IE66500B1 - 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 enzyneactivities and the synthetic capacity of microorganismsand higher plants, which is characterised in that thesame are brought into contact with inactivated elicitor-containing microorganisms, fragments of the same orsecretions of elicitor-containing microorganisms, withthe proviso that, in order to enhance enzyne activitiesand the synthetic capacity of higher plants, inactivatedelicitor-containing bacteria, fragments of the same orsecretions of elicitor-containing bacteria are used.

As is known, elicitors are microbial or plant activeingredients which, when brought into contact withtissues of higher plants, enhance the latterfs enzyneactivities and synthetic capacity. The components soaccumulated in those plants are termed phytoalexins ifthey are antimicrobial (Naturwissenschaften, 58, 1981,447ff, Adv. Enzymol. 55 . 1983, Iff and Spektrum derWissenschaft U,, 1985, 85ff).

At present, more than 100 compounds that satisfy thedefinition of phytoalexins have been isolated fromvarious plant species. They belong to various groups ofnatural substances, such as terpenoids, linolanic acidderivatives, acetylenes and polyacetylenes, bibenzyls,stilbenes, phenanthrenes and dihydrophenanthrenes,benzofurans and phenolbenzofurans, furocumarins, avenalu-mines, flavans, phenvibenzofurans, benzoxazinones,alkaloids, isoflavonoids (Brooks and Watson, Nat. Prod.Reports 2, 1985, 427).

Hitherto, that elicitor action has not found any indus-trial application, for several reasons: with a few excep-tions, it has hitherto not been possible to propagatecells of higher plants in submerged cultures undereconomically acceptable conditions. A priori. it seemed 2 pointless to use elicitors in fermentation by means ofmicroorganisms because, on the basis of the prevailingexpert opinion regarding the mode of action of elicitors(Albersheim P. and Darvill A.G. Spektrua der wissen- 5 schaft, 11 1985 „ 85) it had to toe assumed that elicitors did not influence enzyme activity and the metabolicprocesses in microorganisms.

It should also be noted that, according to the acceptedexpert opinion, bacteria, for example representatives of 10 the genus Erwinia, can induce the formation of phyto- alexins in plants only by freeing elicitors (oligogalact-uronids) from the plant cell wall by means of specificenzymes (pectinases), which elicitors, as endogenouselicitors, stimulate the formation of phytoalexins. 15 it has now been found that compounds and cell prepara- tions of microorganisms, which are referred to in thefollowing as elicitors, are, after all, surprisinglycapable of enhancing enzyme activities in microorganismsand of enhancing the latter's synthetic capacity. In 20 addition, it has been found that there are also bacteria that contain elicitors that are not enzymes or nutritivefactors.

In principle, the method according to the invention canbe carried out by means of isolated or synthesised 25 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 of mechanically disrupted or chemically or enzymaticallylysed cells, or cell components precipitated by auxiliar-ies, such as, for example, ethanol or acetone. Inactiv-ated microorganisms within the context of the inventionare those which have lost their viability permanently.

If the microorganism releases elicitors into the culturemedium, or forms we ter-"soluble el icitor—containing cellcomponents after lysis or sterilisation, those elicitor-containing secretions of the microorganisms can also be 5 used to carry out the method according to the invention.

Any form of elxcxtor-containing product of microorganismsis suitable for carrying out the method according to theinvention.

