IE890079L - Method for increasing enzyme activities and synthesis performance 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.

Description

The invention relates to a method of enhancing enzyme activities and the synthetic capacity of microorganisms and higher plants,, which is characterised in that the same are brought into contact with inactivated elicitor-containing microorganisms, fragments of the same or secretions of elicitor-containing microorganisms, with the proviso that, in order to enhance enzyme activities and the synthetic capacity of higher plants,, inactivated elicitor-containing bacteria, fragments of the sasae or secretions of elicitor-containing bacteria ara used.

As is known, elicitors are microbial or plant active ingredients which, when brought into contact with tissues of higher plants, enhance the latter's enzyme activities and synthetic capacity. The components so accumulated in those plants are termed phytoalexins if they are antimicrobial (Naturwissenschaften, 68, 1981, 447ff, Adv. Enzymol. 55. 1983, Iff and Spektrum der Wissenschaft IX, 1985, 85ff).

At present, more than 100 compounds that satisfy the definition of phytoalexins have been isolated from various plant species. They belong to various groups of natural substances, such as terpenoids, linolanic acid derivatives, acetylenes and polyacetylenes, bibenzyls, stilbenes, phenanthrenes and dihydrophenanthrenes, benzofurans and phenolbenzofurans, furocumarins, avenalu mines, flavans, phenylbenzofurans, benzoxaainones, alkaloids, isoflavonoids (Brooks and Watson, Nat. Prod. Reports 2„ 1985, 427).

Hitherto, that elicitor action has not found any industrial application, for several reasons: with a few excep tions, it has hitherto not been possible to propagate cells of higher plants in submerged cultures under economically acceptable conditions. A priori.. it seesaed pointless to use elicitors in fermentation by means of microorganisms because, on the basis of the prevailing expert opinion regarding the mode of action of elicitors (Albersheia P. and Darvill A.G. Spektrua der wissen-schaft, JJL* 1985 , 85) it had to toe assumed that elicitors did not influence enzyme activity and the metabolic processes in microorganisms - It should also be noted that, according to the accepted expert opinion, bacteria, for example representatives of the genus Erwinia, can induce the formation of phytoalexins in plants only by freeing elicitors (oligogalact-urorsids) from the plant cell wall by means of specific enzymes (pectinases), which elicitors, as endogenous elicitors, stimulate the formation of phytoalexins• It has now been found that compounds and cell preparations of microorganisms , which are referred to in the following as elicitors, are, after all, surprisingly capable of enhancing enzyme activities in microorganisms and of enhancing the latter's synthetic capacity. In addition, it has been found that there are also bacteria that contain elicitors that are not enzymes or nutritive factors.

In principle, the method according to the invention can be carried out by means of isolated or synthesised elicitors, but that is generally far too complicated. It is sufficient to use inactivated elicitor-containing microorganisms, or fragments of those microorganisms, such as£, for example, cell wall fractions, cell fragments cf mechanically disrupted or chemically or enzymaticallv lysed cells, or cell components precipitated by auxiliaries, such as, for example, ethanol or acetone. Inactivated microorganisms within the context of the invention are those ?*itich have lost their viability permanently. - 3 - If the microorganism releases elicitors into the culture medium, or forms water-soluble elicitor-containing cell components after lysis or sterilisation,, those eliciror-containing secretions of the microorganisms can also be 5 used to carry out. the method according to the invention „ Any form of elicitar-containing product of microorganisms is suitable for carrying out the method according to the invention. 10 Microorganisms that are known to possess elicitors are, inter alia, the fungal strains and yeasts listed in the following Table 1: Table 1 Alternaria carthami Botrytis cinerea (ATCC 48345) Ceratocystis fimbriata Ceratocystis ulmi Chondrostereum purpureuin Cladosporium fulvum (ATCC 4 4 961) Colletotrichum lindemuthianum (ATCC 52471) Fusariuni sol ani Fusarium solanifspntori (ATCC 449J4 ) Ganoderma applanation Glomerella cingulata Hslminthosporium carbonum {ATCC 52471) Monilinia fructicola Nectria haeroatococca Phoma exigua Phytophthora cannabivora Phytophthora capsici (ATCC 52771) Phytophthora i nf eetam-. (ATCC 4 4 776 ) Phytophthora megaspermd var tjlycinea Phytophthora infectans Phytophthora megasperma Phytophthora nicotians Puccinia coronata Pyricularia oryzae (ATCC 15923) Saccharomyces cerevlslae Verticillium albo-atrum Verticil1lum dahliae (ATCC 26289) Arch. Bioch. Biop. 229. 1984, 136 Physiol, Plant Pathol. f 1977, 207 Phytochemistry 21, 1984, 759 Phytochemistry U, 1984, 38 3 J. Cham. Soc. Psrkin Trans I, 1984, 14 341 Physiol. Plant Pathol. ]J( 1980, 391 Eur. J. Biochem. 122t 1983, 593 Plant Physiol. 76, 1984, 833 Nat. Prod. Rep. 2, 1985, 439 Phytochemistry 22, 1983, 1039 Physiol, Plant Pathol. 21, 1982, 171 Z< Naturforsch. Sect. C 18, 1983, 899 J. Am. Cham. Soc., §4, 1962, 1919 ' Phytochemistry 2J, 3.983, 2291 * Phytochem. H, 1902, 1818 , Z. Naturf. Sect. C. IS, 1984, 217 Physiol. Plant Pathol, 18, 198.1, 379 Phytochem. 23, 1984, 537 Arch. Bioch. Bioph. , 1984, 136 Phytopathol. Z, 27, 1956, 237 J. Biol. Chem. 259, 1904, 11.341 Phytopathology 7J, 1901, 864 Physiol. Plant Pathol. 29, 1982, 189 Agric. Biol. Chem. 43, 1984, 253 Plant Physiol. §2, 1978, 107 Nat. Prod. Rep. g, 1905, 429 ATCC catalogue, 16th ed. 1984 In our own tests, cell wall preparations, purified proteolytically by trypsin, of gram-positive bacterial strains of the genera Bacillus,, Corynebacterium, Brevi-bacterium, Celluloraonas, Lactobacillus, Pimelobacter, Rhodococcus and Staphylococcus and microorganisms of those genera heat-sterilised in water and the filtrates thereof were investigated to establish whether they possess elicitors™ Table 2 below lists the bacterial strains in which elicitors were detected.

