer MPGS glycerol cell 0 C15 A
orientalis C2726 vancomycin Layer MPGS glycerol + proline cell 0
C16 A orientalis C2726 vancomycin Layer MPGS water broth 20.3 C17 A
orientalis C2726 vancomycin Layer MPGS glucidex broth 95.2 C18 A
orientalis C2726 vancomycin Layer MPGS glucidex + proline broth
120.9 C19 A orientalis C2726 vancomycin Layer MPGS glycerol broth
142.3 C20 A orientalis C2726 vancomycin Layer MPGS glycerol +
proline broth 207.9 C21 A orientalis C2726 vancomycin Plug SV2
water cell 0 C22 A orientalis C2726 vancomycin Plug SV2 glucidex
cell 0 C23 A orientalis C2726 vancomycin Plug SV2 glucidex +
proline cell 14.6 C24 A orientalis C2726 vancomycin Plug SV2
glycerol cell 6.6 C25 A orientalis C2726 vancomycin Plug SV2
glycerol + proline cell 36.9 C26 A orientalis C2726 vancomycin Plug
SV2 water broth 15.1 C27 A orientalis C2726 vancomycin Plug SV2
glucidex broth 9.1 C28 A orientalis C2726 vancomycin Plug SV2
glucidex + proline broth 73.5 C29 A orientalis C2726 vancomycin
Plug SV2 glycerol broth 110.8 C30 A orientalis C2726 vancomycin
Plug SV2 glycerol + proline broth 86.9 C31 A orientalis C2726
vancomycin Plug MPGS water cell 0.0 C32 A orientalis C2726
vancomycin Plug MPGS glucidex cell 0.0 C33 A orientalis C2726
vancomycin Plug MPGS glucidex + proline cell 8.2 C34 A orientalis
C2726 vancomycin Plug MPGS glycerol cell 6.8 C35 A orientalis C2726
vancomycin Plug MPGS glycerol + proline cell 15.1 C36 A orientalis
C2726 vancomycin Plug MPGS water broth 0.0 C37 A orientalis C2726
vancomycin Plug MPGS glucidex broth 17.4 C38 A orientalis C2726
vancomycin Plug MPGS glucidex + proline broth 43.9 C39 A orientalis
C2726 vancomycin Plug MPGS glycerol broth 51.1 C40 A orientalis
C2726 vancomycin Plug MPGS glycerol + proline broth 36.1 CONTROL
C41 A orientalis C2726 vancomycin Layer SV2 SV2 cell 29.3 C42 A
orientalis C2726 vancomycin Layer SV2 SV2 broth 0 C43 A orientalis
C2726 vancomycin Layer MPGS MPGS cell 0 C44 A orientalis C2726
vancomycin Layer MPGS MPGS broth 0 C45 A orientalis C2726
vancomycin Plug SV2 SV2 cell 0 C46 A orientalis C2726 vancomycin
Plug SV2 SV2 broth 0 C47 A orientalis C2726 vancomycin Plug MPGS
MPGS cell 0 C48 A orientalis C2726 vancomycin Plug MPGS MPGS broth
0 C49 A orientalis C2726 vancomycin Shaken BFMS culture 307
[0108] The results show that the apparatus supports the production
of vancomycin by this actinomycete, specifically in the broth of
layer cultures and more generally over plug cultures. The generally
poorer performance of water as a replacement medium indicates the
importance of a carbon source or a carbon and nitrogen source in a
specified ratio, to enhance the production of vancomycin.
[0109] In the eight control cultures C41 to C48 performed in
apparatus as described above, vancomycin is only detectable in one
culture C41. These results indicate that a nutrient replacement
procedure to media containing a carbon or carbon and nitrogen
source is essential to consistently produce vancomycin from the
primary growth media SV2 and MPGS.
[0110] HPLC chromatograms for broths exemplified in FIGS. 9a, 9b
and 9c, for samples C16, C17 and C19 respectively, show flatter
baselines, fewer components and better peak separation than the
control cell extract exemplified by sample C41, whose HPLC
chromatogram is illustrated in FIG. 9d. In addition, comparison of
the HPLC chromatograms for individual spectra exemplified by
samples C16, C17 and C19 show differences in vancomycin titre and
subtle differences in the overall pattern of peaks.