Microorganisms that are known to possess elicitors are, 10 inter alia, the fungal strains and yeasts listed in thefollowing Table l: 4 ci ’S" Alternaria carthami Arch. Bioch. Biop. 229, 1984, 136 =-: en r- =3 rM Gt O' σ o «a. σ» A· CO n- co 03 m ♦"§ m vO *3 © ft» Gt r- fn «> Gt ·, «ft rt =-t - ¢-5 ·. (*» f·. o m A3 CO Gt Cft =4 n-3 in η- co σι CO Gt *—3 Θ «· ω ns σ» %* σ m Gt Gt (rt » ΟΊ o* rt & •®> ¢-4 61 rj M =4 m Gt η «-Μ Gt ft" =3 o n <· rM - 6Ί ra m eg n n m O "»31 03 03 ¢3 Gt 03 t-i «0 X? o 03 6¼ - p—3 ea «· co .-3 ft· ¢-3 G Θ Gt C9 f ί cat ο» os c« «. ft. σι e* «-Ι •ft F-a ¢-11 l". •0 3 > 44 © . a >i44 XS S3 >3 <«. O 44 Λ 8S3 » >44 O . a © G Is . G .G =4 & ;~5 c o Si © K rw Ss ϋ1 c .—.J © 3 <3 44 J.J O (3 o O « 44 (3 «2 . 44 &) © os . . © O (S - o cs O' ¢=3 c© 0 M o Ή CO e-Hl iU 6 ¢=3 £ »"9 JS *-3 ¢-3 ¢=3 O a •eH CO ft -rt m • •H o © «=3 • . a » O o o O 0.1 o «3 - 3 g 6 ca >*O g O «3 - © © · « £ a © « is a © © m . © -=» 4J · -m · © x o 0 Η X £ β H - ft. S-a xs i-3 s £S x; 3 ¢-8 £ (S © =-J eg =! a Is © ο a o 0 ο η η. o a >00000 a o a a - ®.o - ΰ O i - 44 o —! fij g Ο O (5 --5 Ο · Ο - O - (! 4.! CO ii u M · K . Q >, > >, > ts (S 3J >j >5 >j >, > >·) O > >i !-j (3 ϋ U x: x: £ · xs 3 ^ eg £*n. cl, & h a h e, 2 ¢. w b n " h & & < C-i κ i-- ft" nj in r-a η- © CJ —- © U na -ο in Γ- -=3 =-, —.· < σι =3 Γ- O CA ft· U n- ft· > —ft CO V0 ft· a n- ft- -, *ft^ ns Λ O' ε E-3 ns o' Λ3 10 in □ O < in o Gt ns ft" ft1 c o -»= e u Ut co R3 a e= © ^4 O σ> u •«a < ε u < > ft" s u £ •— 3 (S3 u © E-3 3 Ei 4J G U < © © o © S "C U f3 Φ < 3 -=i O O — vt Ξ W £ © -=4 3 — O 44 t.3 « ε U .Q > £ li E la C < © la &3 (S 3 0 ε © $4 © (3 -=3 © © © 0» © -=-3 44 © es£ ) a a "v ε 3 44 ©=-3 0 a o 4J a 44 a—3 > © © M 3 C a 44 (0 Ο Ο O (S3 -=3 ο ω o tn 44 0) © a -=a ej -H 3 > ϋ) , o >— © ks <3.1 3 ε <3-3 ε c c =4 C -=3 O 44 O o — s -=s ε c c la © Ό Φ 3 3 fO (S3 a·^ ka 3 © 0 0 CO ss ω ω Φ S £ r-9 =—j a o O la S Ο <3 © © © © © © la © ε ε -r4 —4 -H U 3 0 0 O (S3 a«-a φ 3 u b U la la h h 0 © 0 3 3 a 44 44 -=3 -=a οι ω 44 a Ua «3 .-a O © XS —a £ £ £ £ £ £ £ G =3 J w > > (0 o 44 K E E ¢-3 X 44 44 44 44 44 44 © © O ¢-3 =3 --CJ ϋ o a o 3 S u © 4J G, © βΧΧΛΛΧΛΧ •=3 p—i 44 o o ί-a W 44 -rt —a © £a GS a Ge Go Sd £Xa Qa £=d 3 © u o >a4J 44 Ό O (II 34 Ό Cl •Ή r—§ >>>>>,>, o (J li M O ffl ® £ ·-; o 3 3 (0 ¢-. Gl O © a a £ £ £ £ £ £ 3 > © © © ss a O U U u a a ϋ a s^saaaaaaaa0ia«i>> 5 In our own tests,, cell wall preparations„ purifiedproteolytically by trypsin, of gram-positive bacterialstrains of the genera Bacillus, Corynebacterium, Brevi-bacteriua, Cellulomonas, Lactobacillus, Pimelobacter,Rhodococcus and Staphylococcus and microorganisms ofthose genera heat-sterilised in water and the filtratesthereof were investigated to establish whether theypossess elicitors. Table 2 below lists the bacterialstrains in which elicitors were detected- Table 2 Bacillus licheniformis ATCC 9945 Bacillus pumilus ATCC 7061 Brevihacterxum toutanicum ATCC 21196 Brevibacterium flavum ATCC 13826, ATCC 14067 Brevibacterium lactofermentum ATCC 13655 Brevibacterium glutamingenes ATCC 13747 Brevibacterium ammaniagenes ATCC 6872 Corynebacterium hydrocarboc las turn ATCC 15592 Corynebacterium nephridii ATCC 114 25 CorynebacteriuM paurometabolum ATCC 8368 Corynebacteriuxa lilium ATCC 15990 Corynebacterium striatum ATCC 6940 Corynebacteriuxa xerosis ATCC 373 Corynebacterium diphtherias (strain Hass. 8/BehringWerke) Corynebacterium melassecola ATCC 17965 Corynebacteriuss glutamicum ATCC 13032 Corynebacterium uratoxidans ATCC 21749 Lactobacillus casei subsp. rhamnosus ATCC 7469 Lactobacillus plantarum DSM 20174 Pimelobacter tumescens AJ 1460 Rhodococcus fascians ATCC 12975 Rhodococcus fascians 1 - isolate of Prof. Or. Stolp,Univ. BayreuthRhodococcus fascians 2 -Univ. Bayreuth isolate of Prof. Or. Stolp,, In the context of the present invention, only bacterialstrains of a £@w genera of grass-positive eubacteria havehitherto been investigated to establish whether theypossess elicitors and, as far as can be established fromprior publications, of the fungal strains including theyeasts, generally only those which were known to bephytopathogenic have been investigated for the presenceof elicitor activities- It is therefore to be expectedthat a large number of other Microorganisms, such as, forexample, bacteria of the genera Mycobacterium, Nocardia,Nocardioides or Pseudonocardia, can also be found thatlikewise possess elicitors.