Table 2 Bacillus licheniforalis ATCC 9945 Bacillus puxnilus ATCC 7061 Brevibacteriua butanicuas ATCC 21196 Brevibacterium flavum ATCC 13826, ATCC 14067 Brevibacterium lactofermentmn ATCC 13655 Brevibacteriuxa glutamingenes ATCC 13747 Brevibacteriu® asononiagenes ATCC 6872 Corynebacterium hydrocarboc 1 as turn ATCC 15592 Corynebacteriura nephridii ATCC 11425 Corynebacteriumi pauroiaetabolum ATCC 8368 Corynebacterium 1 ilium ATCC 15990 CorynebacteriuM striatum ATCC 6940 Corynebacterium xerosis ATCC 373 Corynebacteriura diphtheria® (strain Mass. 8/Behring Werke) Corynebacterium saelassecola ATCC 17965 Corynebacteriusa gluraraicuffl ATCC 13032 CorynebacteriuM uracoxidans ATCC 21749 Lactobacillus casei subsp. rhaianosus ATCC 7469 Lactobacillus plantarum DSM 20174 Pimelobacter tumescens AJ 1460 Rhodococcus fascians ATCC 12975 Rhodococcus fascians 1 - isolate of Prof. Dr. Stolp, Univ. Bayreuth Rhodococcus fascians 2 - isolate of Prof. Dr. Stolp,, Univ. Bayreuth - 6 - In the context; of the present invention, only bacterial strains of a few genera of gram-positive eubacfceria have hitherto been investigated to establish whether they possess elicitors and, as far as can b© established from 5 prior publications, of the fungal strains including the yeastse generally only those which were known to be phytopathogenic have been investigated for the presence of elicitor activities. It is therefore to be expected that a large number of other *aicroorganisas, such as, for 10 example, bacteria of the genera Mycobacterium, Nocardia, Nocardioides or Pseudonocardia, can, also be found that likewise possess elicitors.

The examination of microorganisms for elicitor activity can be readily carried out by means of the usual screen-15 ing tests familiar to the person skilled in the art.

For example, in test series the microorganisms the enzyme activity or synthetic capacity of which is to be enhanced can be grown in submerged cultures, inactivated microorganisms of different species or sub-species can 20 be added to the individual cultures and, when fermenta tion is complete , it is possible to determine analytically in which cultures enzyme activities or synthetic capacity have been enhanced. An enhancement of the microorganisms' enzyme activities is recognised, for 25 example, by the fact that an increased rate of formation - or an increased yield - of process product is achieved in the fermentative conversion of substrates. Accordingly, an enhanced synthetic capacity of the microorganisms can be recognised, for example, by an increased rate of - 30 formation - or an increased yield - of components of the microorganism.