EXAMPLE D
Nocardiopsis sp. 5997E
[0111] This actinomycete bacterium produces K252a. The results of
the procedures applied to the microorganism are set out in Table
12. TABLE-US-00013 TABLE 12 Inoculum Growth Replacement Extract
Conc. Ref: Organism Metabolite Type Medium Medium Type (mg/l) TEST
D1 Nocardiopsis sp 5997E K252a Layer SV2 water cell 15 D2
Nocardiopsis sp 5997E K252a Layer SV2 glucidex cell 0 D3
Nocardiopsis sp 5997E K252a Layer SV2 glucidex + proline cell 13 D4
Nocardiopsis sp 5997E K252a Layer SV2 glycerol cell 46 D5
Nocardiopsis sp 5997E K252a Layer SV2 glycerol + proline cell 32 D6
Nocardiopsis sp 5997E K252a Layer SV2 water broth 0 D7 Nocardiopsis
sp 5997E K252a Layer SV2 glucidex broth 0 D8 Nocardiopsis sp 5997E
K252a Layer SV2 glucidex + proline broth 0 D9 Nocardiopsis sp 5997E
K252a Layer SV2 glycerol broth 0 D10 Nocardiopsis sp 5997E K252a
Layer SV2 glycerol + proline broth 0 D11 Nocardiopsis sp 5997E
K252a Layer MPGS water cell 1962 D12 Nocardiopsis sp 5997E K252a
Layer MPGS glucidex cell 1991 D13 Nocardiopsis sp 5997E K252a Layer
MPGS glucidex + proline cell 2342 D14 Nocardiopsis sp 5997E K252a
Layer MPGS glycerol cell 2275 D15 Nocardiopsis sp 5997E K252a Layer
MPGS glycerol + proline cell 2435 D16 Nocardiopsis sp 5997E K252a
Layer MPGS water broth 0 D17 Nocardiopsis sp 5997E K252a Layer MPGS
glucidex broth 0 D18 Nocardiopsis sp 5997E K252a Layer MPGS
glucidex + proline broth 0 D19 Nocardiopsis sp 5997E K252a Layer
MPGS glycerol broth 0 D20 Nocardiopsis sp 5997E K252a Layer MPGS
glycerol + proline broth 0 D21 Nocardiopsis sp 5997E K252a Plug SV2
water cell 0 D22 Nocardiopsis sp 5997E K252a Plug SV2 glucidex cell
0 D23 Nocardiopsis sp 5997E K252a Plug SV2 glucidex + proline cell
0 D24 Nocardiopsis sp 5997E K252a Plug SV2 glycerol cell 0 D25
Nocardiopsis sp 5997E K252a Plug SV2 glycerol + proline cell 0 D26
Nocardiopsis sp 5997E K252a Plug SV2 water broth 0 D27 Nocardiopsis
sp 5997E K252a Plug SV2 glucidex broth 0 D28 Nocardiopsis sp 5997E
K252a Plug SV2 glucidex + proline broth 0 D29 Nocardiopsis sp 5997E
K252a Plug SV2 glycerol broth 0 D30 Nocardiopsis sp 5997E K252a
Plug SV2 glycerol + proline broth 0 D31 Nocardiopsis sp 5997E K252a
Plug MPGS water cell 0 D32 Nocardiopsis sp 5997E K252a Plug MPGS
glucidex cell 0 D33 Nocardiopsis sp 5997E K252a Plug MPGS glucidex
+ proline cell 0 D34 Nocardiopsis sp 5997E K252a Plug MPGS glycerol
cell 0 D35 Nocardiopsis sp 5997E K252a Plug MPGS glycerol + proline
cell 0 D36 Nocardiopsis sp 5997E K252a Plug MPGS water broth 0 D37
Nocardiopsis sp 5997E K252a Plug MPGS glucidex broth 0 D38
Nocardiopsis sp 5997E K252a Plug MPGS glucidex + proline broth 0
D39 Nocardiopsis sp 5997E K252a Plug MPGS glycerol broth 0 D40
Nocardiopsis sp 5997E K252a Plug MPGS glycerol + proline broth 0
CONTROL D41 Nocardiopsis sp 5997E K252a Layer SV2 SV2 cell 0 D42
Nocardiopsis sp 5997E K252a Layer SV2 SV2 broth 0 D43 Nocardiopsis
sp 5997E K252a Layer MPGS MPGS cell 2284 D44 Nocardiopsis sp 5997E