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

For example, in test series the microorganisms theenzyme activity or synthetic capacity of which is to beenhanced can be grown in submerged cultures, inactivatedmicroorganisms of different species or sub-species canbe added to the individual cultures and, when fermenta-tion is complete, it is possible to determine analyti-cally in which cultures enzyme activities or syntheticcapacity have been enhanced. An enhancement of themicroorganismss enzyme activities is recognised, forexample, by the fact that an increased rate of formation- or an increased yield - of process product is achievedin the fermentative conversion of substrates. According-ly, an enhanced synthetic capacity of the microorganismscan be recognised, for example, by an increased rate offormation - or an increased yield - of components of themicroorganism.

As shown by the tests carried out hitherto, which are 7 described in detail in the worked Examples, the methodaccording to the invention for enhancing enzyme activit-ies or the synthetic capacity of microorganisms seems tobe very versatile. For example, it was possible tostimulate the dyestuff formation of Streptomyces lividans(actinorhodin, prodigiosin) and the dyestuff formation ofStreptomyces coelicolor, Streptomyces griseoruber,Streptomyces latericius, Streptomyces purpurascens andStreptomyces violaceus by adding cell wall preparationsof microorganisms listed in Tabla 2. It was also possibleto stimulate the formation of S-lactam antibiotics ofStreptomyces clavuligerus and the alkaloid synthesis ofClaviceps paspali.

Such an enhancement of the synthetic capacity of micro-organisms can be achieved not only by means of the cellpreparations of the bacteria listed in Table 2 but it isto b® expected that an enhancement of enzyme activitiesand the synthetic capacity of microorganisms can beachieved also by means of elicitor-containing fungi andyeasts such as listed in Table 1.

Using cell wall preparations of microorganisms listed inTable 2 it was also possible to obtain a significantincrease in alkaloid formation in cell cultures of higherplants,, such as Eschscholtzia californica or Rauvolfiaserpentina- It was also possible using those cell wall preparationssignificantly to enhance the capacity of Bacillus lentusto dehydrogenate steroids in the 1,,2-position and thecapacity of Rhodotorula glutinis to reduce 17-ketosteroids selectively and the capacity of Penicillin®raistrickii to hydroxylate steroids in the l5a-position.Hitherto, only the attempt to enhance the capacity ofCurvularia lunata to hydroxylate steroids in the 118" 8 position using those cell wall preparations has beenunsuccessful- Those tests appear to justify the hope that it will bepossible using the method according to the invention also 5 to stimulate the formation of numerous other industrially applicable microbial components and to enhance otherenzyme activities of microorganisms that are susceptibleof industrial application» Such microbial components are, for example, antibiotics, 10 such as the penicillins, cephalosporins, cyclosporins, actinomycins, gramicidins, neomycins, geataaycins,nystatins, tetracyclines, nicomycins or lincomvcin,erythromycin, chloramphenicol, griseofulvin or fusidicacid, inter ajJLa, ergot alkaloids, such as ergocryptine, 15 ergotamine, ergosine, ergocristine, ergooomine, agro- clavin, chanoclavin, festuclavin, paspalic acid, orlysergic acid derivatives, vitamins, sues as vitamin B12, riboflavin or fi-carotene, enzymes, such as theamylases, glucose isomerases, proteases, pectinases, 20 cellulases, lipases, penicillin acylases, chitinase or lactase, nucleosides, such as guanylic acid or inosylicacid or, for example, also amino acids, such as cysteine,glutamic acid, tryptophan or lysine.

It should in principle also toe possible to enhance the 25 endogenous or exogenous protein formation of genetically modified microorganisms. Such useful proteins are notonly the enzymes and antibiotics already mentioned butalso, for example, interferon, insulin, erythropoietinand TNF. 30 Microorganisms that are used industrially owing to their enzyme activities are described, for example, in the following publications^ 9 W. Charney and H. Herzog: Microbial Transformations ofSteroidsj Academic Press, Kew York etc. 1967K. Kieslich: Microbial Transformations of Hon-steroidalCyclic Compounds; Georg Thieiae Publ. Stuttgart (Germany),, 5 1976 and K. Kieslich: Biotransf oraations ? in H.J. Rehm and G. Reed(editors): Biotechnology; Weinheim (Germany) etc. V©1. 6a, 1984.

Such microorganisms are, inter &li&, those which bring10 about steroid transformations such as 11α-, I IS- or 15c- hydroxylation, Aj-dehydrogenatien, 17c-, X7£-keto reduc-tions or the side chain decomposition of sterols ortransformations' of antibiotics, such as penicillincleavage. '5 It is not improbable that it would be possible using themethod according to the invention to find new indus-trially utilisable microbial components, such ase forexample, new antibiotics, by adding inactive elicitor-containing microorganisms to the microorganisms to be 20 tested. That hope is not without foundation because it isknown that numerous higher plants form phytoalexins inappreciable quantities only when they are infected withelicitor-containing raxcroorganisms.

Conventional methods familiar to the person skilled in 25 the art are used to determine what eliciter-containingmicroorganism enhances the enzyme activities or the synthetic capacity of a specific microorganism.