As shown by the tests carried out hitherto, which are - 7 - described, in detail in the uorked Examples, the method according to the invention for enhancing enzyme activities or the synthetic capacity of microorganisms seeas to be very versatile. For example, it was possible to 5 stimulate the dyestuff formation of Streptomyces lividans (actinorhodin, prodigiosin) and the dyestuff formation of Streptomyces coelicolor, Streptomyces griseoruber, Streptomyces latericius, Streptomyces purpurascens and Streptomyces violaceus by adding cell wall preparations 10 of Microorganisms listed in Table 2. It was also possible to stimulate the formation of S-lactaro, antibiotics of Streptomyces clavuligerus and the alkaloid synthesis of Claviceps paspali.

Such an enhancement of the synthetic capacity of raicro-15 organisms can be achieved not only by means of the cell preparations of the bacteria listed in Table 2 but it is to b® expected that an enhancement of enzyme activities and the synthetic capacity of microorganisms can be achieved also by means of elici tor-containing fungi and 20 yeasts such as listed in Table 1.

Using cell wall preparations of microorganisms listed in Table 2 it was also possible to obtain a significant increase in alkaloid formation in cell cultures of higher plants,, such as Eschscholtsia californica or Rauvolfia 25 serpentina- It was also possible using those cell wall preparations significantly to enhance the capacity of Bacillus lentus to dehydrogenate steroids in the 1„2-position and the capacity of Rhodotorula glutinis to reduce 17-keto 30 steroids selectively and the capacity of Penicillium raistrickii to hydro: Those tests appear to justify the hop® t£at it will be possible using the method according to tite invention also to stimulate the formation of numerous other industrially applicable microbial components and to enhance other enzyme activities of microorganisms that are susceptible of industrial application.

Such Eiicrolsial components are, for example, antibiotics, such as the penicillins, cephalosporins, cyclosporins, actinomveins, gramicidins, neomycins „ gemtamycins, nystatins, tetracyclines,, nicomycins or lincomycin, erythromycin, chloramphenicol, griseofulvin or fusidic acid, inter alia, ergot alkaloids, such as ergocryptine, ergotamine, ergosine, ergocristine, ergooornine, agro-clavin, chanoclavin, festuclavin, paspalic acid, or lysergic acid derivatives, vitamins, suca as vitamin B 12, riboflavin or fi-carotene? enzymes, such as the amylases» glucose isomerases, proteases, pectinases, cellulases, lipases, penicillin acylases, chitinase or lactase, nucleosides, such as guanylic acid or inosylic acid or, for example, also ajeino acids,, such as cysteine, glutamic acid,, tryptophan or lysine- It should in principle also be possible to enhance the endogenous or exogenous protein formation of genetically modified microorganisms. Such useful proreins are not only the enzymes and antibiotics already sserdtion«2d but also, for example, interferon, insulin, erythropoietin and TNF.

Microorganisxs that are used industrially owing to their enzyme activities are described, for example, in the following publications: W. Charney and Ho Herzog: Microbial Transformations of Steroids? Academic Press, Kew York etc. 1967 K. Kieslich: Microbial Transformations of non-steroidal Cyclic Compounds; Georg Thieme Publ. Stuttgart (Germany), 1976 and K. Kieslich: Biotransformations; in H-J. Reha and G. Heed (editors): Biotechnology: Weinheim (Germany) etc. Vol. 6a, 1984.

Such microorganisms are, inter &UL&, those which bring about steroid transformations such as lla-, 11B- or lSs~ hydroxylation, Aj-dehydrogenation, 17a-, 176-keto reductions ©r the side chain decomposition of sterols or transformations' of antibiotics, such as penicillin cleavage- It is not improbable that it would be possible using the method according to the invention to find new industrially utilisable microbial components, such ss8 for example, new antibiotics, by adding inactive elicitor-containing microorganisms to the microorganisms to be tested.-, That hope is not without foundation because it is known that numerous higher plants form phytoalexins in appreciable quantities only when they are infected with elicitor'-containing microorganisms.

Conventional methods familiar to the person skilled in the art are used to determine what elicitor-containing microorganism enhances the enzyme activities or the synthetic capacity of a specific microorganism.

