Method of using an elicitor to increase production of metabolites in biological cells

A method for culturing fungal and plant cells in a culture medium containing an elicitor to increase production of a metabolite produced by the cells. For example, tobacco leaves produce the metabolite nicotine and the nicotine is produced in significantly greater amounts when the tobacco is cultured in a elicitor-containing medium. The elicitor is an oxidized Bovine Serum Albumin (BSA), or a glycosylated BSA, or an oxidized lysozyme, or glycosylated lysozyme or azetidine-2-carboxylic acid. Further, metabolite production is increased 1000% over the production of the metabolite when an elicitor is not added during the culturing method.

FIELD OF THE INVENTION 
The present invention relates to a method for the formation of useful 
metabolites in biological cells, and in particular a method for promoting 
or inducing the formation of such useful metabolites, by contacting the 
cells in culture media with elicitors. 
BACKGROUND OF THE INVENTION 
Plant-derived secondary metabolites form the basis of important segments of 
the good, pharmaceuticals, pesticide and cosmetic industries. Interest in 
such metabolites will continue to grow as e.g. plant sources of new and 
useful drugs are discovered. Moreover, it is becoming increasingly known 
that fungi, bacteria, algae and animal cells are also sources of 
potentially useful metabolic products. The recovery of useful metabolites 
from natural sources is in many instances constrained, however, by the 
remote location of such sources, and even more by the enormous quantities 
of source material which may be required for the isolation of utilizable 
quantities of the desired products, which problems are of course reflected 
in the often very high prices of the latter. 
The problems of recovery of useful secondary metabolites from natural 
sources may potentially be circumvented by cell culture. Whereas in 
plants, for example, production of the desired products is often confined 
to a few specialized cells and specific differentiated tissues, as well as 
to limited stages in the development of the cells, under suitable cell 
culture incubation conditions, on the other hand, it should be possible to 
obtain a higher yield in the production of the metabolites, and 
independently of the metabolic periodicity often found in nature. In 
practice, however, cultured plant cells tend to lose their competence for 
the production of the desired metabolites, for various reasons, and to the 
inventors' knowledge, the only such commercial process at the present time 
is the production of shikonin from Lithospermum erythrorhizon cells in a 
two-stage process. Attempts to overcome such problems by cloning, 
selecting and subculturing, or by manipulating the culture medium, have so 
far not met with notable success. 
If, as seems to be agreed, culture productivity is predominantly a function 
of the cells' own control mechanism, it should be more useful to be able 
to induce the synthesis of enzymes which produce useful metabolites at the 
gene level. It is known that enzymes may be induced by fungal elicitors 
after infection of plants or cultured cells by pathogens to produce 
phytoalexins. Elicitors are agents which induce plants to synthesize the 
mRNA's and enzymes required for the synthesis of the induced products 
(phytoalexins); extracts of mycelium or cell walls from the pathogens may 
serve as effective elicitors, which analysis has shown to comprise oligo- 
or polysaccharides and low molecular weight compounds. However, many 
pathogenic organisms which are sources of elicitors are relatively 
difficult to cultivate on a large scale. The present invention is based 
upon the discovery of elicitors which do not depend on the cultivation of 
pathogens. 
These elicitors have been found to promote and induce the formation of 
useful metabolites in biological cells. By the term "promote" in this 
context, it is intended to convey that the production of useful 
metabolites is enhanced. However, the production of such metabolites is 
also induced in cell cultures which are otherwise inactive in this 
request. 
It is accordingly an object of the present invention to provide substances 
which promote or induce the formation of useful metabolites in biological 
cells from diverse groups of organisms. 
A further object of the invention is the provision of a method for the 
promotion or induction of the formation of useful metabolites in 
biological cells. 
Yet a further object of the invention is the provision of such a method 
which utilizes elicitors. 
Another object of the invention is to provide such a method by which the 
desired cell metabolic products may be produced in economically viable 
yields. 
Yet another object of the invention is to provide such a method in which 
the elicitors are preformed. 
Still another object of the invention is to provide such a method in which 
the elicitors are formed in situ. 