It is not difficult for the person skilled in the art to 30 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 syntheticcapacity of which is to be enhanced is grown under the 10 known conditions; the inactivated elicitor-containingmicroorganisms, fragments of the same, cell extracts ofthe saxe or secretions of the same are then added to theculture and fermentation is continued in the customary 5 nanner. The addition of the inactivated microorganisms, the fragments or extracts of those higher plants or thesecretions of elicitor-containing microorganisms can heeffected at the beginning of the fermentation process.

The optiwuaa time for addition naturally depends on the 10 type of microorganism grown, especially on the course of its 'exponential growth phase, and must be determined ineach individual case. For example, it often provesadvantageous in the case of bacteria to effect theaddition from 4 to 30 hours after the commencement of 15 fermentation. In the case of the addition of inactivated Microorganisms or fragments of the same, from l to 1000 g(preferably from 10 to 200 g) of inactivated micro-organise or from 0J to 100 g (preferably from 1 to 30 g)of the fragment of that organism are customarily used per 20 j cubic wetre of fermentation broth. If secretions of elicitor-containing microorganisms are used, it willgenerally be sufficient to use from 1 to 50 litres of thesecretion solution per cubic metre of fermentationvolume- 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 additionof the substrate will generally he started from 0 to 10hours after the addition of the elicitor-containinginactivated microorganism or its fragments or secretions - 30 ψΐιβ optimum fermentation conditions depend on the type of microorganism used, the nutrient medium used, thefermentation time, the type and amount of the elicitor-contaxning material etc; they have to he determined ineach 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-contain-ing microorganisms, the latter are grown under customaryconditions j, then separated from the culture medium bycentrifugation or filtration, if desired washed andisolated again. Various processes can be used to inactiv-ate the microorganisms.

Possible methods of inactivation consist in causingtypical substances lethal to cells, such as ethyleneoxides, formaldehyde, ©sons, mercury compounds or organicsolvents, such as methanol, ethanol or acetone, to act onthe microorganisms or in killing the microorganisms byheating to from 90"C to 140"c, by the influence ofextreme pressure differences (disintegration), the actionof high-frequency electric fields or by UV irradiation,irradiation with V-rays or the action of ultrasound. Theconditions under which inactivation can be carried outare well known to the person skilled in the art (K.H.Wallhauser, H. Schmidt: Sterilisation, Desinfektion,Konservierung, Chemotherapie, Georg Thierae Verlag,Stuttgart (Germany), 1967).

Fragments of elicitor-containing microorganisms can beobtained, for example, by lysis of the microorganisms bythe action of osmotic shock or temperature shock, byautolysis of the microorganisms, by treatment of thecells with ultrasound or by trituration of the micro-organisms with glass beads, powdered glass or quartzsand followed by differential centrifugation (Hughes, D.E., Wimpenny, J.W.T. and Lloyd, 0.: The disintegrationof micro-organisms. In: Methods in Microbiology, Vol. 13,(Norris, d.R. and Ribbons, D.W., eds.) pp. 1-54, AcademicPress, New York, London, 1971).

Purified cell wall fractions can be obtained from those 12 cell fragmentst for example,, by trypsin treatment. Thecell wall fractions already mentioned and used in thefollowing worked Examples were prepared in accordancewith the method described by Schleifer and Kandler (Arch.Mxkrobiol. §7, 1967, 335-365).

On the other hand, it is, however, also possible toprepare elxcitor-containing precipitates from water-soluble cell constituents by precipitation, for examplewith ethanol or acetone (Kocourek, J- and Ballou, C.S., J. Bacterial. 100, 1969, 1175-1181).

Secretions of el icitor-containing microorganisms areactively released cell constituents obtained by lysing, making "leaky", extracting with supercritical liquefiedgases (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, ifnecessary, be further purified, for example by extractionof lipophilic substances, adsorption of strongly colour-ing substances, etc..

It has already been mentioned that the use of enzyme-freeelicitor-containing material from bacteria to enhance thesynthetic capacity of components of higher plants hasbeen demonstrated experimentally; this could be ofimportance for the use of plant cell cultures in thepreparation of the active ingredients of medicaments(M.H. Zenk in: Pharmazie heute, 103. 1982, Volume 3,131-138).

The following worked Examples serve to illustrate theinvention in detail. 13 Example. 1 Stimulation of the synthesis of coloured components(actinorhodin, prodigiosin) in the case of Streptomyceslividans (ATCC 1S344) hv cell wall preparations. 80 ml of a nutrient medium comprising 103 g 10 g 10 ..,12 g0.25 g0.1 g 800 ml saccharose glucose magnesium chloride hexahydrate potassium sulphate casamino acids (Difco Labs, Detroit/USA)distilled water are sterilised (20 minutes, 120'C) and supplemented understerile conditions with the following freshly preparedsolutions. 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) 0.2 ml trace element solution - containing per 1 itre 40 mg 2inc(II) chloride 200 mg iron(III) chloride hexahydrate10 mg copper (XX) chloride dihydrate10 mg manganese ( XI) chloride tetrahydrate10 mg disodium tetraborate dihydrate10 mg hexaasraionium heptamolybdate tetrahydrate 0.5 ml IN sodium hydroxide solution 1.8 ml of that nutrient solution are introduced under 14 sterile conditions into each of the 24 3-sal-capacitychambers of a polystyrene multidish 'Multidish„ manufac-tured by Nunc, S200 Wiesbaden 12). 2 mg of the cell wallpreparations to be tested for their elicitor content arestex’ilised for 20 minutes at 120'C in bi-distilled waterand the resulting suspensions are added to the chambers. 2 Chambers receive no additions and are used as controls-The volume in all the chambers is Uoniformly adjusted to2 ml with bi-distxlled water under sterile conditions.Each chamber is inoculated identically with 5 μΐ of aspore suspension of Streptcmyces lividans (ATCC 19844).The test batch is incubated under aerobic conditions(Tablar shaker; 100 revolutions per minute) at 26"C.