It is not difficult for the person skilled in the art to carry out the method according to the invention as far a® fermentation by means of microorganisms is concerned. The microorganism the en2yme activity or the synthetic capacity of which is to be enhanced is grown under the - 10 - known conditions? the inactivated elicitor-containing microorganisms, fragments of the same, ceil extracts of the sase or secretions of the same are then added to the culture and fermentation is continued in the customary 5 manner. The addition of the inactivated microorganisms, the fragments or extracts of those higher plants or the secretions of elicitor-containing microorganisms can be effected at the beginning of the fermentation process. The optimum tine for addition naturally depends on the 10 type of microorganism grown, especially on the course of its; exponential growth phase, and must be determined in each individual case. For example, it often proves advantageous in the case of bacteria to effect the addition from 4 to 30 hours after the commencement of 15 fermentation. In the case of the addition of inactivated microorganisms or fragments of the sams, from l to 1000 g Cpreferably ffroia 10 to 200 g) of inactivated microorganism or from 0.1 to 100 g (preferably from 1 to 30 g) of the fragment of that organism are customarily used per 20 j cubic mtetre of fermentation broth. If secretions of elicitor-containing microorganisms are used, it will generally be sufficient to use from 1 to 50 litres of the secretion solution per cubic metre of fermentation vol^aae. If the method according to the invention is used 2 5 to enhance the enzyme activity of a microorganism used for the enzymatic conversion of substrates, the addition of the substrate will generally be started from 0 to 10 hours after the addition of the elicitor-containing inactivated microorganism or its fragments or secretions. 30 The optimum fermentation conditions depend on the type of jnicroorganisM used, the nutrient medium used, the fermentation time, the type and amount: of the elicitor-containing material etc; they have to be determined in each individual case by preliminary tests such as are 35 familiar to the person skilled in the art.

- IX - For the preparation of the inactivated elicitor-containing Microorganisms, the latter are grown under customary conditions,, than separated from the culture medium by centrifugation or filtration, if desired washed and 5 isolated again. Various processes can be used to inactiv ate the microorganisms.

Possible methods of inactivation consist in causing typical substances lethal to cells, such as ethylene oxides, formaldehyde,, ©sons,, mercury compounds or organic 10 solvents, such as methanol, ethanol or acetone., to act on the microorganisms or in killing the microorganisms by heating to fraro 90"C to 140"c, by the influence of extreme pressure differences (disintegration), the action of high-frequency electric fields or by Cjv irradiation, 15 irradiation with Y-rays or the action of ultrasound. The conditions under which inactivation can be carried out are well known to the person skilled in the art (K.H. Wallhauser, H- Schmidt: Sterilisation, Desinfektion, Konservierung, Chemotherapie, Georg Thierae Verlag, 20 Stuttgart (Germany), 1967).

Fragments of elicitor-containing microorganisms can be obtained, for example, by lysis of the microorganisms by the action of osmotic shock or temperature shock, by autolysis of the microorganisms, by treatment of the 2 5 cells with ultrasound or by trituration of the microorganisms with glass beads, powdered glass or quartz sand followed by differential centrifugation (HughesP D.E., Wimpenny, J.W.T. and Lloyd, D.: The disintegration of micro-organisms. In: Methods in Microbiology, Vol- 13, 30 (Norris, J.R. and Ribbons, D.W., eds.) pp. 1-54, Academic Press, Mew York, London, 1971).

Purified cell wall fractions can be obtained from those cell fragments, for example, by trypsin treatment. The cell wall fractions already Mentioned and used in the following worked Examples were prepared in accordance with the method described by Sclileifer and Handler (Arch. Hikrobiol. 52, 1967, 335-365).

On the other hand, it is, however, also possible to prepare elicitor-containing precipitates froa water-soluble cell constituents by precipitation, for example with ethanol or acetone (Kocourek, J- and Ballou, c. E-, J. Bacterial. 100, 1969, 117S-1181).

Secretions of elicitor-containing microorganisms are actively released cell constituents obtained by lysing, making "leaky*, extracting with supercritical liquefied gases (for example carbon dioxide) or heat-sterilising cells in water, water-soluble culture media, or culture media obtained by removing the microorganisms and higher plants by filtration or centrifugation. These can, if necessary, toa further purified, for example by extraction of lipophilic substances,, adsorption of strongly colouring substances, etc..

It has already been mentioned that -he use of enzyme-free elicitor-containing material from bacteria to enhance the synthetic capacity of components of higher plants has been demonstrated experimentally; this could be of importance for the use of plant cell cultures in the preparation of the active ingredients of medicaments (M.H. Sank in: Phsrwazie heutef 103. 1982, Voiu®® 3, 131-138).