K252a Layer MPGS MPGS broth 644 D45 Nocardiopsis sp 5997E K252a
Plug SV2 SV2 cell 0 D46 Nocardiopsis sp 5997E K252a Plug SV2 SV2
broth 0 D47 Nocardiopsis sp 5997E K252a Plug MPGS MPGS cell 0 D48
Nocardiopsis sp 5997E K252a Plug MPGS MPGS broth 0 D49 Nocardiopsis
sp 5997E K252a Shaken K252/P1 culture 2108
[0112] The results show that metabolite K252a is most effectively
produced in cell extracts of layer cultures transferred to
replacement medium following growth in MPGS medium. Titres of K252a
in these culture samples D11 to D15 are not significantly different
from the control culture D43. However, comparison of HPLC spectra
for samples D11 and D15, as illustrated in FIGS. 10a and 10b, show
that cell extracts for those samples contain fewer, well defined
peaks than shown in the HPLC chromatogram for control sample D43
(FIG. 10c), indicating the existence of simpler solutions.
[0113] Again this example shows that although the titres are low,
the described procedure induces production of K252a in SV2 medium
when none is produced under control conditions. This demonstrates
that the apparata can be used to produce secondary metabolites
through the use of only a limited number of media, whereas up to
ten media would previously have been required.
EXAMPLE E
Streptomyces citricolor C2778
[0114] This actinomycete bacterium produces the compound
aristeromycin. The results of the procedures applied to the
microorganism are set out in Table 13. TABLE-US-00014 TABLE 13
Inoculum Growth Replacement Conc. Ref: Organism Metabolite Type
Medium Medium Extract Type (mg/l) TEST E1 S. citricolor C2778
aristeromycin Layer SV2 water cell 5 E2 S. citricolor C2778
aristeromycin Layer SV2 glucidex cell 3 E3 S. citricolor C2778
aristeromycin Layer SV2 glucidex + proline cell 9 E4 S. citricolor
C2778 aristeromycin Layer SV2 glycerol cell 3 E5 S. citricolor
C2778 aristeromycin Layer SV2 glycerol + proline cell 10 E6 S.
citricolor C2778 aristeromycin Layer SV2 water broth 22 E7 S.
citricolor C2778 aristeromycin Layer SV2 glucidex broth 20 E8 S.
citricolor C2778 aristeromycin Layer SV2 glucidex + proline broth
28 E9 S. citricolor C2778 aristeromycin Layer SV2 glycerol broth 16
E10 S. citricolor C2778 aristeromycin Layer SV2 glycerol + proline
broth 41 E11 S. citricolor C2778 aristeromycin Layer MPGS water
cell 3 E12 S. citricolor C2778 aristeromycin Layer MPGS glucidex
cell 9 E13 S. citricolor C2778 aristeromycin Layer MPGS glucidex +
proline cell 16 E14 S. citricolor C2778 aristeromycin Layer MPGS
glycerol cell 12 E15 S. citricolor C2778 aristeromycin Layer MPGS
glycerol + proline cell 5 E16 S. citricolor C2778 aristeromycin
Layer MPGS water broth 23 E17 S. citricolor C2778 aristeromycin
Layer MPGS glucidex broth 36 E18 S. citricolor C2778 aristeromycin
Layer MPGS glucidex + proline broth 48 E19 S. citricolor C2778
aristeromycin Layer MPGS glycerol broth 68 E20 S. citricolor C2778
aristeromycin Layer MPGS glycerol + proline broth 37 E21 S.
