IL88953A - Method for increasing enzyme activity of organisms - Google Patents

Abstract

In the process disclosed, the organisms are brought into contact witn inactivated elicitor-containing microorganisms, fragments of the latter, or excretions of elicitor-containing microorganisms. Only inactivated elicitor-containing bacteria, fragments of the latter, or excretions of elicitor-containing bacteria may be used to activate enzymes and promote synthesis in non-microbial organisms. The process can be used, for example, to promote synthesis in microorganisms or plants which produce pigments, antibiotics, alkaloids or phytoalexins or to activate enzymes in microorganisms capable of transforming steroids. [EP0325933A1]

Description

•METHOD TOR INGKCAQING ENBYME ACTIVITIES AND SYNTHESIS PERFORMANCE OF ORGANISMS Background of the Invention This invention relates to a process for increasing enzyme activities and synthesis performance of organisms .

As is known, elicitors are microbial or vegetable active agents which, when brought into contact with tissues of higher plants, increase enzyme activities and synthesis performance of the latter. The ingredients thus accumulated in these plants are called phytoalexins if they are antimicrobial. (Naturwissenschaften 68, Ϊ981, 447ff; Adv. Enzymol. 55, 1983, Iff; Spektrum Der Wissenschaft1 · 11, 1985, 85ff) .

At present, more than 100 compounds meeting the phytoalexin definition have been isolated from various types of plants. They belong to various groups of natural substances, such as terpenoids, linolenic acid derivatives, acetylenes and polyacetylenes, bibenzyls, stilbenes, phenanthrenes and dihydrophenanthrenes , benzofurans and phenolbenzofurans, furocoumarins, avenalumins, flavanes, phenylbenzofurans, benzoxazinones, alkaloids, isoflavonoids, etc. (Brooks and Watson, Nat. Prod. Reports 2 : 427, 1985).

Thus far, this elicitor effect has not been exploited industrially, for several reasons: Except for a few exceptions, it has not been possible heretofore to grow cells of higher plants in submerged cultures under economically feasible conditions. Heretofore, it has seemed useless a priori to employ elicitors in fermentation by means of microorganisms since it had to be assumed, pursuant to the prevailing viewpoint taught regarding the mechanism of elicitor action (Albersheim, P. and Darvill A.G. : "Spektrum der Wissenschaft" 11 : 85, 1985) , that these elicitors do not affect enzyme activity and the metabolic processes in microorganisms. It is also worth noting that, according to prevalent opinion, bacteria can trigger phytoalexin formation in plants only by releasing, by means of certain enzymes, elicitors from the vegetable cell membrane, stimulating phytoalexin formation as endogenous elicitors.

Summary of the Invention It has now been discovered that compounds and cell preparations, called "elicitors" hereinbelow, are surprisingly capable, after all, of increasing enzyme activities in microorganisms and the synthesis performance of the latter. Moreover, it has been found that there are also bacteria which contain elicitors that are not enzymes or nutritive factors and thus which can provide exogenous elicitors to other organisms.

Thus, this invention involves increasing (enhancing) enzyme activities and synthesis performance of organisms, i.e., enhances the ability of an organism to affect its environment by its enzymatic activity, e.g., by providing enzymes thereto, e.g., to catalyze reactions and enhances the ability of an organism itself to synthesize products. This is achieved by bringing the organisms into contact with inactivated elicitor-containing microorganisms, fragments thereof, or excretions of elicitor-containing microorganisms, with the proviso of using, for increasing enzyme activities and synthesis performance of nonmicrobial organisms, - 3 - 88953/2 inactivated elicitor-containing bacteria, fragments thereof, or excretions of elicitor-containing bacteria.

Thus, the present invention provides a method for enhancing the product synthesizing performance of a culture of a first microorganism or a cell culture of a higher plant, comprising contacting said culture with an elicitor in the form of an inactivated elicitor-containing second microorganism, an elicitor-effective fragment thereof, or an elicitor-effective excretion of an elicitor-containing second microorganism, with the proviso that when said culture is a cell culture of a higher plant, said elicitor-containing second microorganism is a bacterium.

It will normally be much too expensive to isolate elicitors proper, or to synthesize them in order to utilize them thereafter for influencing the metabolism of other organisms. Fortunately, per this invention, it is usually sufficient to use inactivated elicitor-containing microorganisms or fragments of these microorganisms. Suitable fragments will be any which contain effective amounts of the elicitor (s), such as, for example, cell wall fractions, cell fragments of mechanically processed or chemically or enzymatically lysed cells, or cell components precipitated ij auxiliary agents, such as, for example, ethanol or acetone, among other fragments. Of course, use of isolated or synthesized elicitors is also within the scope of this invention.

In the case where the microorganism releases (e.g., excretes) elicitors into the culture broth, or forms water-soluble elicitor-containing cell ingredients after lysis or sterilization, then these elicitor-containing excretions of the microorganisms can likewise be utilized for conducting the process of this invention. In essence, the elicitor can be used in any form in which it is derived from the microorganism such that it is effective for the purpose of this invention.