A further object of the invention relates to elicitors which are novel 
substances. 
Yet other objects of the invention will appear from the description which 
follows. 
SUMMARY OF THE INVENTION 
The present invention accordingly provides in one embodiment a method for 
promoting or inducing the formation of useful metabolites in biological 
cells, which comprises containing biological cells in a culture medium 
with an effective amount of at least one promoting or inducing elicitor 
selected from the group consisting of (1) proteins and polypeptides 
comprising sulfoxide moiety-containing amino acid units; (2) proteins and 
polypeptides comprising glycosylated amino acid units; (3) proteins and 
polypeptides comprising amino acid units of which at least part are 
sulfoxide moiety-containing and at least part are glycosylated; (4) 
modified proteins produced in situ by the action on biological cells of 
antimetabolites having the capacity to produce modified proteins; and (5) 
combinations thereof; the biological cells being plant cells (including 
calli and plant cells in suspension), fungi cells, bacteria cells, algae 
cells and/or animal cells. 
Preferably, the method of the invention is effected in the presence of at 
least one of the following additional ingredients, namely: 
at least one substance selected from 
(a) vanadium compounds and other substances effective to neutralize the 
effect of ATP-dependent and possible other proteases produced by the cells 
as a defence reaction to the presence of the at least one elicitor, and 
(b) particles of at least one substance selected from carborundum and other 
substances having sufficient hardness to abrade the surface of the cells 
for the purpose of improving contact between the latter and the at least 
one elicitor.

DETAILED DESCRIPTION OF THE INVENTION 
In an embodiment of the method of the invention, the elicitor may, for 
example, comprise at least one member selected from naturally occurring 
thioether moiety-containing proteins wherein at least part of the 
thioether content has been replaced by sulfoxide in vitro and such 
proteins which have in addition been glycosylated in vitro. In another 
embodiment of the method of the invention there may be used glycosylated 
proteins which need not contain thioether or sulfoxide moieties. 
In a particular embodiment of the method of the invention, the elicitor may 
comprise at least one sulfoxide moiety-containing protein isolated from 
natural sources, or a derivative thereof made by glycosylation in vitro. 
In yet another embodiment of the method of the invention, the elicitor 
comprises at least one thioether moiety-containing polypeptide not 
occurring in nature or/and such polypeptide which has been glycosylated, 
and in which at least part of the thioether content has been replaced by 
sulfoxide. 
In still another embodiment of the method of the invention, the elicitor 
comprises polymethionine in which at least part of the thioether content 
has been replaced by sulfoxide or/and polymethionine which has been 
glycosylated and in which at least part of the thioether content has been 
replaced by sulfoxide. 
The elicitors may be introduced into the cell culture media in 
concentrations of up to (e.g.) about 300 .mu.g./ml. 
While the present invention is not to be restricted by any theory of 
action, nevertheless it is presently believed that the elicitors elicit 
the transcription of new mRNA's followed by the formation of enzymes which 
lead to the production of various cell-specific products. In some cases, 
abnormal proteins of promoting or inducing potential were synthesized in 
situ by the presence of antimetabolites having the capacity to produce 
modified proteins such as azetidine-2-carboxylic acid; it will be apparent 
to those skilled in the art that other equivalent antimetabolites having 
the capacity to produce modified proteins could potentially be utilized 
for a similar purpose. 
The use of particles of added carborundum, or other abrading substance 
(such as e.g. tungsten carbide or boron carbide) of sufficient hardness to 
abrade the surface of the cells for the purpose of improving contact 
between the latter and the at least one elicitor, in accordance with an 
embodiment of the invention, has already been mentioned; it is especially 
useful in cultures of cells of higher plants and of algae. Also mentioned 
above, was the optional use of a vanadium compound, preferably of 
pentavalent vanadium such as a metavanadate or orthovanadate, e.g. sodium 
metavanadate or orthovanadate. The vanadium compound can in some cases be 
replaced by other nonspecific protease inhibitors such as heparin, poly 
(glut-tyr) (at a 1:1 ratio) and polyanions. 