citricolor C2778 aristeromycin Plug SV2 water cell 0 E22 S.
citricolor C2778 aristeromycin Plug SV2 glucidex cell 0 E23 S.
citricolor C2778 aristeromycin Plug SV2 glucidex + proline cell 0
E24 S. citricolor C2778 aristeromycin Plug SV2 glycerol cell 0 E25
S. citricolor C2778 aristeromycin Plug SV2 glycerol + proline cell
0 E26 S. citricolor C2778 aristeromycin Plug SV2 water broth 0 E27
S. citricolor C2778 aristeromycin Plug SV2 glucidex broth 0 E28 S.
citricolor C2778 aristeromycin Plug SV2 glucidex + proline broth 0
E29 S. citricolor C2778 aristeromycin Plug SV2 glycerol broth 0 E30
S. citricolor C2778 aristeromycin Plug SV2 glycerol + proline broth
0 E31 S. citricolor C2778 aristeromycin Plug MPGS water cell 0 E32
S. citricolor C2778 aristeromycin Plug MPGS glucidex cell 0 E33 S.
citricolor C2778 aristeromycin Plug MPGS glucidex + proline cell 0
E34 S. citricolor C2778 aristeromycin Plug MPGS glycerol cell 6 E35
S. citricolor C2776 aristeromycin Plug MPGS glycerol + proline cell
0 E36 S. citricolor C2778 aristeromycin Plug MPGS water broth 4 E37
S. citricolor C2778 aristeromycin Plug MPGS glucidex broth 2 E38 S.
citricolor C2778 aristeromycin Plug MPGS glucidex + proline broth 0
E39 S. citricolor C2778 aristeromycin Plug MPGS glycerol broth 16
E40 S. citricolor C2778 aristeromycin Plug MPGS glycerol + proline
broth 0 CONTROL E41 S. citricolor C2778 aristeromycin Layer SV2 SV2
cell 52 E42 S. citricolor C2778 aristeromycin Layer SV2 SV2 broth 1
E43 S. citricolor C2778 aristeromycin Layer MPGS MPGS cell 40 E44
S. citricolor C2778 aristeromycin Layer MPGS MPGS broth 51 E45 S.
citricolor C2778 aristeromycin Plug SV2 SV2 cell 0 E46 S.
citricolor C2778 aristeromycin Plug SV2 SV2 broth 0 E47 S.
citricolor C2778 aristeromycin Plug MPGS MPGS cell 0 E48 S.
citricolor C2778 aristeromycin Plug MPGS MPGS broth 0 E49 S.
citricolor C2778 aristeromycin Shaken GAM6.6 culture 21
[0115] The results show that the apparatus supports the production
of aristeromycin by this actinomycete, specifically in layer
cultures and more generally over plug cultures. In layer cultures
and for both SV2 and MPGS media significantly higher levels of
aristeromycin are found in the broth samples from cultures produced
in accordance with the invention. The titres of aristeromycin in
those cultures are comparable to the controls (no transfer to
replacement medium) but HPLC chromatograms reveal that broth
samples in those cultures are much simpler chemically than samples
from the controls and contain a very much higher proportion of
aristeromycin relative to other sample components. This is
illustrated in FIGS. 11a and 11b with reference to E16 and E19,
with their corresponding control sample E44 illustrated in FIG.
11c.
[0116] The examples set out above demonstrate that metabolite
titres achieved in the apparatus of the specific embodiments of the
invention approach those which are achievable in a traditional
liquid shaken culture system which would use an optimised medium
for a specific microorganism. The present invention as exemplified
by the preceding procedures makes use of generalised growth media
and replacement media which are nutrient deficient, rather than
specialised media. By using generalised media, large scale trials
with a plurality of different microorganisms can be made much more
cost effective.
[0117] In all the examples where the secondary metabolite is
secreted into the nutrient deficient medium, the proportion of
metabolite relative to the other components, as indicated by HPLC,
is very significantly enhanced over controls. This enables the
sample to be concentrated by solvent evaporation to further
increase the concentration of the specific metabolite without
raising the concentration of non-specific components to a level
where they would cause interference if the sample is tested in a
biological assay. This equally applies to analysis by Matrix
Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF)
mass spectrometry (and other analytical systems) where the
measurement of a desired analyte can be significantly enhanced by
the removal of potentially interfering substances.