After 96 hours,, the cells are isolated by centrifugation,washed with physiological saline solution and dried invacuo over calcium chloride to give the cell yieldslisted in the Table. The supernatants obtained bycentrifugation are adjusted to a pH of 7, diluted to 4 mlwith water and their absorption spectra are determinedbetween 180 and 800 nm. The relative amounts of thesynthesised dissolved secondary substances act i nor hod inand prodigiosin are determined approximately by assessingthe absorption peaks of the spectra recorded automatical-ly.

The following Table 3 shows the results obtained in thattest series. 15 TABUS 3 Tested bacterial cell walls of dry cell yield (mg) dyestuff content rel. absorp __txon units without (control) 22 1 B. ammoniagenes (ATCC 6872) 25 38 B„ glutasiingenes (ATCC 13747) 32 24 Ba. pumilus (ATCC 7061) 34 2 B. linens (ATCC 19391) 23 1 c, diphtherias (Mass. 8) 24 34 C- saelassecola (ATCC 17965) 26 34 C. glutamicum (ATCC 13032) 43 50 C. lilium (ATCC 15990) 33 40 Ce. cellassa (ATCC 14359) 36 2 L. piantarum (DSM 20174) 32 31 S. aureus strain H 44 1 B = Brevibacterium Ba = Bacillus C = Corynebacterium Ce = Cellulosnonas L = Lactobacillus S = Staphylococcus Example 2 Stimulation of the synthesis of coloured components(actinorhodin, prodigiosin) in the case of Streptomyceslivxdans (ATCC 19844) by cell wall extracts.

Under the conditions of Example lf but using the sterilefiltrates of the 2-rag cell wall preparations sterilisedfor 20 minutes at 120eC in bi"distilled water, a stimula- tian of dyestuff formation is achieved in Streptomyceslividans (ATCC 19844) that is almost as strong as whenusing suspensions of those sterilised cell walls.

...J Stimulation of the synthesis of coloured components(actinorhodin, prodigiosin) in the case of Streptomyceslividans (ATCC 19844) by cell extracts.· Under the conditions of Example 2, but using 20 mg ofbiomass instead of 2 mg of cell wall preparation, astimulation of dyestuff formation is achieved that isapproximately equally as strong as when using suspensionsof the sterilised cell walls. gj£§BSig__4 Stimulation of the synthesis of coloured components inthe case of Streptomyces coelicolor A3(2) or (ATCC 13405).

Under the conditions of Example 1, but using Streptomycescoelicolor A3(2), a marked stimulation of dyestuffformation (probably likewise actinorhodin) is achieved inthe case of this bacterium.

Stimulation of the synthesis of coloured components inthe case of Streptomyces griseoruber (DSH 40275).

Under the conditions of Example 1, but using Streptomycesgriseoruber (DSH 40275), a very marked increase indyestuff formation (presumably anthracvclin antibiotics)is achieved in the case of this bacterium also. 17 EMsaalS-A Stimulation of the synthesis of coloured components inthe case of Streptomyces purpurascens (DSH 40 310), Under the conditions of Example 1, but using Streptomyces5 purpurascens (DSH 40 310), a distinct increase in dyestuff formation (presumably likewise anthracyclinantibiotics) is achieved in the case of this bacteriumalso, Ε2£δΒΚΐ£_Ζ 10 Stimulation of the synthesis of coloured components inthe case of Streptomyces latericius (DSH 40 163).

Under the conditions of Example 1, but using Streptomyceslatericius (DSH 40 163), a significant increase indyestuff formation is achieved in the case of this bac- 15 terium also.

Example-.¾ Stimulation of the synthesis of coloured components inthe case of Streptomyces violaceus (DSH 40 082), Under the conditions of Example 1, but using Streptomyces20 violaceus (DSH 40 082), a significant increase in dyestuff formation is achieved in the case of thisbacterium also. 18 Example 9 Stimulation of the formation of B-lactam antibiotics(cephalosporins, penicillin K) by Streptomyces clavuli-gerus (ATCC 27064). 5 g 3-(M-morpholino)-propanesulphonic acid (MOPS) 3.5 g .dipotassium hydrogen phosphate0.5 g magnesium sulphate heptahydrate 2 g L~aspsragin@ 10 g glycerol 1 g yeast extract (Oxxd, Wesel, Germany) 1 ml trace element salt solution ~ containing per litre 1 g iron(II) sulphate heptahydrate1 g manganese(II) chloride tetrahydrate1 g zinc chloride heptahydratel g calcium chloride are wade up to 1 litre with distilled water and sterili-sed (20 minutes; 120'"c) . 1.8 ml of that nutrient solution are introduced understerile conditions into each of the 3-ml~capacitychambers of a sterile polystyrene multidish (Multidish;manufactured by Munc, 52 Hiesbaden 12). 2 mg of the cellwall preparations to be tested for elicitor activity or10 mg of the cells to be tested are added in the form ofhomogeneous suspensions sterilised in bi-distilled water(20 and 45 minutes, respectively, 120"C). Two chambersserving as control receive no additions. The volume inall the chambers is then uniformly adjusted to 2 ml withbi-distilled water under sterile conditions. Each chamberis inoculated identically with 5 μΐ of a spore suspensionof Streptomyces clavuligerus (ATCC 27064).