Microorganisms known to exhibit elicitors include, inter alia , the fungal strains and yeasts listed in Table 1 below.

In recently conducted tests, cell wall preparations, purified proteolytically by means of trypsin, of gram-positive bacterial strains' of the genera Bacillus, Corynebacterium, Brevibacterium, Cellulomonas, Lactobacillus, Pimelobacter, Rhodococcus and Staphylococcus, and microorganisms of these genera heat-sterilized in water, and their filtrates were investigated for elicitor content. Bacterial strains proven to contain elicitors are set forth in Table 2 below.

T A B L E 1 Arch. Bioch.Biop. 221. 1984. 136 D ο t r y t : 5 c 1 n Therefore, there Will be numerous additional microorganisms that likewise have elicitors, such as, for example, bacteria of the genera Mycobacterium, Nocardia, Nocardioides or Pseudonocardi . Testing of microorganisms for elicitor activity can be effected without problems, using the customary, fully conventional screening tests familiar to those skilled in the art.

Thus, in typical series tests, the microorganisms or other organisms whose enzyme activity or synthesis performance is to be increased can be grown, for example, in submerged cultures, e.g., using the microorganisms of the examples. Inactivated candidate microorganisms of varying species or sub-species can be added to the individual cultures, and, after fermentation has taken place, an analysis can be made as to which cultures demonstrate an increase in enzyme activity (e.g., increase in yield or rate of production of a product produced by enzymatic action provided in a medium by a microorganism) or synthesis performance (e.g., increase in yield of product produced by a microorganism per se or its rate of production) , all using conventional methodologies.

As shown by the experiments conducted thus far, described in greater detail in the examples, the process of this invention is applicable with great versatility for increasing enzyme activities or synthesis performance of microorganisms. Thus, for example, by adding cell wall preparations of microorganisms listed in Table 2, the chromogenesis of Streptomyces lividans (actinorhodin, prodigiosin) as well as the color production of Streptomyces coelicolor, of Streptomyces griseoruber, of Streptomyces latericius, of Streptomyces purpurascens, and of Streptomyces violaceus could be stimulated. It was furthermore possible to stimulate formation of β-lactam antibiotics of Streptomyces clavuligerus and the alkaloid synthesis of Claviceps paspali. Such increases in the synthesis performance of microorganisms can be achieved not only by means of cell wall preparations of the bacteria listed in Table 2 but an increase in enzyme activities and synthesis performance of microorganisms can also be attained by means of elicitor-containing fungi and yeasts as set forth in Table 1.

It was furthermore possible to obtain a significant increase in alkaloid formation with the aid of cell wall preparations of microorganisms set forth in Table 2 in cell cultures of higher plants, such as Eschscholtzia californica or Rauwolfia serpentina.

Furthermore, a marked increase has been achieved using elicitors, e.g., with the aid of these cell wall preparations, in the enzyme activity of microorganisms, e.g., in the capability of Bacillus lentus to dehydroge-nate steroids in the 1, 2-position, and in the capability of Rhodotorula glutinis to selectively reduce 17-keto steroids, and in the capability of Penicillium raistrickii to hydroxylate steroids in th§ 15 -position. Thus far, only an attempt to increase, with the aid of these cell wall preparations, the ability of Curvularia lunata to the Ιΐβ-hydroxylate steroids has remained unsuccessful.

Determination of whether a given elicitor-containing microorganism will be effective to increase enzyme activity or synthetic capabilities of a given desired organism will be carried out routinely as will a determination of which elicitor-containing microorganisms do effect such an increase. - These experiments demonstrate that the process according to this invention will also stimulate the formation of numerous other commercially exploitable microbial ingredients and those of other organisms, and increase other enzyme activities of (micro) organisms that are industrially useful.

Such microbial ingredients include, for example, antibiotics such as the penicillins, cephalosporins, cyclosporins, actinomycins, gramicidins, neomycins, gentamycins , nystatins, tetracyclins, nikomycins or lincomycin, erythromycin, chloramphenicol, griseofulvin, or fusidic acid, etc. ; ergot alkaloids, such as ergocryptin, ergotamine, ergosine, ergocristine, ergocornine, agroclavine, chanoclavine, festuclavine, paspalic acid, or the lysergic acid derivatives; vitamins, such as vitamin B12, riboflavin, or β-carotene; enzymes, such as the amylases, glucoseisomerases, proteases, pectinases, lipases, penicillinacylases, chitinase or lactase; nucleosides, such as guanylic acid or inosylic acid; amino acids, such as, for example, cysteine, glutamic acid, tryptophan, or lysine; and many more.

Thus, this invention will be very useful to enhance the effect of microorganisms utilized industrially for their enzyme activities including, for example, those effecting steroid transformations, such as 11 -, ΐΐβ- or 15 -hydroxylation, -^-dehydrogenation, 17 ,17B-keto reduction, the side chain degradation of sterols, or antibiotic transformations, such as penicillin cleavage.