[0118] The enhanced resolution of peaks in HPLC chromatograms of
samples as shown in FIGS. 7a, 8a and 8b, 9a, 9b and 9c, 10a and
10b, and 11a and 11b in comparison with FIGS. 7, 8c, 9d, 10c and
11c respectively demonstrates that the present method as
exemplified herein permits easier separation of desired secondary
metabolites from other chemicals than possible with previous
fermentation apparatus and methods.
[0119] The invention allows for separation of the microorganism
under investigation from the growth medium in which mycelial
biomass is generated, in such a manner that secondary metabolism of
the microorganism can be carefully controlled. Secondary metabolism
can be carried out in a medium which is designed to promote
production of a particular metabolite. In that way, specific
components may be included in the replacement medium, as an inducer
or precursor to the mechanism by which metabolites are produced.
For example, test sample A25 demonstrates that mannitol has a
stimulatory effect on the production of GR 195359 as a secondary
metabolite of Phoma sp. F16006.
[0120] Further specific embodiments of the apparatus in accordance
with the present invention will now be described with reference to
FIGS. 12 to 18 of the accompanying drawings. It will be understood
that the apparatus described below makes use of the same principles
as the apparatus previously described, and so it can be used to
generate secondary metabolites in the same manner. However, the
apparatus described below has specific advantages which will become
apparent from the following description.
[0121] With reference to FIG. 12, fermentation apparatus 100 in
accordance with a fourth embodiment of the invention comprises a
fermentation receptacle 110 of generally cylindrical shape. A lid
112 is threadingly engaged to one end thereof. The lid 112 has a
throughbore 114, from which a peripheral flange 113 extends into
the receptacle 110. A fermentation vessel 128 of generally
cylindrical shape has an end taper-fitted to the flange 113. The
opposite end of the vessel 128 is terminated at an acute angle to
the longitudinal axis of the vessel 128, thereby forming a surface
of elliptical shape. That end of the vessel 128 has two membranes
134, 136 formed thereacross, each being of 0.6 micrometers pore
size hydrophilised melt cast polypropylene. The outer membrane 134
is fixed to the body of the vessel 128, and the inner membrane 136
is laid across the outer membrane 134. In that way, the inner
membrane 136 can be removed from the vessel 128. A polystyrene foam
filter 116 is placed in the bore 114.
[0122] By fitting the vessel 128 to the lid 112, the vessel 128 can
be transferred into and out of the receptacle easily while
maintaining aseptic conditions.
[0123] FIG. 13 illustrates the fermentation vessel 128 in more
detail. This shows the elliptical shape of the bottom end of the
vessel 128, comprising the membrane 134.
[0124] The apparatus illustrated in FIG. 12 can be used to generate
mycelial biomass, by including a quantity of a growth medium 118 in
the receptacle 110. The tip of the vessel 128 dips into the growth
medium, and the two membranes 134, 136 act as a wick, growth medium
being drawn up into the membranes 134, 136 by capillary action. The
inner membrane 136 is inoculated with a microorganism, which grows
at the air/growth medium interface provided by the wicking
membranes.
[0125] FIG. 14 illustrates further use of the apparatus illustrated
in FIG. 12. In this arrangement, the apparatus is shown after the
growth medium 118 has been replaced by a replacement medium 120,
deficient in particular nutrients so as to stimulate secondary
metabolism. In this case, the apparatus 100 is tilted such that the
replacement medium 120 makes contact with the entire outer membrane
134. Again, the inner and outer membranes 134, 136 act as wicks,
but it is advantageous to have as much of the area of the membranes
in contact with the liquid as possible, so as to promote secretion
of secondary metabolites into the medium 120.
[0126] In both FIGS. 12 and 14, the apparatus can be agitated
either by shaking or stirring as indicated by arrows 122, to
promote aeration of the medium 118, 120.