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

The formation of antibiotics is examined in comparisonwith the controls using the plate diffusion test» Thedetector organisms suspended in soft nutrient agar areMicrococcus luteus and Bacillus subtilis (106 cells perml). 25 μΐ of centrifuged (48,000 x g) culture mediumfrom the test chambers are applied to each ©f a number ofstandard filter plates (0,9 cm diameter). After adiffusion time of 4 hours at 4*C, the biotest is incub-ated for 24 hours at 30’c.

In the case of the cultures that were grown with theaddition of killed cells of Brevibacterium flavum ATCC13826 or of cell wall preparations of that bacterium orof cell wall preparations of Corynebacterium diphtherias(strain Mass. 8), clearly enlarged inhibiting areolasexhibited an increased formation of B-lactam antibiotics(penicillin M, cephalosporins) by streptomyces clavuli-gerus compared with the controls.

Exajaale-IO Stimulation of the formation of alkaloids (sanguinarine,chelirubine, marcarpine and chelerythrine) by cultures ofEschscholtzia califarnica - Tissue cultures of Eschscholtzia californica are grown ineach of 24 1-ral chambers of a polystyrene multidish(manufactured by Nunc, 6200 Wiesbaden 12) under theconditions described as optimum by J. Berlin e,t a_l. (Z.Naturforsch., 38c, 1983, 346-352). One of the chambersdoes not receive any further additions and is used as thecontrol, one chamber receives 266 rag/1 of heat-extracted 20 and ethanol-precipitated yeast elicitor (prepared inaccordance with Kocourek J. and Balleu„ C.E. „ J- Bacter-ial Iflfii, 1969, 1175-1181), and the others each receive266 mg/1 of cell wall preparation of the bacteria listedin Table 3. The cultures are then incubated for 72 hoursat 24 "C and then the alkaloid content of the cultures isdetermined photometrically, the alkaloid content inducedby the yeast elicitor being evaluated as 100 %.

Table 4 below shows the results obtained in this test series. TABLE 4 Tested bacterial cell walls % elicitor activi£v Brevibacterium butanicus ATCC 21196 19 Brevibacterium flavum ATCC 13826 16 Brevibacterium flavum ATCC 14067 30 Brevibacterium glutamingenes ATCC 137 113 Brevibacterium lactofermentum ATCC 13655 43 Brevibacterium ammoniagenes ATCC 6872 40 Corynebacterium hydrocarboclastum ATCC 15592 8 Corynebacterium nephridii ATCC 11425 123 Corynebacterium paurometabolum ATCC 8368 16 Corynebacterium liliua ATCC 15990 108 Corynebacterium striatum ATCC 6940 17 Corynebacterium petrophilum ATCC 19080 0 Corynebacterium xerosis ATCC 373 102 Corynebacterium diphtherias strain Mass. 8 137 Rhodococcus fascians ATCC 12975 27 Rhodococcus fascians 1 11 isolate of Prof. Dr. Stolp* Univ. Bayreuth Rhodococcus fascians 2 isolate of. Prof... -Dr Qni,y^ ftavEeush. 21 Exafflels-ll Stimulation of the formation of indole alkaloids (valles-iacotaaine) in cultures of Rauvolfia serpentina. A suspension culture of Rauvolfia serpentina (Stockist, J., A. Pfitzner and J. Firl: Plant Cell Rep. 1, 36-39(1981) is cultivated in ULnsmaier and Skoog (LS)-medium(Physiol. Plantaruis 18. 100-127 (1965) en rotary shakers(100 revolutions per minute) at 23 "C vith permanentlight (6Θ0 lux). For elicitation# 200 g of call freshveight/1 LS medium are inoculated. Cell wall preparationsof the bacteria listed in Table 4 are used at a concen-tration of 130 mg/1 medium as elicitor-containingfragments of microorganisms.

After 5 days' incubation, the biomass has doubled both inthe elicited cultures and in the controls. The cells areharvested and extracted with methanol.

The amount of the indole alkaloid vallesiacotamine is determined by separating the extracts by HPLC. Whereasthe untreated control cultures contain only 1.16 mg/1 ofmedium, the yield in the case of the elicited cultures isa maximum of 58 mg/1. That corresponds to a 50-foldincrease by the elicitor.