Of course, this invention is not limited to any specific type of microorganism-mediated transformation but will be generally applicable to all such transformations. See, e.g., W. Charney and H. Herzog, "Microbial Transformations of Steroids", Academic Press, New York,: etc. , 1967;' K. Kieslich, "Microbial Transformations of Non-steroidal Cyclic Compounds", Georg Thieme Publ. Stuttgart (DE) , 1976; and K.

Kieslich, "Biotransformations" in H.J. Rehn and G. Reed (Editors), "Biotechnology" Weinheim (DE) etc. Vol. 6A, 1984.

It will also be possible with the aid of the process according to this invention to discover novel, industrially useful microbial components, such as, for example, novel antibiotics, by adding inactive elicitor-containing microorganisms to microorganisms to be tested. This is especially likely because it is known that numerous higher plants form phytoalexins in appreciable quantities only if they are infected with elicitor-containing microorganisms .

The performance of the process according to this invention, especially insofar as it concerns fermentation, e.g., by means of microorganisms as well as other cell cultures, poses no problems to a person skilled in the art. The microorganism or other cells whose enzyme activity or synthesis performance is to be increased is grown under conventional conditions; then the culture is combined with the inactivated elicitor-containing microorganisms, fragments thereof, cell extracts thereof, or excretions thereof, and fermentation is continued as usual. The amount of elicitor added will vary from system to system and will be routinely determinable using conventional considerations especially in conjunction with the guidance of this specification.

The addition of the elicitor, e.g., the inactivated microorganisms, the fragments or extracts of these organisms, or the excretions of elicitor-containing microorganisms, can take place as early as at the beginning of the fermentation. The optimum time of addition is, of course, dependent on the type of microorganism or other cells that are incubated, especially on the curve of its exponential growth phase, and can be determined routinely in an individual case.

Thus, it is frequently expedient, for example, in case of bacteria to effect this addition 4-30 hours after the onset of fermentation. When adding inactivated microorganisms or fragments thereof, then usually 1 - 1000 g (preferably 10 - 200 g) of inactivated microorganism or 0.1 - 100 g (preferably 1 - 30 g) of the fragment of this organism, is utilized per cubic meter of fermentation broth. When using excretions of elicitor-containing microorganisms, it will normally be sufficient to use, per cubic meter of fermentation volume, 1 - 50 1 of the excretion solution. When the process of this invention is used for increasing the enzyme activity of a microorganism employed for the enzymatic conversion of substrates, then the addition of the substrate will usually start 0 - 10 hours after the elicitor-containing inactivated 1 microorganism or its fragments or excretions have been added.

For non-microbial organisms, e.g., cell cultures (including tissue cultures) of plant cells, animal cells, including mammalian such as human cells, similar relative amounts of inactivated microorganisms, fragments or excretions can be used.

The optimum fermentation conditions will vary as usual depending on the type of microorganism or culture media utilized, on the type and quantity of elicitor-containing material, etc.; they can routinely be determined in an individual case by routine preliminary tests highly familiar to those skilled in the art.

To prepare the inactivated form of the elicitor-containing microorganisms, they can first be incubated under their usual conditions, then separated conventionally from the culture broth by centrifuging or filtration, optionally washed, and again isolated.

Various methods can be employed for inactivating the microorganisms, i.e., effecting a permanent loss of viability.

Possible inactivation methods include exposing these microorganisms to typical cytotoxins, such as ethylene oxides, formaldehyde, ozone, mercury compounds, organic solvents, such as methanol, ethanol or.acetone, or killing the microorganisms by heating to 90° to 140°C, by the effect of extreme pressure differences (disintegration) , by the effect of high-frequency electric fields, by UV irradiation or irradiation with gamma-rays, or by the effects of ultrasound. The conditions under which inactivation can be conducted are well known to a person skilled in the art. ( .H.

Wallhfluser, H. Schmidt: "Sterilization, Disinfection, Preservation, Chemotherapy" , Georg Thieme Publishers, Stuttgart, Germany, 1967) .

Fragments of elicitor-containing microorganisms can be obtained, for example, by lysing the microorganisms by the effects of osmotic shock or temperature shock, by autolysis of the microorganisms, by treating the cells with ultrasound, or by trituration of the microorganisms with glass beads, ground glass or quartz sand and subsequent differential centrifugation (Hughes, D.E., Wimpenny, J.W.T., and Lloyd, D. : "The Disintegration of Micro-Organisms" in: Methods in Microbiology vol 13 [Norris, J.R. and Ribbons, D.W. , eds.] pp. 1-54, Academic Press, New York, London, 1971) . : Purified cell wall fractions can be prepared from these cell fragments, for example, by trypsin treatment. The above-mentioned cell wall fractions utilized in the subsequent examples were produced in accordance with the method disclosed by Schleifer and Kandler (Arch.

Mikrobiol. 57 : 335-365, 1967).