[0127] FIG. 15 shows a fifth specific embodiment of the apparatus
in accordance with the invention. The apparatus 200 is of similar
construction to the apparatus illustrated in FIG. 12. To the extent
that the apparatus 200 includes a receptacle 210, a lid 212 with
associated bore 214 and flange 213, and a foam plug 216, as
described with reference to FIG. 12, no further description of
those parts is necessary. However, the apparatus further includes a
fermentation vessel 228 of different construction to the
fermentation vessel illustrated in FIG. 12. In this case, the
vessel 228 is formed with an outer membrane 234 extended
substantially down the entire length of the vessel 228 except for a
short length at which the vessel is taper-fitted to the flange 213.
Furthermore, the outer membrane 234 extends over the opposite end
of the vessel 228, which is illustrated dipped in a quantity of a
growth medium 218. This provides a large area of membrane for
growth of microorganism thereover. As in FIG. 12, the outer
membrane 234 has an inner membrane 236 laid thereover, on which
microorganism can be grown. At the end of the membrane 234 adjacent
the portion of the vessel 228 to be taper fitted, the vessel 228 is
provided with a radially inwardly extending dam 229.
[0128] FIG. 16 illustrates the vessel 228 in further detail. The
apparatus of FIG. 15 can be used to generate mycelial biomass in
the same manner as is described in relation to FIG. 12. Moreover,
the apparatus can be used to stimulate secondary metabolism. FIG.
17 illustrates an arrangement whereby the apparatus is being used
with replacement medium 220 to stimulate such secondary metabolism.
In this case, since the membranes 234, 236 extends substantially
longitudinally of the vessel 228, the apparatus 200 can be laid
horizontally to achieve full contact of secondary medium 220 with
the membranes 234, 236. This can be advantageous since the
apparatus can be stored on a simple rack. The dam 229 prevents
ingress of liquid into the vessel 228 when in the horizontal
position.
[0129] Although the apparatus 200 is shown in a horizontal position
in FIG. 16, in practice it is unlikely that the quantity of liquid
in the receptacle 210 will be exactly the amount to produce the
arrangement illustrated in FIG. 16. However, the orientation of the
apparatus can be deviated slightly from the horizontal in order to
achieve as much contact as possible between the membranes 234, 236
and the secondary medium 220.
[0130] In each of the embodiments described in FIGS. 12 to 17, it
is clear that the microorganism is isolated from the exterior of
the fermentation vessel 128, 228, so that spores generated by the
microorganism cannot pass into the medium contained in the
receptacle 110, 210. Accordingly, secondary metabolites introduced
into secondary medium 120, 220 are separated from the biomass by
which they are produced.
[0131] By virtue of the isolation, and the definition of an inner
chamber within the vessel 128, 228, a pressure differential can be
created across the membrane 132, 232 so as to urge medium
therethrough. By controlling the pressure differential, or another
mechanism such as humidity gradient, the rate at which medium is
supplied to the microorganism can be controlled, thereby allowing
the control of metabolism, growth and cellular differentiation.
[0132] It will be appreciated that in the embodiments illustrated
in FIGS. 12 to 16, the outer membrane 134 can be augmented or . . .
replaced by an outer polypropylene sheet, with pore size up to 0.3
microns. Such a sheet 134, 234 would be capable of preventing
biomass transfer out of the vessel into the medium contained on the
receptacle. In practice, a vessel constructed in that way would
still be capable of presenting medium to a microorganism inoculated
on the inner membrane 136, since medium would soak through the
polypropylene sheet by virtue of pressure differential, humidity
gradient, or both mechanisms. Thereafter, medium which has soaked
through will wick up the inner membrane 136, 236 to the
microorganism.
[0133] It will be apparent that the invention is not limited to
vessels 128, 228 described above. For example, FIG. 18 illustrates
a component 300 comprising a wicking material with substantial
rigidity, which could be used as a fermentation vessel in the
apparatus previously described. In that component, microorganism
could be allowed to grow over the entire internal surface area of
the component 300, thus maximising the biomass thereof.