Examples of particular preformed elicitors (to which the invention is of 
course not limited) which may be utilized within the scope of the 
invention are: 
(a) bovine serum albumin, lysozyme, collagen and hemoglobin in which at 
least some of the thioether moieties in the methionine residues have been 
replaced by sulfoxide, as e.g. by oxidation with oxidizing agents such as 
hydrogen peroxide; 
(b) polymethionine (which is commercially available as the poly-L-form in 
molecular weight ranges of e.g. 5-15, 16-36, 30-50 and 
100-200.times.10.sup.3), in which the thioether moieties in the methionine 
residues have been replaced by sulfoxide, as e.g. by oxidation with 
oxidizing agents such as hydrogen peroxide; 
(c) purified protein of pea and other legume seeds, which contain 
methionine residues of which about 20% have been oxidized (i.e. the 
thioether moieties are in sulfoxide form); 
(d) Amadori protein products of lysozyme, protamine and hemoglobin, which 
have been submitted to a prolonged glycosylation process; and 
(e) the products specified in (a), (b) and (c), which have been subjected 
to glycosylation. 
It is presently contemplated that the invention will be applicable to the 
production of (e.g.) the following classes of plant-derived alkaloids, 
flavanoids, glycosides, naphthoquinones, polyphenols, steroids, tannins 
and terpenoids, and more particularly to the products of commerce 
mentioned below. 
Food Ingredients 
Colors: anthocyanins, betacarotene, betacyanins, canthaxanthin and saffron. 
Flavors: strawberry, grape, vanilla, tomato, celery, asparagus. 
Oils: mint, rose, vetiver, jasmine, patchouly, sandalwood, lemon, onion, 
garlic. 
Sweeteners: stevioside, thaumatin, miraculin, monellins. 
Agricultural Chemicals 
Pyrethrins, rotenone, azadirachtin, neriifolin, solanine, alleopathic 
chemicals. 
Pharmaceuticals 
Codeine, morphine, scopolamine, atropine, colchicine, cocaine, vinblastine, 
L-dopa, hyoscyamine, diosgenin, digitoxin, digoxin, quinidine, quinine, 
vincristine, shikonin, ajmalicine, serpentine, physostigmine, pilocarpine, 
tubocurarine, eicosapentaenoic acid. 
Unclassified Pigments 
Phycobiliproteins such as phycocyanins, phycoerythrins, allophycocyanine 
and phytochrome. 
It is moreover within the ambit of the present invention to produce useful 
metabolites from animal cells, e.g. cytokines from blood cells. 
Lignin is the most abundant organic material next to cellulose and is in 
any case the most abundant waste material. It is a complex polymeric 
material containing phenylpropanoid subunits, so that it is potentially 
the source of aromatic chemicals, although in practice it has so far been 
found resistant to biodegradation. A fungal enzyme, ligninase, has been 
isolated, and this can depolymerize lignin, but its exploitation has so 
far been limited by a number of factors including its scarcity. According 
to the prior art, the enzyme-carrying fungus requires a 100% oxygen 
atmosphere for ligninase formation. It has surprisingly been found in 
accordance with a particular embodiment of the present invention, that by 
utilizing elicitors as disclosed herein, a notable increase in ligninase 
yield from the fungus Phanerochaete chrysosporium is obtained in absence 
of a 100% oxygen atmosphere. 
The method of the present invention as applied to the production of 
ligninase is believed to pen new vistas for lignin treatment. The 
potential availability of ligninase on a industrial scale, applied to the 
wood utilization industries in place of the current chemical methods 
should improve quality by causing less damage to cellulose fibers, 
reducing yellowing and cutting energy costs by perhaps 30%. It is believed 
that enzyme treatment could be used in existing paper plants. Isolation 
from lignin treatment of useful aromatic compounds such as vanillin, and 
other flavorings and fragrances, would also be a potential gain derived 
from this particular application of the method of the present invention. 
Ligninase is also active in the detoxification of hazardous waste material 
such as chlorinated lignin, polycyclic aromatic pollutants, TNT, etc. 
The term "ligninase" as used herein is intended as a functional term and is 
not intended to imply any particular limitation as to the chemical 
structure thereof. 