On the other hand, however, it is also possible to prepare elicitor-containing precipitates from water-soluble cell components by precipitation, for example, with ethanol or acetone (Kocourek, J., and Ballou, C.E., J. Bacteriol. 100 : 1175-1181, 1969).

Suitable excretions of elicitor-containing microorganisms are actively released cell components obtained by lysing, "rendering leaky", extraction with supercritical liquefied gases (e.g., carbon dioxide),, or sterilization of cells, water-soluble culture broths or culture broths obtained by filtering off or centrifuging the organisms. These can be further purified if required, for example by extraction of lipophilic compounds, absorption of strongly coloring substances, etc.

In essence, the elicitors can be used in any form as long as they retain their efficacy in accordance with this invention. All such forms are contemplated as equivalents for use in this invention.

According to this invention, analogous to the foregoing, elicitors derived from bacteria, e.g., inactivated elicitor-containing bacteria, fragments thereof or excretions of elicitor-containing bacteria, can also be employed for increasing enzyme activities and synthesis performance in tissues, tissue cultures, cell cultures, etc., derived from higher organisms, such as plants, animals, e.g., mammals, including humans, especially plants. As has been mentioned above, use of enzyme-free elicitor-containing material of bacteria will increase the performance of the synthesis of products by higher plants as has been confirmed by experiments; this will be of importance for the utilization of vegetable cell cultures for the preparation of active medicinal agents (M.H. Zenk in: "Pharmazie heute" [Today's Pharmacy] 103, vol 3 : 131-138, 1982). The elicitor-containing material of bacteria will likewise serve per this invention for increasing the performance of the synthesis of ingredients in cultures of animal tissues or cells, including human and other mammalian tissues or cells, and will be useful in therapy — for example, in treatment of wounds by increasing the rate at which cells produce wound-treating components.

While this application primarily discusses the enhancement of the production of non-proteinaceous products, it is fully applicable to the enhancement of enzymatic activity and synthesis performance related to any enzymatic process in which the affected organism is involved. Consequently, this invention will also enhance production of proteinaceous materials by addition of the elicitor-containing medium to an organism used in preparing a given protein endogenously or exogenously. Consequently, this invention will be useful in biological production of polypeptides, e.g., proteins, including enzymes, antibodies, interferon, TNF, erythropoietin, etc., using the well known methods of genetic engineering, including culturing/fermenting of genetically engineered microorganisms.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description; utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.

The entire texts of all applications, patents and publications, if any, cited above and below, and of corresponding application German P 38 01 023.2, filed— January 13, 1088-, are hereby incorporated by reference.

Exam le 1 Stimulation of Synthesis of Colored Ingredients (Actinorhodin , Prodigiosin) in Streptomyces lividans (ATCC 19844) by Cell Wall Preparations 80 ml of a nutrient medium consisting of 103 g sucrose 10 g glucose 10.12 g magnesium chloride hexahydrate 0.25 g potassium sulfate 0.1 g Casaminoacids (Difco Labs, Detroit/USA) 800 ml distilled water is sterilized (20 minutes, 120° C) and supplemented under sterile conditions with :.the following, freshly prepared solutions. 1 ml 0.5% strength potassium dihydrogen phosphate solution 8 ml 3.68% strength calcium chloride dihydrate solution 1.5 ml 20% strength L-proline solution 10 ml 5.73% strength TES buffer solution (pH 7.2) 0.2 ml trace element solution -- containing, per liter, 40 mg zinc(II) chloride 200 mg iron (III) chloride hexahydrate 10 mg copper (II) chloride dihydrate 10 mg manganese (II) chloride tetrahydrate 10 mg disodium tetraborate dihydrate 10 mg hexaamonium heptamolybdate tetrahydrate 0.5 ml IN sodium hydroxide solution Respectively 1.8 ml of this nutrient solution is introduced under sterile conditions into the 24 chambers of a polystyrene multidish with a: volume of respectively 3 ml (Multidish, Nunc, 6200 Wiesbaden 12). Respectively 2 rag of the cell wall preparations to be tested for elicitor content is sterilized in twice-distilled water for 20 minutes at 120° C, and the resultant suspensions are added to the chambers. Two chambers contain no additives; they serve as controls!. In all chambers, the volume is uniformly adjusted .to 2 ml under sterile condi--. tions with twice-distilled water. Each chamber is inoculated under sterile conditions identically with 5 yl of a spore suspension of Streptomyces lividans (ATCC 19844) . The incubation of the test batch takes place aerobically ("Tablar" shaker; 100 rpm) at 26°. C.

After 96 hours, the cells are removed by centrifuging, washed with physiological sodium chloride solution, dried under vacuum over calcium chloride, and the cell yields listed in the table are thus obtained. The supernat ant portions obtained during centrifuging are adjusted to a pH of 7, diluted with water to 4 ml, and their absorption spectra are determined between 180 and 800 nm.

The relative quantities of the synthesized dissolved secondary substances actinorhodin and prodigiosin are determined in an approximation by weighing the absorption peaks of the automatically recorded spectra.._ Table 3 below shows the results obtained in the test series of Example 1.