[0134] A further demonstration of the nutrient replacement
technique to substantially remove growth medium components and
enable the direct detection of secreted secondary metabolites by
MALDI-TOF mass spectrometry is demonstrated in the following sixth
example of use of the apparatus, with reference to FIGS. 17 to 20
of the drawings.
[0135] Two unidentified fungi F1 and F2 are used in the example.
For the purpose of the example, organism F1 is known to produce a
family of metabolites called verticillins while F2 is known to
produce another family of metabolites called enniatins.
[0136] Both organisms are grown in a fermentation apparatus 100 as
illustrated in FIG. 14, under previously described conditions for
fungi using FS as the growth medium and the crystalline sugar
mannitol (10 g/l) as the replacement medium. In the apparatus, a
precise volume of medium (25 ml) is placed in contact with the
maximum surface area of the membrane, as shown in that drawing. The
membranes 134, 136 are replaced by a single membrane constructed
from hydrophilic polypropylene fibre (Kimberly-Clark) with an open
structure which acts to support organism growth but not physically
prevent penetration. In each case, the apparatus is inoculated
using an agar plug containing actively growing mycelium. The growth
phase FS is maintained for 10 days and then incubation of the
replacement medium is allowed to proceed for 10 days. The
temperature under both phases of growth is controlled at 22.degree.
C.
[0137] Despite penetration of the membrane support to the medium
side, both fungi remain almost entirely attached, facilitating easy
aseptic transfer to a second vessel containing the replacement
medium. The fungal mycelium remains attached to the membrane
support while incubated on the replacement medium allowing easy
separation from the fungal biomass at the end of incubation. The
nutrient replacement medium containing secreted fungal metabolites
is retained for analysis.
[0138] In control experiments run alongside the above described
example for reasons of composition, the organisms are allowed to
grow on FS medium with no medium replacement, for a period of 20
days. A sample of the FS medium free of any fungal mycelium is
retained for analysis.
[0139] Experimental and control samples are then analysed by
MALDI-TOF mass spectrometry as follows:
[0140] 300 .mu.l of the experimental samples are dried down under
vacuum and concentrated threefold by resuspending in 100 .mu.l 50%
methanol in deionised water containing 0.1% trifluoroacetic acid.
The aqueous control samples are analysed directly without the
concentration step. 0.5 .mu.l of sample is mixed with 0.5 .mu.l of
matrix (20 mg/ml 2,5-dihydroxybenzoic acid in deionised water) on a
mass spectrometer slide and allowed to dry. The slide is then
inserted into the instrument. The mass spectrometer is operated in
reflectron mode with an extraction voltage of 40 kV. The laser is
tuned to an optimal level for the analysis of each sample.
[0141] In the spectra of the experimental samples, peaks
corresponding to the verticillins (organism F1) are prominent and
clearly identifiable (FIG. 19a). A fragment of the particular area
of interest of FIG. 19a is expanded in FIG. 19b.
[0142] For example, a peak which is prominent in FIG. 19b, has a
mass/charge ratio of 755.5 corresponds with verticillin B,
potassium adduct (M.sub.B+3H+ K.sup.+) Another peak, prominent at a
mass/charge ratio of 771.1 corresponds with verticillin C, sodium
adduct (M.sub.C+0.3H+ Na.sup.+). These correspondences are provided
in libraries of data which are in the public domain.
[0143] Similarly, the experimental samples generated from organism
F2 are analysed by MALDI-TOF mass spectrometry and identify members
of the enniatin family. These are shown in the spectra illustrated
in FIGS. 21a and 21b.
[0144] As shown in FIG. 21b, a peak is prominent at a mass/charge
ratio of 663.2. This corresponds with enniatin B, sodium adduct
(M.sub.B+Na.sup.+). A peak at mass/charge ratio 677.1 corresponds
with enniatin B, potassium adduct (M.sub.B+K.sup.+), a peak at
691.6 corresponds with enniatin D, potassium adduct
(M.sub.D+K.sup.+) and a peak at mass/charge ratio of 706.8
corresponds with enniatin A, sodium adduct
(M.sub.A+2H+Na.sup.+).