The following worked Examples serve no illustrate the invention in detail. - 13 - Example 1 Stimulation of the synthesis of coloured, components (actinorhodin, prodigiosin) in the case of Streptomyces lividans (ATCC 19844) by cell wall preparations. 5 80 ml of a nutrient medium comprising 103 g saccharose 10 g glucose 10 „ 12 g magnesium chloride hexahydrate 0.25 g potassium sulphate 10 0.1 g casaxaino acids (Difco Labs, Detroit/USA) 800 ml distilled water are sterilised (20 minutes, 120*C) and supplemented under sterile conditions with the following freshly prepared solutions. 3 5 1 ml 0.5 % potassium dihydrogen phosphate solution 8 ml 3.68 % calcium chloride dihydrate solution 1.5 ml 20 % L-proline solution 10 ml 5.73 % TES buffer solution (pH 7.2) 20 0.2 ml trace element solution - containing per litre 40 mg 2inc(II) chloride 200 mg iron(lll) chloride hexaftydrate 10 mg copper (II) chloride dihydrate 25 10 mg manganese(II) chloride tetrahydrate 10 rag disodium tetraborate dihydrate 10 mg hexa&wmon iurn heptamolyfodate tetrahydrate 0.5 ml IN sodium hydroxide solution 30 1.8 ml of that nutrient solution are introduced under sterile conditions into each of the 24 3-mi-capacity chambers of a polystyrene multidish (Multidish, manufactured by Nunc, 6200 Wiesbaden 12). 2 mg of the cell wall preparations to ba tested for their elicitor content: are sterilised for 20 minutes at 120 "C in bi-distilled water and the resulting suspensions are added to the chambers. 2 Chambers receive no additions and are used as eostrols,. The volume in all the chambers is uniformly adjusted to 2 ml with Iai-dist:illed water under sterile conditions. Each chamber is inoculated identically with 5 pi of a spore suspension of Streptomyces lividans (ATCC 19844). The test batch Is incubated under aerobic conditions (Tablar shakers 100 revolutions per minute) at 26~C- After 96 hours,, the cells are isolated by centrifugation, washed with physiological saline solution and dried in vacuo over calcium chloride to give the cell yields listed in the Table. The supernatants obtained by centrifugation are adjusted to a pH of 7, diluted to 4 ml with water and their absorption spectra are determined between 180 and 800 na. The relative amounts of the synthesised dissolved secondary substances sctlr.orhodin and prodigiosin are determined approximately by assessing the absorption peaks of the spectra recorded auto'matical-ly.

The following Table 3 shows the results obtained in chat test series. - 15 - TABLE 3 Tested bacterial dry cell dyestuff cell walls of yield (mg) content rel. absorp- 5 ... tion u: without (control) 22 1 B. amnioniagenes (ATCC 6872) 25 38 B, glutassingenes (ATCC 13747) 32 24 Ba - piutilus (ATCC 7061) 34 2 B. linens (ATCC 19391) 23 1 C. diphtheriae (Mass. 8) 24 34 C. raelassecoia (ATCC 17965) 26 34 c. glutamicuffi (ATCC 13032) 43 50 c. liliun (ATCC 15990) 33 40 ce . cellasea (ATCC 14359) 35 2 L. piantarum (DSM 20174) 32 31 S. aureus strain H 44 1 B = Brevibacteriura Ba = Bacillus 2o C = Corynebacter iuia Ce = Cellulosnonas L = Lactobacillus S = Staphylococcus Exaianle 2 2 5 Stimulation of the synthesis of coloured components (actinorhodin, prodigiosin) in the case of Streptomyces lividans (ATCC 19844) by cell wall extracts.

Under the conditions of Example l, but using the sterile filtrates of the 2-rag cell wall preparations sterilised 30 for 20 minutes at 120"C in bi-distilled water,, a stimula - ifi - tion of dyestuff formation is achieved in Streptomyces lividans (ATCC 19844) that is almost as strong as when using suspensions of those sterilised cell walls.

Example 3 5 Stimulation of the synthesis of coloured components (actinorhodin„ prodigiosin) in the case of Streptomyces lividans (ATCC 19844} lay cell extracts.• Under the conditions of Example 2, but using 20 mg of biomass instead of 2 mg of cell wall preparation, a 10 stimulation of dyesrtuff formation is achieved that is approximately equally as strong as whan using suspensions of the sterilised cell walls. £2£3bib1ILJL Stimulation of the synthesis of coloured components in 15 the case of Streptomyces coelicolor A3(2) or (ATCC 13 405).

Under the conditions of Exaiaple 1, but using Streptomyces coelicolor A3(2)p a marked stimulation of dvestufr formation (probably likewise actinorhodin) is achieved in 20 the case of this bacterium.

Example 5 Stimulation of the synthesis of coloured components in the case of Streptomyces griseoruber (DSH 40275).