Bacterial Cell Walls Dry ;Cell Yield Color Content Tested of I (mg) Rel. Absorption Units Without (Control) 22 1 B. ammoniagene s (ATCC 6872) 2$ 38 B . glutarningenes (ATCC 13747) 32 24 B . pumilus (ATCC 7061 ) 34 2 B. linens (ATCC 19391) 23 1 C. diphtheriae (Mass. 8) ^ 34 C. melassecola (ATCC 17965) 26 34 C. glutamicurn (ATCC 13032 ) 43 50 C. lilium (ATCC 15990) 33, 40 Ce. cellasea (ATCC 14359) 36 2 L. plantarum (DSM 20174) 32 31 S. aureus Strain H 44 1 8 = Brevibacteriu = Corynebacterium Ce= Cellulomona s L = Lactobacillus S - Staphylococcus Example 2 Stimulation of Synthesis of Colored Ingredients (Actinorhodin, Prodigiosin) in Streptomyces lividans (ATCC 19844) by Cell Wall; Extracts ; Under the conditions of Example 1, but using sterile filtrates of 2 mg- of cell wall preparations sterilized in double distilled water for 20 minutes at 120° C, an almost equally, strong stimulation of pigment formation in Streptomyces lividans (ATCC 19844) is obtained as in the use of suspensions of these sterilized cell walls.

Example 3 Stimulation of Synthesis of Colored Ingredients (Actinorhodin, Prodigiosin) in Streptomyces lividans (ATCC 19844) by Cell Extracts Under the conditions of Example 2, but using respectively 20 mg of cell material instead of 2 mg of cell wall preparation, an approximately equally strong stimulation of pigment formation is obtained as in the case of using suspensions of the sterilized, cell walls.

Example 4" Stimulation of Synthesis of Colored Ingredients in Streptomyces coelicolor (ATCC :13405) Under the conditions of Example 1, but with the use of Streptomyces coelicolor (ATCC 13405) , a significant increase in pigment formation (supposedly likewise actinorhodin) is achieved also in case of this bacterium.

Example 5 Stimulation of Synthesis of Colored Ingredients in Streptomyces griseoruber (D$fl 40275) Under the conditions of Example 1, but using Streptomyces griseoruber ( DSM 40275 ) , a very pronounced increase in color formation (probably anthracyclin antibiotics) is obtained also in: case of this bacterium.

Example 6 Stimulation of Synthesis of Colored Ingredients in Streptomyces purpurascens (DSM 40310) Under the conditions of Example 1, but with the use of Streptomyces purpurascens ( DSM 40310) , a strong increase in color formation (presumably also anthracyclin antibiotics) is likewise obtained in this bacterium.

Example 7 Stimulation of Synthesis of Colored Ingredients in Streptomyces latericius ( DSM 40163) Under the conditions of Example 1, but using Streptomyces latericius ( DSM 40163) . , a significant increase in color production is likewise achieved in case of this bacterium.

Example 8- Stimulation of the Synthesis of Colored Ingredients in Streptomyces violaceus (DSM *»0082) Under the conditions of Example 1, but with the use of Streptomyces violascens (DSM l»0082) , a significant increase in pigment production is obtained also with this bacterium.

Example 3 Stimulation of the Formation of 8-Lactam Antibiotics (Cephalosporins, Penicillin N) by Streptomyces clavuligerus (ATCC 27064) 5 : 9 3- (N-morpholino) ropanesulfonic acid (MOPS) 3. 5 9 dipotassium hydrogen phosphate 0. 6 g magnesium sulfate heptahydrate 2 L-asparagine 10 glycerol 1 g yeast extract (Oxid, Wesel, Germany) 1 ml trace element salt solution - containing, per liter, 1 g iron (II) sulfate heptahydrate 1 g manganese (II) chloride tetrahydrate 1 g zinc chloride heptahydrate 1 g calcium chloride are filled up to 1 liter with distilled water and sterilized (20 minutes; 120° C) .

Respectively 1.8 ml of this nutrient solution are introduced under sterile conditions into chambers, volume 3 ml, of a sterile polystyrene multidish (Multidish; Nunc, 62 Wiesbaden 12). Respectively 2 mg of the cell ;^all preparations to "be tested for elicitor activity, or 10 mg of the cells to be tested are added as homogeneous suspensions sterilized in double distilled water (20 and, respectively, 45 minutes at 120° C) . Two chambers, serving as controls, do not receive any additives.

The volume is then set uniformly in all chambers with double distilled water under sterile conditions to be 2 ml. Each chamber is inoculated identically with 5 yl of a spore suspension of Streptomyces clavuligerus (ATCC 27064) .

Incubation of the test series is conducted under aerobic conditions ("Tablar" shaker; 160 rpm) at 26° C.

The incubation period is 24-48 hours.