Experiments conducted by the inventors have shown the following 
non-limitative examples of advantageous results, when applying the process 
of the invention by use of S-oxidized and glycosylated proteins or 
polypeptides. 
1. In cultured cells of Nicotiana tabacum (tobacco) the yield of nicotine 
was enhanced up to 100-1000 fold; the yield of other unidentified 
metabolites was also increased similarly. 
2. In cultured undifferentiated Atropa belladona leaf cells the yield of 
atropine was enhanced 100-1000 fold; the yield of other unidentified 
metabolites was also increased similarly. This result is of particular 
significance since in Belladona only cultures roots produce detectable 
atropine levels. 
3. In cultures cells of Dioscorea deltoides (Mexican yam) the yield of 
diosgenin was increased 5 to 10 times. 
4. In cultured cells of Catharanthus roseus (rosy periwinkle) the yield of 
anticancer alkaloids was increased almost 100 times. 
5. Modified proteins were found to stimulate the production of useful 
metabolites (such as those indicated in parentheses) in the microalgae 
Porphyridium cruentum and Monodus subterraneous (eicosapentaenoic acid, 
arachidonic acid); and Haematococcus sp. (canthaxanthin). 
6. In cultures cells of white rot fungus (Phanerochaete chrysosporium) the 
yield of ligninase was increased about 20 times, in comparison with 
untreated cells. 
The invention will now be illustrated by the following non-limitative 
examples. 
EXAMPLE I 
Nicotiana tabacum cv. Xanthi (tobacco) leaf cells (about 300-400 mg. fresh 
weight per flask) were grown in the dark in suspension in a growth medium, 
as described by Murashige and Skook (Physiol. Plantarum 1962 15: 473), 
containing 1 mg./l. 1-naphthaleneacetic acid, 0.1 mg./l. kinetin and 3% 
sucrose in 125 ml. Erlenmeyer flasks on a rotary shaker (120 rpm, 
25.degree. C.). The volume of growth medium per flask was 30 ml., which 
contained about 10-20 mg. fine mesh carborundum. Comparative runs were 
carried out with and without addition to the medium of modified protein in 
the form of oxidized BSA (20 .mu.g./ml.). After 4 weeks, the cells were 
harvested, weighted, frozen and lyophilized. The dried cells were 
extracted with methanol in a Soxhlet extractor and the extract was 
evaporated to dryness at 40.degree. C. with a Buchi Rotoevaporator. The 
residue was dissolved in 0.5N HCl and extracted with ether. The aqueous 
layer was brought to pH 9.5 with NaOH, extracted twice with ether and the 
combined two ether extracts were dried with K.sub.2 CO.sub.3, prior to 
adding HCl and then evaporating to dryness. The residue was dissolved in 
0.5N HCl and aliquots were applied to TLC plates of silica gel G (Merck). 
The plates were developed in 85:14:1 chloroform-ethanol-ammonia, dried in 
a stream of N.sub.2, and alkaloids were visualized by Dragendorff's 
reagent. Quantitatively the alkaloids were determined by GLC analysis. In 
absence of elicitor there were obtained 2.8.times.10.sup.-3 % (dry weight) 
nicotine, compared with 2.2% (dry weight) in presence of elicitor, the 
latter representing a 7.8.times.10.sup.3 fold enhancement of yield. The 
dry weight of cells at the end of the run was 3.2 g. per flask. 
EXAMPLE II 
Belladona atropa cells (about 200 mg. fresh weight per flask) were grown in 
suspension in a medium described by Wood and Brown (P.N.A.S. 1961 47:1907) 
and Sharma and Khanna (Ind. J. Pharm. Sci. 1981 43: 175), containing 0.22 
mg./l. 2,4-dichlorophenoxyacetic acid and 2.0 mg./ml. kinetin. The volume 
of growth medium per flask was 30 ml., which contained about 10-20 mg. 
fine mesh carborundum. Comparative runs were carried out with and without 
addition to the medium of elicitor in the form of oxidized BSA (20 
.mu.g./ml.). After 5-6 weeks, the cells were harvested, weighted, frozen 
and lyophilized. Further processing of the harvested cells and 
determination of the alkaloids by TLC and GLC, was carried out as 
described in Example I. In absence of elicitor 1.8.times.10.sup.-4 % (dry 
weight) atropine were obtained, compared with 0.2% (dry weight) in 
presence of elicitor, the latter representing a 1.1.times.10.sup.3 fold 
enhancement of yield. The dry weight of cells at the end of the run was 
2.1 g. per flask. 