Under the conditions of Example 1, but using Streptomyces 25 griseoruber (DSH 40275), a very marked increase in dyestuff formation (presumably anthracyclin antibiotics) is achieved in the case of this bacterium also. - 17 - Exa«nle_6.

Stimulation of the synthesis of coloured components in the case of Streptomyces purpurascens (DSH 40 310), Under the conditions of Example i, but using Streptomyces 5 purpurascens (DSH 40 310) , a distinct increase in dyestuff formation (presumably likewise anthracyclin antibiotics) is achieved in the case of this bacterium also* 10 Stimulation of the synthesis of coloured components in the case of Streptomyces latericius (DSH 40 163)- Under the conditions of Example 1, but using Streptomyces latericius (DSH 40 163), a significant increase in dyestuff formation is achieved in the case of this bac-15 teriu® also.

Example § Stimulation of the synthesis of coloured components in the case of Streptomyces violaceus (DSK 40 082).

Under the conditions of Example 1, but using Streptomyces 20 violaceus (DSM 40 082), a significant increase in dyestuff formation is achieved in the case of this bacterium also. - 18 - Example 9 Stimulation of the formation of G-lactaia antibiotics (cephalosporins,, penicillin N) by Streptomyces clavuli-gerus (ATCC 27064). 5 g 3-(M—marpholino)-propanesulphonic acid (HOPS) 3»5 g - dipotassiun hydrogen phosphate 0.6 g magnesium sulphate heptahydrate 2 g L~asparagin@ 10 g glycerol 1 g yeast extract (Oxide, Wesel, Germany) 1 ml trace element salt solution - containing per litre l g iron(XI) sulphate heptahydrate 1 g manganese(II) chloride tetrahydrate 1 g zinc chloride heptahydrate l g calcium chloride are made up to 1 litre with distilled water and sterilised (20 minutes? 120*C) - 1.8 ml of that nutrient solution are introduced under sterile conditions into each of the 3-ml~capacity chambers of a sterile polystyrene multidish (Multidish; manufactured by Nunc, 62 Wiesbaden 12). 2 mg of the cell wall preparations to be tested for elicitor activity or 10 mg of the cells to be tested are added in the form of homogeneous suspensions sterilised in bi-distilled water (20 and 45 minutes, respectively, 120"C). Two chambers serving as control receive no additions„ The volume in all the chambers is then uniformly adjusted to 2 ml with bi-distilled water under sterile conditions. Each chamber is inoculated identically with 5 nl of a spore suspension of Streptomyces clavuligerus (ATCC 27064).

The incubation of the test series is carried out under - 19 - aerobic conditions (Tablar shaker? 160 revolutions per minute) at 26"C. The incubation time is fross 24 to 48 hours.

The formation of antibiotics is examined in comparison with the controls using the plate diffusion test:. The detector organisms suspended in soft: nutrient agar are Micrococcus luteus and Bacillus suhtilis (3.0® cells per si). 25 /*1 of centrifuged (48,000 x g) culture medium from the test chambers are applied to each of a number of standard filter pistes (0,9 cm diameter}- After a diffusion time of 4 hours at 4*c, the biotest is incubated for 24 hours at 30"c.

In the case of the cultures that were grown with the addition of killed cells of Brevibacterium flavura ATCC 13826 or of cell wall preparations of that bacterium or of call wall preparations of Corynebacterium diphtherias (strain Mass. 8), clearly enlarged inhibiting areolas exhibited an increased formation of B-l&ctam antibiotics (penicillin M, cephalosporins) by Streptomyces clavuli-gerus compared with the controls.

Example 10 Stimulation of the formation of alkaloids (sanguinarine, chelirubine, marcarpine and chelerythrine) by cultures of Eschscholtzia californica.

Tissue cultures of Eschscholtzia californica are gro'^r, in each of 24 1-ial chambers of a polystyrene multidish (manufactured by Nunc, 6200 Wiesbaden 12) under the conditions described as optimum by J. Berlin al. (Z. Naturforsch., 38 Table 4 below shows the results obtained in this test j0 series.