The antibiotics production is tested in comparison with the controls using the plate diffusion test. The detector organisms suspended in soft nutrient agar are Micrococcus luteus and, respectively, Bacillus subtilis (10^ cells per ml). On standard filter plates (0.9 cm diameter), respectively 25 μΐ of centrifuged (48,000 x g) culture broth from the test chambers are applied. After a diffusion period of 4 hours at 4° C, the biotest is incubated for 24 hours at 30° C.

In the cultures incubated with the addition of killed cells of Brevibacterium flavum ATCC 13826, or of cell wall preparations of this bacterium and, respectively, cell wall preparations of Corynebacterium diphtheriae (strain Mass. 8), clearly enlarged inhibition halos as compared with the controls demonstrated increased formation of β-lactam antibiotics (penicillin N, cephalosporins) by Streptomyces clavuligerus.

Example 10 Stimulation of Production of Alkaloids (Sanguinarine , Chelirubin, Marcarpine and Chelerythrine) by Culture's of Eschscholtzia californica Under the conditions described to be optimal by J. Berlin et al. (Z. Naturforsch. [Journal of Natural Sciences] 38c : 346-352, 1983), tissue cultures of Eschscholtzia californica are grown individually in 24 ml chambers of a polystyrene multidish (Nunc, 6200 Wiesbaden 12) . One of the chambers, being the control, remains without any further addition; one chamber receives 266 mg/1 of heat-extracted and ethanol-precipitated yeast elicitor (prepared according to Kocourek, J., and Ballou, C.E. , J. Bacterid. 100 : 1175-1181, 1969) ; the remaining chambers each receive 266 mg/1 of cell wall preparation of the bacteria listed in Table 3.

Then the cultures are incubated for 72 hours at 24° C and thereafter the alkaloid content of the cultures is determined by photometry, the alkaloid content induced by the yeast elicitor being rated as 100%.

Table 4 below shows the results achieved in this test series.

T A B L E Bacterial Cell Walls % Elicitor Activity Tested Brevibacterium butanicum ATCC 21196 19 Brevibacterium flavum ATCC 13026 16 Brevibacterium fiavum ATCC H067 30 Brevibacterium glutamingenes ATCC 137 113 Brevibacterium lactofermentum ATCC 13655 43 Brevibacterium ammoniagenes ATCC 6872 40 Corynebacterium hydroca rbocla stum ATCC 15592 8 Corynebacterium nephridii ATCC 11425 123 Corynebacterium paurometabolum ATCC 8368 16 Corynebacterium lilium ATCC 15990 108 Corynebacterium striatum ATCC 6940 Π Corynebacterium petrophilum ATCC 19080 0 Corynebacterium xerosis ATCC 373 102 Corynebacterium diphtheriae StrainMass . β 137 Rhodococcus fasciens ATCC 12975 27 Rhodococcus fascians 1 11 Isolate by Prof. Dr. Stolp. Univ. Bayreuth Rhodococcus fascians 2 7 Isolate by Prof. Dr. Stolp. Univ. Bayreuth - 2k - Example Π Stimulation of the Production of Indole Alkaloids (Vallesiacotamine) in Cultures of Rauvolfia serpentina A suspension culture of Rauvolfia serpentina (Stockigt, J., ' A. Pfitzner and J. Firl: Plant Cell Rep. 1 : 36-39, 1981) is. cultivated in Linsmaier and Skoog (LS) medium (Physiol. Plantarum 18 : 100-127, 1965) on rotary shakers (100 r'pm) at 23° C and constant light (600 lux) . For eliciting, 200 g of cell fresh weight per liter of LS medium is used for inoculation. Ce11 wal1 preparations of the microorganisms listed in Table are utilized as el i ci tor-containing fragments of microorganisms, in a concentration of 130 mg/1 of medium.

After an incubation of 5 days, the cellular mass has doubled in the elicited cultures as well. as in the controls. The cells are harvested and extracted with methanol .

The amount of the indole alkaloid vallesiacotamine is determined by way of an HPLC separation of the extracts. While the untreated control cultures con-tain only 1.16 mg/1 of medium, the yield in the elicited cultures is maximally 58 ml/1. This corresponds to an increase of 50 times 'by the elicitor.

Example ,12 - Stimulation of the 17-Keto Steroid Reductase Activity of Rhodotorula glutinis IFO 0389 (a) A 2-liter Erlenmeyer flask with 500 ml of sterile nutrient medium containing 5 % glucose monohydrate 2 % corn steep liquor - set at pH 6.5 -is inoculated with a smear of an agar slant of Rhodotorula glutinis IFO 0389 and incubated " for 40 hours at 30° C with 190 rpm. (b) A 500 ml Erlenmeyer flask with 100 ml of sterile nutrient medium containing 1 % corn steep liquor 5 % "Nurupan" (manufacturer:; Nurupan GmbH, 4000 Dusseldorf 1,1 Germany) 1 % "Metarin" (manufacturer: Lucas Meyer, 2000 Hamburg 28, Germany) - set at pH 6.2 -is inoculated with 10 ml of the Rhodotorula subculture prepared according to Example 13 (a) and incubated for 7 hours at 30° C and 180 rpm.