[0145] The corresponding control samples (FIG. 20 for F1 and FIG.
22 for F2) generate very poor spectra under MALDI-TOF mass
spectrometry. In these spectra, there is no evidence of a peak
corresponding to either of the verticillin (FIG. 20) or the
enniatin (FIG. 22) species identified in respect of the
experimental samples.
[0146] For optimal results in applying MALDI-TOF mass spectrometry
to a sample, it is desirable that the sample crystallises with the
matrix on the slide prior to analysis. Crystallinity is not
apparent on the slides containing control samples which explains
the poor analytical results. This problem arises from the higher
concentrations of medium components in each control sample, which
cause a syrup to be formed when the control sample is
dehydrated.
[0147] It can be seen from the foregoing example that the nutrient
replacement process clearly generates samples of fungal origin
which can be analysed directly by MALDI-TOF mass spectrometry
without extensive pre-preparation.
[0148] A further example will now be used to demonstrate that the
procedures and apparatus are applicable to the secretion of
proteins into a "clean" medium, allowing for ease of isolation. In
this example, another unidentified fungus, F3 is grown under
identical conditions as F1 and F2 and experimental and control
samples analysed by MALDI-TOF mass spectrometry. The spectrum
corresponding to the experimental sample is illustrated in FIG. 23,
and that corresponding to the control sample is illustrated in FIG.
24. In the experimental samples, a characteristic peak
corresponding to a small unidentified protein with an m/z
(mass/charge ratio) of 6239.1 is clearly visible (FIG. 23). Again,
the corresponding control spectrum shown in FIG. 24 is poor and no
protein peaks are detectable.
[0149] The nutrient replacement process therefore provides a means
of culturing organisms to produce samples containing secreted
proteins which can be detected directly by MALDI-TOF mass
spectrometry (a technology used extensively for protein and peptide
analysis).
[0150] Combining the nutrient replacement process with MALDI-TOF
analysis therefore enables the direct screening of organisms for
secreted protein products. The organisms may be wild type strains
or genetically modified by the insertion of a gene (expressing a
known or unknown protein) into a suitable host. The presently
described procedures and apparatus allow such protein expression to
be conducted and analysis to be applied directly to the generated
samples, without the need for intermediate steps to increase the
purity or cleanness of the sample. Purity and cleanness are
concerned with the level of impurities in the sample--the
concentration of the desired biochemical in the sample is of less
importance than the need to ensure that other chemicals do not
prevent operation of or obscure the spectrum of the chemical or
chemicals under investigation.
[0151] Once metabolites have been produced by the methods described
above in accordance with the apparatus illustrated in the
accompanying drawings, they can be isolated and prepared in
accordance with known methods to produce pharmaceuticals for
medical or veterinary use, or to produce agrochemicals such as
fungicides or other pesticides. Moreover, the metabolites can be
extracted to establish their chemical structures, as a precursor to
identify alternative methods of production thereof, such as by
non-biological chemical processes.
[0152] In particular, samples of secondary metabolites can be
produced by methods as described above in accordance with specific
embodiments of inventions, for development of new biochemicals,
such as pharmaceuticals (both medical and veterinary) and
agrochemicals (e.g. pesticides, fungicides, herbicides and growth
regulators). A large array of different metabolites can be produced
with ease. Each metabolite can then be tested for efficacy, for
instance as a pharmaceutical or agrochemical, and any metabolites
demonstrating useful effects can then be selected for further
development. Further development includes the steps of identifying
a method by which metabolite can be produced for commercial
exploitation thereof. This may be by large scale fermentation in
accordance with the described procedures, or alternatively it could
involve identifying the molecular structure of a metabolite so that
it can be synthesised.
[0153] It will be appreciated by the reader that the term
metabolite is being used in its broadest sense, i.e. a biochemical
the product of a biosynthesis process within, or associated with, a
microorganism. In that sense, a metabolite would include one of the
secondary products associated with metabolism in a fungus, and may
also include metabolic products such as enzymes, proteins and
peptides.
* * * * *