Sxa®aia=ia Stimulation of the 17-keto steroid reductase activity ofRhodotorula glutinis XFO 0389. a) A 2-litre Srlenmeyer flask with. 500 ml of sterilenutrient medium containing5 % glucose monohydrate 22 2 % cornsteep liquor - adjusted to pH 6.5 - is inoculated with a smear from a slant agarculture of Rhodotorula glutinis IF0 0389 andcultivated for 40 hours at 30" C and. at 190 revolu-tions per minute. b) A SOO-ni Erlenmeyer flask with 100 ml of sterilenutrient aieditm containing 1 % corasteep liquor 5 % Hurupan (R) (aanufacturer Murupan GnbK, 4000DQsseldorf 1? Gerssany) 1 % Hetarin W (manufacturer Lucas Meyer; 2000 Hamburg 2S; Genaany) - adjusted to pH 6.2 - is inoculated with 10 ml of the Rhodotorula pre-culture prepared in accordance with Example 12a andcultivated for 7 hours at 30"C and 180 revolutionsper minute . 10 mg of 3-hydraxy-l, 3,5( 10) ,7~oestratetraen-17-oneare then added to the culture and fermentation iseffected for a further 210 hours. The culture isthen extracted with methyl isobutyl ketone, theextract is concentrated and the resulting crudeproduct is purified by chromatography on a silicagel column to yield 5.9 mg of 1,3,5( 10) ,7-oestra-tetraene-3„ 17a~diol = 59 % of the theoretical yield. c) 10 mg of 3-hydroxy-l, 3,5 (10), 7-oestratetraen- 17-one are fermented with a culture of Rhodotox-ula glutinisunder the conditions of Example 12b except that 5 mlof a sterile suspension of 50 mg of a cell wallpreparation of Bacillus licheniforssis (ATCC 9945) inwater are added to the culture immediately beforethe addition of the substrate. After working up the 23 culture, 6.8 mg of 1,3,5(10),7"oestratetraene-3,17-diole = 68 % of the theoretical yield are obtained. £KawlS_U.

Stiaulation of the steroid Δχ-dehydrase activity of5 Bacillus lentus (ATCC 13805). a) A 2-litre Erleimeyer flask with 500 al of sterilenutrient solution containing0.5 % cornsteep liquor Q.05 % glucose nonohydrate0.1 % yeast extract ~ adjusted to pS 7.0 - is inoculated with a rinse of Bacillus lentus (ATCC13 805) and shaken for 48 hours at 30'c and at 190revolutions per minute. 20 25 30 b) A SQO-ffnl Erleruaeyer flask with 100 sal of sterilenutrient solution containing3.0 % soya powder 0.5 % cornsteep liquor 0.1 % yeast extract 0.05 % glucose raonohydrate~ adjusted to pH 7.3 - is inoculated with 10 ml of the Bacillus lentuspreculture and shaken for 7 hours at 30"C and at 180revolutions per minute. A sterile-filtered solutionof 40 sa of Se^a-dif luoro-llB^lVe-dihydroxy-ieffi-methyl-'J-pregnene-l ,20-dione in 4 ml of dimethyl-formamide is then added to the culture and the wholeis incubated for a further 41 hours.

The culture is then extracted with methyl isobutylketone, the extract is concentrated in vacuo andthe residue is purified by chromatography on a 24 silica gel column to yield 16 sg of 6cf9c:~dii luoro-1XB,17a-dihydroxy"16c-methyl-l „ 4-pregnadiene-3,20-dione (= 40 % of the theoretical yield)» c) 40 mg of 6ci,9G-difluoro-llBl)17G-dihydroxy-l6G" methyl-4-pregnene-3,20-dione are fermented with aculture of Bacillus lentus under the conditions of Example 13b except that 5 al of a sterile suspensionof 50 mg of cell wall preparation of Corynebacteriumdiphtherias (strain Mass» 8) in water are added tothe culture immediately before the addition of the substrate. After working up the culture, 21 mg of Se e 9®-dif luora-llfi g I7c-dihydroxy»16ec-methyl-i , 4-pregnadiene~3»2G~dione (= 52.5 % of the theoretical yield) are obtained. S3£3JiEl£_ll Stimulation of the formation of alkaloids (lysergic acidamide and isolysergic acid amide) by Claviceps paspali(ATCC 13895). a) A 500-ml Erlenmever flask with 50 ml of a starilanutrient solution containing4 % sorbitol (industrially pure) 1 % glucose monohydrate 2 % succinic acid 0.6 % ammonium sulphate 0.5 % yeast extract (Difco(^) manufactured tv Difco Labs. Detroit/USA) 0.1 % potassium dihydrogen phosphate 0.03 % magnesium sulphate heptahydrate-adjusted to pH 5.2 with sodium hydroxide solution-is inoculated with a culture of Claviceps paspali(ATCC 13895) deep-frozen to -70‘c and shaken for 5days at 24’C and 240 revolutions per minute. 25 10 15 b) A 500"®l Erlenmeyer flask with 50 sal of a sterilenutrient solution containing3 t sorbitol (industrially pure) δ % succinic acid 0-9 % ammonium sulphate 0.1 i calciua nitrate tetrahydrate 0.05 % dipotassiu® hydrogen phosphate 0.03 ϊ magnesium sulphate heptahydrate 0.02 t yeast extract (Difc©^ manufactured by Difco Labs., Detroit/USA) 0.0007 % iron(II) sulphate heptahydrate0.0QQ6 % zinc sulphate heptahydrate-adjusted to pH 5.2 with sodium hydroxide solution-is inoculated with 5 si of a pre-culture of Clavi-ceps paspali and shaken for 250 hours at 24’c and at240 revolutions per minute.