Thereafter, the culture is combined with 10 mg of 3-hydroxy-l , 3 , 5 ( 10 ), 7-estratetraen-17-one and fermentation is continued for 210 hours. The culture is then extracted with methyl isobutyl ketone, the extract is concentrated, and the thus-obtained crude product is purified by chromatography over a silica gel column.

In this way, 5.9 mg of 1 , 3 , 5 ( 10) , 7-estratetraene-3 , 17a-diol is obtained = 59% of theory. (c) Under the conditions of Example 13(b), 10 mg of 3-hydroxy-l, 3, 5 (10) , 7-estratetraen-17-one is fermented with a culture of Rhodotorula glutinis but with the difference that this culture is combined directly prior to substrate addition with 5 ml of a sterile suspension of 50 mg of a cell wall preparation of Bacillus licheniformis (ATCC 9945) in water. After the culture has been worked up, 6.8 mg of 1 , 3 , 5 ( 10 ) , 7-estratetraene-3 , 17-diol is obtained = 68% of theory.

Example 13 Stimulation of Steroid A^-Dehydrase Activity of Bacillus lentus (ATCC 13805) (a) A 2-liter Erlenmeyer flask with 500 ml of a sterile nutrient solution containing 0.5 % corn steep liquor 0.05 % glucose monohydrate 0.1 % yeast estract - adjusted to pH 7.0 -is inoculated with a supernatant broth of Bacillus lentus (ATCC 13805) and shaken at 190 rpm for 48 hours at 30° C. (b) A 500 ml Erlenmeyer flask with 100 ml of sterile nutrient solution containing 3. 0 % soybean powder 0. 5 % corn steep liquor 0. 1 % yeast extract 0. 05 % glucose monohydrate - set at pH 7.3 -is inoculated with 10 ml of the Bacillus lentus subculture and shaken at 180 rpm for 7 hours at 30° C. Then a sterile-filtered solution of 40 mg of 6a , 9a-difluoro-11$ , 17a-dihydroxy-16a-methyl-4-pregnene-3 , 20-dione in 4 ml of dimethylformamide is added to the culture and the latter incubated for another 41 hours.

Then the culture is extracted with methyl isobutyl ketone, the extract is concentrated under vacuum, and the residue is purified by chromatography over' a silica gel column, thus obtaining 16 mg of 6a, 9a-difluoro-113- , 17a-dihydroxy-16a-methyl-l , 4-pregnadiene-3, 20-dione (= 40% of theory) . (c) Under the conditions of Example 14(b), 40 mg of 6a , 9 -difluoro-ΙΙβ , 17a-dihydroxy-16a'-methyl-4-pregnene-3 , 20-dione is fermented with a culture of Bacillus . lentus , but with the difference that this culture is combined, directly prior to addition of substrate, with 5 ml of a sterile suspension of- 50 mg of cell wall preparation of Corynebacterium diphtheriae (strain Mass. 8) in water. After the culture has been worked up, 21 mg of 6a , 9a-difluoro-ΙΙβ , 17a-dihydroxy-16a-methyl-l , 4-pregnadiene-3 , 20-dione is obtained (= 52.5% of theory) .

Example I k Stimulation of Production of Alkaloids (Lysergic Acid Amide and Isolysergic Acid Amide) by Claviceps paspali (ATCC 13895) (a) A 500 ml Erlenmeyer flask with 50 ml of a sterile nutrient solution containing 4 % sorbitol (industrially pure) 1 % glucose monohydrate 2 % succinic acid 0.6 % ammonium sulfate 0.5 % yeast extract ("Difco" of Difco Labs, Detroit, USA) 0.1; % potassium dihydrogen phosphate 0.03 % magnesium sulfate heptahydrate - adjusted to pH 5.2 with sodium hydroxide solution is inoculated with a culture, deep-frozen to -70° C, of Claviceps paspali (ATCC 13895) and shaken for 5 days at 24° C with 240 rpm. (b) A 500 nil Erlenmeyer fltask with bO ml of a sterile nutrient solution containing 8 sorbitol (industrially pure) 6 % succinic acid 0. 9 ammonium sulfate 0. 1 % calcium nitrate tetrahydrate 0. 05 dipotassium hydrogen phosphate 0. 03 magnesium sulfate heptahydrate 0. 02 yeast extract ("Difco" of Difco Labs, Detroit, USA) 0. 0007% iron (II) sulfate heptahydrate 0. 0006% zinc sulfate heptahydrate adjusted to pH 5.2 with sodium hydroxide solution -is inoculated with 5 ml of a subculture of Claviceps paspali and shaken at 240 rpm for 250 hours at 24° C.

Then the culture is combined with such an amount of sodium hydroxide solution that a pH of at least 10 is reached; the culture is extracted with methyl iso-butyl ketone, the extracts are concentrated under vacuum and purified by chromatography over a silica gel column.