TABUS 4 Tested, bacterial cell vails % elicitor activity Brevibacterium bwtsnicws ATCC 21196 19 15 Brevibacterium flavum ATCC 13826 16 Brevibacterium £lavum ATCC 14067 30 Brevibacterium glutamingenes ATCC 137 113 Brevibacterium lactofena@ntum ATCC 13655 43 Brevibacterium anunoniagenes ATCC 6872 4 0 20 Corynebacterium hydrocarboc 1 ast urn ATCC 15592 8 Corynebacteriurn nephridii ATCC 11425 123 Corynebacteriurn paurometabolum ATCC 8368 16 Corynebacterium liliua ATCC 15990 108 Corynebacterium striatum ATCC 6940 17 25 Corynebacter ium petrophilua ATCC 19080 0 Corynebacter ium xerosis ATCC 373 102 Corynebacter ium diphtherias strain Mass. 8 137 Rhodococcus fascians ATCC 12975 27 Rhodococcus fascians 1 11 30 isolate of Prof. Dr. Stolp, Univ. Bayreuth Rhodococcus faseians 2 7 isolate of Prof. Dr. Stolo. Univ. Bavreuth - 21 - Example 1.1 Stimulation of the formation of indole alkaloids (valles-iacotamine) in cultures of Rauvolfia serpentina.

A suspension culture of Rauvolfia serpentina (Stockist,, 5 J., A. Pfitzner and J. Firl: Plant Cell Rep. JL, 36-3® (1981) is cultivated in Linsnaier and Skoog (LSJ-nediun (Physiol. Plantar-urn UL 100-127 (1965) on rotary shakers (100 revolutions per minute) at 23"C with permanent light (60S lux) _ For elicitation, 20© g of cell fresh 10 weight/1 LS siediua are inoculated. Cell wall preparations of the bacteria listed in Table 4 are used at a concentration of 130 sag/1 iaadius as elicitor-containing fragments of microorganisms™ After 5 days41 incubation, the biomass has doubled both in 15 the elicited cultures and in the controls. The cells are harvested and extracted with Methanol.

The amount of the indole alkaloid vallesiacotamine is determined by separating the extracts by KPLC. Whereas the untreated control cultures contain only 1.16 mg/1 of 20 medium, the yield in the case of the elicited cultures is a naxiroum of 58 sag/1. That corresponds to a 50-fold increase by the elicitor.

Stimulation of the 17-keto steroid reductase activity of Rhodotorula glutinis XFO 0389. a) A 2-litre Erleniaeyer flask with 500 ml of sterile nutrient medium containing 5 % glucose monohydrate - 22 - 2 % cornsteep liquor - adjusted to pH 6.5 - is inoculated with a smear from a slant agar culture of Rhodotorula glutinis IFG 0389 and cultivated for 40 hours at 30"C and at 190 revolutions per minute.

A SOO-ssi Erlenmeyer flask with 100 ml of sterile nutrient medium containing 1 % cornsteep liquor 5 % Nurupan (R) (manufacturer Nurupan GabK, 4000 Dusseldorf 1? Germany) 1 % Meter in (R) (manufacturer Lucas Meyer; 2000 Hamburg 28; Germany) - adjusted to pH 6.2 - is inoculated with 10 ml of the Rhodotorula pre-culture prepared in accordance with Example 12a and cultivated for 7 hours at 30*C and 180 revolutions per minute. 10 tag of 3-hydroxy-l,3,5(10) ,7-oestratetraen-i7-one are then added to the culture and fermentation is effected for © further 210 hours. The culture is then extracted with methyl isobutyl ketone, the extract is concentrated and the resulting crude product is purified by chromatography on a silica gel column to yield 5.9 mg of 1,3,5(10) ,7-oestra-tetraene-3 „ 17a~diol = 59 % of the theoretical yield. 10 sg of 3-hydroxy-l ,3,5(10), 7-oestratetraen-17-one are fermented with a culture of Rhodotorula glutinis under the conditions of Example 12b except that 5 ml of a sterile suspension of 50 mg of a cell wall preparation of Bacillus licheniformis (ATCC 9945) in water are added to the culture immediately before the addition of the substrate. After working up the culture, 6.8 lag of 1,3,5(10),7-oestratetraene-3,17- diole = 68 % of the theoretical yield are obtained.

SXSffiai£__L3.

Stimulation of the steroid A ^-dehydrase activity of Bacillus lentus (ATCC 13805). a) A 2-litre Erlerraeyer flask with 500 al of sterile nutrient solution containing 0.5 % cornsteep liquor 0.05 % glucose raonohydrate 0.1 % yeast extract - adjusted to pH 7.0 - is inoculated with a rinse of Bacillus lentus (ATCC 13 805) and shaken for 48 hours at 30"C and at 190 revolutions per minute. b) A 500—sfol Erienneyer flask with, 100 al of sterile nutrient solution containing 3.0 % soya powder 0.5 % cornsteep liquor 0.1 % yeast extract 0.05 % glucose monohydrate - adjusted to pH 7.3 - is inoculated with 10 ml of the Bacillus lentus preculture and shaken for 7 hours at 30"C and at 180 revolutions per minute. A sterile-filtered solution of 40 tag of 6cr, 9a-dif luoro-llS, l?a-dihydroxy-16c:-methyl-4-pregnene-3,20-dione in 4 »l of dimethyl-formauide is then added to the culture and the whole is incubated for a further 41 hours.