EXAMPLE III 
Dioscorea deltoides (Mexican yam) cells (about 300-400 mg. fresh weight per 
flask) were grown in the dark in suspension in a growth medium, as 
described by Murashige and Skook (Physiol. Plantarum 1962 15: 473), 
containing 0.1 mg./l. 2,4-dichlorophenoxyacetic acid, 1 mg./l. nicotinic 
acid, 10 mg./l. thiamine-HCl, 1 mg./l. pyridoxine-HCl and 100 mg./l. 
inositol, in 125 ml. Erlenmeyer flasks on a rotary shaker (100 rpm, 
28.degree. C.). The volume of growth medium per flask was 30 ml., which 
contained about 10-20 mg. fine mesh carborundum and about 10 mg. sodium 
orthovanadate. An approximately equimolar amount of sodium metavanadate 
could be used instead of the orthovanadate. Comparative runs were carried 
out with and without addition to the medium of elicitor in the form of 
oxidized BSA (20 .mu.g./ml.). After about 3 weeks, the cells were 
harvested, lyophilized, refluxed for 2 hours in 2N HCl, filtered, washed 
with water and dried. The cells were then extracted with chloroform at 
60.degree. C., the extract was filtered and evaporated to dryness, and the 
residue was taken up in chloroform and chromatographed. The spots were 
visualized with antimony chloride in chloroform. The spots were then 
dissolved in chloroform and the O.D. read in a spectrophotometer. Some 
determinations were made by GLC. In absence of elicitor there were 
obtained 0.1% (dry weight) diosgenin, compared with 3.8% (dry weight) in 
presence of elicitor, the latter representing a 38 fold enhancement of 
yield. The dry weight of cells at the end of the run was 1.6 g. per flask. 
EXAMPLE IV 
Catharanthus roseus (rosy periwinkle) cells were grown in suspension in 
Linsmaier-Skook's medium (Physiol. Plantarum 1965 18: 100) containing 0.22 
mg./l. 2,4-dichlorophenoxyacetic acid and 2.0 mg./ml. kinetin. The volume 
of growth medium per flask was 30 ml., which contained about 10-20 mg. 
fine mesh carborundum. Comparative runs were carried out with and without 
addition to the medium of elicitor in the form of oxidized BSA (20 
.mu.g./ml.). After 4 weeks, the cells were harvested, and the alkaloids 
were extracted as described by Smith et al (Plant Cell Rep. 1987, 6: 142) 
and assayed by TLC. The plates were developed by 3:1 ethyl 
acetate/absolute ethanol and were then viewed first under UV, afterwards 
spraying with ceric ammonium sulfate. Qualitative and quantitative 
analyses were also conducted by mass spectroscopy. In absence of modified 
protein there were obtained 1.2.times.10.sup.-3 % (dry weight) alkaloids 
(vinblastine+vincristine+catharantine+ajmalicine), compared with 0.1% (dry 
weight) in presence of elicitor, the latter representing an 83 fold 
enhancement of yield. 
EXAMPLE V 
Cells of white rot fungus (Phanerochaete chrysosporium) were grown in air 
in a chemically defined standard medium (Kirk et al, Enzyme Microb. 
Technol. 1986 8: 27) in absence or presence of Tween 20 (Jaeger et al, 
Appl. Environ. Microb. 1985 50: 1274). The volume of growth medium per 
flask was 30 ml.; no carborundum was used. Comparative runs were carried 
out with and without addition to the medium of elicitor in the form of 
oxidized BSA (10 .mu.g./ml.) After 3-4 days, the cultures were filtered 
through Whatman No. 1 filter paper, and ligninase activity in the solution 
was measured by determining the rate of oxidation of veratryl alcohol to 
veratraldehyde (Tien and Kirk, P.N.A.S. 1984 81: 2280). by following the 
change in optical density at 310 nm per min., per ml. of enzyme solution. 