In this way, 35 mg of a mixture of lysergic acid amide and isolysergic acid amide is obtained (yield 700 mg/1 of culture) . (c) Under the conditions of Example 15(b), a culture of Claviceps paspali is incubated, but with the difference that, after 72 hours, the culture is combined with 5 ml of a sterile suspension of 25 mg of cell wall preparation of Lactobacillus casei subsp. rhamnosus (ATCC 7469) in water. After the culture has been worked up, 45 mg of a mixture of lysergic acid amide and isolysergic acid amide is obtained (yield 900 mg/1 of cul ure) . . - 29 - Example 15 Stimulation of 15a-Hydroxylase Activity of Penicillium raistrickii (ATCC 10490) (a) A 2-liter Erlenmeyer flask with 500 ml of a sterile nutrient medium containing 3 % glucose monohydrate 1 % corn steep liquor 0.2 % sodium nitrate 0.05 % magnesium sulfate heptahydrate 0.05 % potassium chloride 0.002 % iron(II) sulfate hexahydrate 0.1 % potassium dihydrogen phosphate 0.2 % dipotassium hydrogen phosphate - adjusted to pH 6.0 -is inoculated, with a smear of an agar slant of Penicillium raistrickii (ATCC 10490) and incubated for 48 hours at 30° C with 180 rpm. (b) A 500 ml Erlenmeyer flask with 100 ml of a sterile nutrient medium containing 1 % corn steep liquor ; 3 % glucose monohydrate 0.1 % potassium dihydrogen phosphate 0.2 % dipotassium hydrogen phosphate 0·.05 % magnesium sulfate heptahydrate - set at pH 6.0 -is inoculated with 10 ml of the Penicillium subculture-produced in accordance with (a) .

Thereafter, the culture is combined with 300 mg of 13-ethyl-4-gonene-3 , 17-dione> and fermented for 120 hours at 30° C with 180 rpm.

Then the culture is extracted with methyl isobutyl ketone, the extract is concentrated, and the resultant crude product is purified by chromatography over a silica gel column, thus obtaining 180 mg of 13-ethyl-15a-hydroxy-4-gonene-3 , 17-dione. (c) Under the conditions of (b) , 300 mg of 18-methylnorandrostenedione is fermented with a culture of Penicillium raistrickii, but with the difference that 5 ml of a sterile suspension is added to this culture immediately prior to substrate addition. These 5 ml contain 50 mg of a cell wall preparation of Corynebacterium diphtheriae (strain Mass. 8) in water. After the culture has been worked up, 210 mg of 13-ethyl-15 -hydroxy-4-gonene-3 , 17-dione is obtained.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims (11)

- 32 - 88953/2 WHAT IS CLAIMED IS:

1. A method for enhancing the product synthesizing performance of a culture of a first microorganism or a cell culture of a higher plant, comprising contacting said culture with an elicitor in the form of an inactivated elicitor-containing second microorganism, an elicitor-effective fragment thereof, or an elicitor-effective excretion of an elicitor-containing second microorganism, with the proviso that when said culture is a cell culture of a higher plant, said elicitor-containing second microorganism is a bacterium.

2. A method of claim 1, wherein said culture, the performance of which is to be enhanced, is a culture of a first microorganism.

3. A method of claim 2, wherein said elicitor-containing microorganism is effective to increase the product synthesizing capability of said microorganism to be enhanced.

4. A method of claim 2, wherein said first microorganism culture, the performance of which is to be enhanced, is a bacterium, fungus or yeast, and said elicitor-containing second microorganism is a bacterium, fungus or yeast. - 33 - 88953/ 2

5. A method of claim 4, wherein said culture, the performance of which is to be enhanced, is a bacterium, fungus or yeast capable of synthesizing a colored product, an alkaloid, or an antibiotic.

6. A method of claim 2, wherein said elicitor-containing microorganism is effective to increase enzymatic activity of said microorganism to be enhanced.

7. A method of claim 4, wherein said culture, the performance of which is to be enhanced, is a bacterium, fungus or yeast capable of enzymatic steroid transformation.

8. A method of claim 1, wherein said culture, the performance of which is to be enhanced, is a cell culture of a higher plant.

9. A method of claim 8, wherein a cell culture of a higher plant capable of synthesizing a colored productt, an alkaloid or a phytoalexin is brought into contact with inactivated elicitor-containing bacteria, an elicitor-effective fragment thereof, or an elicitor-effective excretion of elicitor-containing bacteria.

10. A method of claim 1, wherein said elicitor is added as an inactivated elicitor-containing second microorganism, wherein said second microorganism is inactivated by heat-sterilization in water, and wherein said elicitor is - 34 - 88953/2 contacted with the culture in the form of the intact inactivated second microorganism, as a fragment thereof or as a filtrate thereof.

11. A method of claim 1, wherein said elicitor is added as a microorganism cell wall preparation. for the Applicant: WOLFF, BREGMAN AND GOLLER by: - {