The culture is then extracted with methyl isobutyl ketone, the extract is concentrated in vacuo and the residue is purified by chromatography on a - 24 - silica gel column to yield 16 mg of 6 c, 9 Stimulation of the formation of alkaloids (lysergic acid amide and isolysergic acid asaide) by Claviceps paspali (ATCC 13895). a) A 500-sul Erlenmeyer flask with 50 ml of a sterile nutrient solution containing 4 % sorbitol (industrially pure) 1 % glucose monohydrate 2 % succinic acid 0.6 % ammonium sulphate 0.5 % yeast extract (DifcoW manufactured by Diffco Labs. Detroit/USA) 0.1 % potassium dihydrogen phosphate 0.03 % magnesium sulphate heptahydrate -adjusted to pH 5.2 with sodiua hydroxide solutionis inoculated with a culture of Claviceps paspali (ATCC 13895) deep-frozen to -70"c and shaken for 5 days at 24"c and 240 revolutions per minuta. - 25 - k 5QQ-k1 Erlermeyer flask with 50 sal of a sterile nutrient: solution containing a t 6 % Sufficient sodium hydroxide solution is then added to the culture to adjust the pH to at least 10, extraction is carried out with kgthy1 isobutyl ketone and the extracts are concentrated in vacuo and purified by chromatography on a silica gel coluffln* 35 mg of a mixture of lysergic acid aside and isolysergic acid asaide (yield 700 ng/1 of culture) are thus obtained.

A culture of Claviceps paspali is grown under the conditions of Example 14b except that 5 ml of a sterile suspension of 25 rag of cell wall preparation of Lactobacillus casei subsp. rhamnosus (ATCC 7469) in water are added to the culture after 72 hours. After working up the culture, 45 mg of a mixture of lysergic acid amide and isolysergic acid 26 - amide (yield 900 ag/1 of culture) are obtained, Stimulation of the 15c:~hydroxylas

Claims (7)

- 28 - Ea&sa£-£laiffl&

1. Method of enhancing enzyme activities and the synthetic capacity of microorganisms and higher plants, characterised in that the sane are brought into contact 5 with inactivated elicitor-containing microorganisms, fragments of the saiae or secretions of elicitor-containing aicroorganissas P with the proviso that, in order to enhance enzyme activities and the synthetic capacity of higher plants,, inactivated elicitor-containing bacteria, 10 fragments of the same or secretions of elicitor-containing bacteria are used.

2. « Method of enhancing enzyme activities and the synthetic capacity of aicroorganisas according to patent clain l, characterised in that the same are brought into 15 contact with inactivated elicitor-containing Microorganisms , fragments of the sawe or secretions of elicitor-containing microorganisms-

3. Method of enhancing the synthetic capacity of nicro-organisms according to patent claiu 2, characterised in 20 that bacteria, fungi and. yeasts capable of forming dyestuffs, alkaloids or antibiotics are brought into contact with inactivated elicitor-containing bacteria, fungi or yeasts, fragments of the same or secretions of those microorganisms. 25

4. Method of enhancing enzyme activities of issicro- organisms according to patent claia 2, characterised in that bacteria,, fungi or yeasts capable of steroid transformation are brought into contact with inactivated elicitor-containing bacteria, fungi or yeasts, fragments 30 of the sasne or secretions of those microorganisms.

5. Method of enhancing the synthetic capacity of higher - 29 - plants according to patent claim 1, characterised in chat; cell cultures of higher plants capable of dyestuff synthesis, alkaloid synthesis or phytoalexin synthesis are brought into contact with inactivated elicitor-5 containing bacteria, fragments of the same or secretions of those bacteria.

6. Method of enhancing enzyme activities and the synthetic capacity of microorganisms and higher plants according to patent claims l to 5, characterised in that SO the sasae are broaght into contact with elicitor-containing aicroorganisES heat-sterilised in water, or with filtrates of the sasse-

7. . A ■ ne shod subscatnc ial ly as bereinbe fore described with reference Co the Exaraples. Dated Chis !31h day of January 1989 CSDXCKSRAHK & CO. Agents for the applicant 1 Holies Scraet Dublin 2