Sufficient sodium hydroxide solution is then added to the culture to adjust the pH to at least 10,extraction is carried out with «ethyl isobutylketone and the extracts are concentrated in yasna and purified by chromatography on a silica gelcolunn. 35 rag of a mixture of lysergic acid aside andisolysergic acid amide (yield 700 sag/I of culture) 25 are thus obtained. c) A culture of Claviceps paspali is grown under theconditions of Example 14b except that 5 ml of asterile suspension of 25 rag of cell wall prepara-tion of Lactobacillus casei subsp. rhawraosus (ATCC 30 7469) in water are added to the culture after 72 hours. After working up the culture# 45 rag of amixture of lysergic acid amide and isolysergic acid 26 amide (yield 900 sag/1 of culture) are obtained.

Exassls-U Stimulation of the 15e-hydroxylase activity of Penicil-lin® raistrickii (ATC'C 10490). a) A 2-litre Erlennever flask with 500 al of sterilenutrient aediua containing 3 % glucose aoncfcydrate 1 % eornsteep liquor 0.2 % sodiuE nitrate 0.05 % magnesia» sulphate heptahvdrate 0.05 t potassium chloride0.002 % iron(II) sulphate hexahydrate0., 1 * potassium dihydrogen phosphate 0.2 i dipotassiua hydrogen phosphate - adjusted to pK 6.0 - is inoculated with a suear from a slant agarculture of Penicilliuia raistrickii (ATCC 1G490) andcultivated for 48 hours at 30 "c and at 180 revolu-tions per ainute. b) A 500—331 Erlenmeyer flask with 100 ml of sterilenutrient medium containing 1 1 cornsteep liquor 3 % glucose monohydrate 0.1 § potassium dihydrogen phosphate 0.2 % dipotassiu® hydrogen phosphate 0.05 % magnesium sulphate heptahydrate - adjusted to pH 6.0 - 27 is inoculated with 10 ml of the Penicillium pre-culture prepared in accordance with a)« 300 sag of l3~ethyl-4-gonene-3,17-dione are thenadded to the culture and the whole is fernented for 5 120 hours at 30 "c and at 180 revolutions per minute.

The culture is then extracted with methyl isobutvlketone, the extract is concentrated and the result-ing credos product is purified toy chroaatography ona silica gel column to yield ISO ag of 13-ethyl-15c- 10 hydroxy-4-gonene-3,17-dione. c) 300 mg of 13-ethyl-4~gonene-3,17-d£one are fermentedwith a culture of Penicillium raistrickii under theconditions of b) except that 5 ml of a sterilesuspension containing 50 mg of a call wall prepara- 15 tion of Corynebacteriua diphtheria® (strain Hass- 8) in water are added to the culture issBediaeelybefore the addition of the substrate- After workingup the culture, 210 mg of 13-efchyl—15c-hydroxy~4-gomene-3,17-dione are obtained.

Claims (7)

28

1. Method of enhancing enzyme activities and the syn-thetic capacity of microorganisms and higher plants,characterised in that the same are brought into contactwith inactivated elicitor-containing nicroorganisus ,fragments of the same or secretions of elicitor-contain-ing microorganisms, with the proviso that, in order toenhance enzyme activities and the synthetic capacity ofhigher plants, inactivated elicitor-cemtaining bacteria,fragments of the same or secretions of elicitor-contain-ing bacteria are used.

2. Method of enhancing enzyme activities and the syn-thetic capacity of microorganisms according to patentclaim 1, characterised in that the same are brought intocontact with inactivated elicitor-containing micro-organisms , fragments of the same or secretions ofelicitor-containing microorganisms»

3. , Method of enhancing the synthetic capacity of nicro-organisns according to patent claim 2, characterised inthat bacteria, fungi and yeasts capable of formingdyestuffs, alkaloids or antibiotics are brought intocontact with inactivated elicitor-containing bacteria,fungi or yeasts, fragments of the same or secretions ofthose microorganisms,.

4. Method of enhancing enzyme activities of micro-organisms according to patent claim 2, characterised inthat bacteria, fungi or yeasts capable of steroidtransformation are brought into contact with inactivatedclicitor-containing bacteria, fungi or yeasts, fragmentsof the same or secretions of those microorganisms.

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

6. , Method off enhancing enzyme activities and the syn-thetic capacity of microorganisms and higher plantsaccording to patent claims 1 to 5, characterised in that 8 0 the same are brought into contact with elicitor-contain-ing microorganism» heat-sterilised in water, or withfiltrates of the same.

7. , A 'taechod subscatttiaily as hereinbefore describedwich reference to the Examples. Dated this 1 3 eh day of January 1989 CRCICKSKABK δ CO. Agents for the Applicant 1 Holies Street Dublin 2