In presence of elicitor there was obtained a 20-fold increase in the 
optical density as compared with a control experiment not using elicitor. 
FIG. 1 shows the variation of the amount of ligninase produced with 
oxidized BSA concentration, in similar experiments to the foregoing. FIG. 
2 shows the variation of the amount of ligninase produced with the 
concentration of glycosylated lysozyme (instead of oxidized BSA), in 
otherwise similar experiments to the foregoing; in this connection, we 
have found that the use of unmodified lysozyme has a negative effect, 
reducing the yield of ligninase to one-third of the control. FIG. 3 shows 
the variation of the amount of ligninase produced with the concentration 
of azetidine-2-carboxylic acid, which acts on the fungus cells to produce 
modified protein in situ (instead of oxidized BSA), in otherwise similar 
experiments to the foregoing. 
PREATION OF ELICITORS 
Preparation A; Conversion to Thioether Moieties in Proteins and 
Polypeptides to Sulfoxide 
Bovine serum albumin fraction V, lysozyme, hemoglobin, collagen and 
poly-L-methionine (MW in the range of 16-36.times.10.sup.3) were each 
(separately) oxidized in 0.25N HCl with 0.3M hydrogen peroxide at 
23.degree. C. for one hour. The mixture was then dialyzed and lyophilized. 
The sulfoxide content of the product was demonstrated by reduction to 
thioether with 0.8M 2-mercaptoethanol for 48 min. at 37.degree. C., the 
sulfone moieties not being reducible under these conditions. Analysis 
showed that 80-90% of the thioether moieties of the protein methionine 
residues had been converted to sulfoxide. 
Preparation B: Glycosylation of Proteins 
Introduction 
Irreversible glycosylation of proteins occurs progressively in a 
protein-glucose mixture, with elimination of the elements of water. This 
process leads to a rearrangement of the nonenzymatic addition product of 
glucose with protein amino groups, the Amadori product. Proteins modified 
by advanced glycosylation continue to accumulate, and in tissue proteins 
serve as biological markers of protein age (Monnier et al, P.N.A.S. 1984, 
81: 583). 
Method 
Protein (either lysozyme or protamine: 1 g.) and glucose (300 mg.) were 
dissolved in water (100 ml.), and the mixture adjusted to pH 8.5 with 
ethanolamine and lyophilized. The residue was transferred to a humidity 
chamber at 50.degree. C. with saturated KI (1 g. KI in 0.5 ml. water at 
100.degree. C. to ensure supersaturation at 50.degree. C.) to obtain 65% 
relative humidity. The protein-glucose mixture was kept in the humidity 
chamber for at least one week (after 3 days, 25-30% glycosylation; after 7 
days, about 50% glycosylation). Thereafter, the protein-glucose mixture 
was dissolved in 100 ml. water, dialyzed against water and finally 
lyophilized. 
In place of the exemplary lysozyme or protamine there may be used one of 
the sulfoxide-containing products of Preparations A or C (below). 
Preparation C: Isolation of Native Proteins from Legumes 
Proteins were extracted from dry seeds of pea cv. Progress No. 9. The seeds 
were crushed, milled and extracted with phosphate buffer (0.2M, pH 6.0). 
The proteins were precipitated from the extract with 10% (v/v) aqueous 
trichloroacetic acid solution. After centrifuging, the precipitate was 
dissolved in water, dialyzed and lyophilized. The amino acid analysis of 
these proteins, after basic hydrolysis, indicated that 20% of their 
methionine residues were present as the corresponding sulfoxide. 
While certain embodiments of the invention have been particularly 
described, it will be appreciated by persons skilled in the art that many 
variations and modifications may be made without departing from the scope 
and spirit of the invention. These embodiments are therefore to be 
construed merely illustratively, and the scope of the invention is to be 
regarded as defined rather by the claims which follow: