Note: Descriptions are shown in the official language in which they were submitted.
13~72~
METHOD FOR INCREASING ENZYME ACTIVITIES
AND SYNTHESIS PERFORMANCE OF ORGANISMS . -
Back~round 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 oP
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, 1981, 447ff; Adv.
Enzymol. 55, 19$3, lff; Spektrum Der Wissenschaft'' 11, 1985,
85~f)-
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 economicaly
feasible conditions.
1 --
~3~7~7
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 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 oxganisms. 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 excretion of elicito-
containing microorganisms, with the proviso of using, for
increasing enzyme activities and svnthesis performance of
nonmicrobial organisms, inactivated elicito-containing
2 --
~3~7~
bacteria, fragments thereof, or excretions of elicitor-
containing bacteria.
Thus, this invention provides a method for enhancing
enzyme activity of or the synthesis performance of an
organism, comprising contacting said organism to be enhanced
with an inactivated elicitor-containing microorganism, an
elicitor-effective fragment thereof, or an eli~-itor-effective
excretion of an elicitor-containing microorganism, with the
proviso that when said organism is nonmicrobial, said
elicitor-containing 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 o~ 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 fraqments of mechanically processed or
chemically or enzymatically lysed cells, or cell components
precipitated by 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 o~
this invention.
In the case where the microorganixm 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 o~
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.
-- 3
~ .7.,
~ ~ ~ 72~
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-
5 positive bacterial strains of the general 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.
~0
_ ~ _
72~7
TABLE 1
Alternaria carthami Arch. 8ioch. Biop. 229,1984, 136
Botrytis cinerea (ATCC 48345) Physiol. Plant Pathol.11, 1977, 287
Ceratocystis fimbriata Phylochemistry 23, 1984, 759
Ceratocystis uimi Phytochemistry 23, t984, 383
Chondrostereum purpureum J. Chem. Soc. Perkin Trans 1. 1984,14341
Claclosporium fulvum (ATCC ~4961) Physiol. Plant Pathol.16,1980, 391
Colletotrichum lindemuthianum ~ATCC 52471) Eur. J. Biochem.129,1983, 593
Fusarium solani Plant Physiol. 76,1984, 833
Fusarium solanifspmori (ATCC 44934) Nat. Prod. Rep. 2, 1985 439
Ganoderma applanatum Phytochemistry _, 1983, 1039
Glomerella Cingulata Physiol. plant. Pathol. 21, 1982,171
Helminthosporium carbonum (ATCC 24962) Z. Naturforsch. Sect. C 38,1983, 899
Monilinia ~ructicola J. Am. Chem. Soc., 84, 1962, 1919
Nectria haematococca Phytochemistry 22, 1983, 2291
Phoma exigua Phytochem. 21,1982, 1818
Phytophthora cannabivora Z. Naturf. Sect. C. 39,1~84, 217
Phytophthora capsici (ATCC 52771) Physiol. Pla~. Pathol.18, ~981, 379
Phytophthora infectans (ATCC 44776) Phytochem. 23, 1984, 537
Phytophthora megasperma var glycinea Arch. Bioch. Bioph. 229, 1984,136
Phytophthora infectans Phytpathol. Z, 27, 1956 237
Phytophthora megasperma J. Biol. Chem. 259,1984,11841
Phytophthora nicotiane Phytopathology 71, 1981, 864
Puccinia coronata Physiol. Plant Pathol. 20,1982, 189
Pyricularia oryzae (ATCC 15923) Agric. ~iol. Chem. 48,1g84 253
Saccharomyces cerevisiae Plant Physiol. 62, 1978, 107
Verticillium albo-atrum Nat. Prod. Rep. 2,1985 429
Verticillium dahliae (ATCC 26289) ATCC-Katalog. 16th ed. 1984
sl ,~
~3~7~l~7
TABLE 2
Bacillus licheniformis ATCC 9945
Brevibacterium butanicum ATCC 21196
Brevibacterium flavum ATCC 13826, ATCC 14067
Brevibacterium lactofermentum ATCC 13655
Brevibacterium glutamingenes ATCC 13747
Brevibacterium ammoniagenes ATCC 6872
Brevibacterium pumilus ATCC 706~
Corynebacterium hydrocarboclastum ATCC 15592
Corynebacterium nephridii ATCG 11425
Corynebacterium paurometabolum ATCC 8368
Corynebacterium lilium ATCC 15990
Corynebacterium striatum ATCC 6940
Corynebacterium xerosis ATCC 373
Corynebacterium diphtheriae (Strain Mass. 8/Behring Werke)
Corynebacterium melassecola ATCC 17965
Corynebacterium glutamicum ATCC 13032
Corynebacterium uratoxidans ATCC 21749
Lactobacillus casei subsp. rhamnosus ATCC 7469
Lactobacillus plantarum DSM 20174
Pimelobacter tumescens AJ ~460
Rhodococcus fascians ATCC 12975
Rhodococcus fascians 1-Isolate by Prof. Dr. Stolp. Univ.
Bayreuth
Rhodococcus fascians 2 Isolate by Prof. Dr. Stolp. Univ.
Bayreuth
:
,; ~,
~7~
Thus far, bacterial strains of only a few genera of
gram-positive eubacteria have been investigated wik~i-n the
scope of the present invention as to whether they have
elicitors. Also among the fungal strains, including the
yeasts, in most instances, those examined for the presence of
elicitor activities -- insofar as this can be derived from
the prior publications -- are those known as phytopathogenic.
Therefore, there will be numerous additional
microorganisms that likewise have elicitors, such as, for
example, bacteria of the genera Mycobacterium, Nocardia,
Nocardioides or Pseudonocardia. 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 microorgainsms or
other organisms whose enzyme activity or synthesis
performance is to be increased can be grown, for example, in
submerged culturss, 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 o*
microorganisms. Thus, for example, by adding cell wall
preparations of microorganisms listed in Table 2, the
-- 7
.~ .
2 ~ 7
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 ~-lactum 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 bacterial 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.
2Q 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 dehydrogenate
steroids in the 1,2-position, and in the capability of
Rhodotorula glutinis to selectively reduce 17-keto steroids,
and in the capahility of Penicillium raistrickii to
hydroxylate steroids in the 15-position. Thus far, only an
attempt to increase, with the aid of these cell wall
preparations, the ability of Curvularia lunata to the 11~-
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
-- 8
- "
~3~72~7
elicitor-containing microorganisms do effect such an
ncrease. ~
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, eryosine, ergocristine, ergocornine, agroclavine,
chanoclavine, festuclavine, paspalic acid, or the lysergic
acid derivatives; vitamins, such as vitamin B12, riboflavin,
or ~carotene; enzymes, such as the amylases,
glucoseisomerases, proteases, pectinases, lipases,
penicillinacylases, chitinase or lactase; nucleosides, such
29 as guanylic acid or inosylic acid; amino acids, such as, for
example, cysteine, glutamic acid, tryptophan, or lysin; 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 ~ or 15-
hydroxylation, l-dehydrogenation, 17, 17~-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,
..^ ~
~3~ 7~ 7
"Microbial Trans~ormations 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 vf 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
espicially 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
con~unction with the guidance of this specification.
The addition of the elicitor, e.g., the inactivated
microorganisms, the frayments or extracts of these organisms,
or the excretions of elicitor-containing microorganisms, can
take place as early as at the beginning of the fermentation.
3Q The optimum time of addition is, of corse, dependent on the
type of microorganism or other cells that are incubated,
especially on the curve of its exponential growth phase,
-- 10 --
., ,i;
-
3~7 ~7
~nd aan ~ d~ermin~l rc~utlnely in ~n indl~idual c:a~e.
q:hu~, it i~; f:r~querltl~ exped~er~t:, for ~ax~ple, in
aa~3e of ba~teria to ef~e~;~ thil3 Addit:i~n 4-30 hours
~iEte~ ~h~ on~iet ol~ ~erment~ltioll~ Wh~n ad~ing
i~açtivat~d mic:roorgani~m~3 or ~ragment~ t~ereof, then
u~ually 1 - 1000 g (prefer~bly 10 ~ ~0~) g) t:~
ina~-tivated microor~ani~m or 0.1 - 100 ~ tpre~erably ~ -
30 g) of th~ fragment of thl~ or~ani~ ; u~ d per
~ubic meter of ~e~rment~tion bro~h. Whe~ u lng
exGr~tion~ of ~licitor-oon~ining miaroorg~ni~m8
will normally be su~ ien~ to u~ per ~Ublc ~eter o~
~erm~ntativn volume, 1 - 50 l o~ t~e exar~tion 301utio
When the proa~ss of ~hi~ inventi~n i~ used ~or
i~crea~in~ ~h~ en~-ym~ activity of a mioro~rg~ni m
employe~ ~or the ~yma~ia conver~ion o~ su~strateæ r
~hen ~he addition o~ the sub~trat~ Will usually ~tar~ O
- 10 h~ur~ er ~he ~licitor-containin~ inactiva~
m$Gr~or~Ani~m or its ~ra~m~n~ or ex~e~i~n~ h~v~ b~n
a~e~.
~0 For ~on-miarobial organi~m~, ~7g~ ~ a~ ul~u~æ
[inaludln~ u~ cultur~s) o~ pl~n~ ~ell~, ~ni~Al
c:ell~, inaludin~ mammalian su~h a~ hum~n ~ imilzllr
~ela~ive amount oP inaa1;ivated ml ::rvc~rgarli~n~,
fr~gmen~ ~r ~xcretion~ c:an be u~ed.
The optimum ferm~ntAtiQn c::ondition~ will v~ry ~;
uGual dependin~ on ~he type ~:f mi~oxganl~m or culture
medi~ utilize~, on th~ type ~nd lauarlti~y of elic:itor-
containing m~terial , etc l; th~y c~an rou~in~ly b~
determine~ in an lndi~ridual ca~3~ by r~u~ine preliminary
t~ t~ highly ~ami~iar to t~se ~kill~d in ~h~ ~r~.
~o pr~p~r~ ~he inaG~ivated ~orm of the ~ ox-
c:~n~a~nlny mic::roor~anism t they carl ~Eirxt be inauba~e~
un~ler thelr u~ual condl~lon~; then ~;epara~d
conventionally f~om ~he culkure ~ h 3by c~n~ri~uylnç~ or
filtration, optionally wa~hed, and a~a~n i~olal~ecl.
Var1o~s mç~thod~ can ~e employed ~or inactivating tha
! .
3~ 72~ ~
mlaroor~fani~3ms , i . ~ ~, e~ating a perman~n~ lo~ of
ility.
Pc~:~ibl~ ina~ti~r~tlon ~n~thod~: ln~lude ~xpo~n~
these mio~oo~anlsms to typia~l oytotoxins, guc:h a~
S e~hyl~ne oxideG, ~orm~ldeh~rde, 020n~, meraury ~ompou~d6,
organlc: 801vent8, ~2uch as me~hanol, elthAnol or acet~n~,
or killin~ the microorg~niG~n~ lby ~e~in~ t~ ~0~ ~o
140C, by the eff~t of extre~n~ pree;sure dif~er~nce~
~di~integr~on), by ~he ef~ t ~f high-~r~quenay
~leatric~ sEields, ~y W irradiatic;~n or irradia~ion with
ray~, or by ~h~ ~Pf~c~ o~ ultra~c~und~ Th~
conditionEt und~r which inactivation c~n ~e c:onducted are
w~ll known to a p~r~on ~3killed in th2 ar~
Wallh~u~:e~, ~, S~hmid~: "$~e~ilizat.ion, Di~in~ctlon,
Pre~ervation, Chemotherapy~i Georg Thiem~ Puhli~he~rs,
S~ut~gart~ ;erma~y, 19~7)~
Fraq~nentf3 o~ eli~itor-~ont~inins~ mic~oorg~ m~ can
~e obtalne~, for ~xample, by 1 ysing ~h~ microorgarlism .
l~y th~ o~ o~mo~ ho~:k or temper ~ure ~:ho¢l~ ~ ~y
dut41y~i~ of ~h~ microor~ani~m~ y treat~ng the aell~
with ultrasound, or by trituratlon ~f the rdiaros;~gani~m~
with gla~ heil~s, ground gl~ or ~ z ~hd ~nd
~ub~equent ~ifferenti~ n~ ug~t.ion (Nugh~, D. E~,
wi~npenn}t, J.W-T., and ~loyd~ "Th~ nt~gra~i~n C?P
Micro-Organiæm~ in: Method~ in Miar~hiblogy vol 13
t~orrl~, J4R. ~d RibbvnR, ~W. ~ ed~ ~ pp~ 1 ~4,
A~ad~ Pre~E~, Nellw York, I.ondon, . ~g71) 0
Puri~ied c:ell wall ~rac:tion~ can ~ prep~r~ m
the~e c~ ragm~nt~, for ex~nple, }~y ~p~3in ~at~nt.
The a~ove-~en~ioned ~:ell w~ rac~tion~: utill~d ~ h~
~u~e~uent exampl~ were produced in acc:orda~noe with t~e
me~h~d di~ale)~:e~ b~ Schl~ r ~nd ~dl~r ~rc:h.
~ikrs~bio}. 57: 33~365, lg67).,
O~ the okher h~nd, h~weYer~ it ~ ;o p~lble
prepare ~licitor~::ont~ininçl pr63cipitat~ ~Eri~m watçar-
~oluble t:ell ~mporle~k~ l~y prec~ipitatic~ or ex~n~ple,
L3~2~
with eth~ l or aae~one (Kocourl3kt ~ nd Elallou, C~. E .,
J. B~ctQriol~ lOP: 1175--llBl, 1~6g) .
5uit~ble exar~tiorln of ~lial~or-con-taining mlaro-
org~nism~ are activ6~1y ~ a~ed a~ll componen~3 ob~c~ine~l
S by lysing, 3~rend~ring 1~A)~Y~ xtrAct~ on with cuper,
critical lique~l~d ga~e~ (eO~, csar}~on diosc~ ), or
~teriliz~ion of c:Rl18~ wat~r- oluble ~ulture broth~ ~r
aul~ure 13roth6; obtained by ~ilte~ing of f ~ ~ent3~ ugin~
the org~nl~m Th~se c~n b~ ~urther purifi~d i~ re-
lo qulred, ~or e~ample by extract~ on o~ lipophili~ avm-
pounds, a~3orption of Rtronqly coloriJ~sr sub3tance~, etc:.
Ih Rssence, ~hç~ el i ~i~ors~ oan ~e used in any ~o~m
a~ long a~; th~y retain ~h~ir ef~içacy in ~c~ord~n~a with
tlli~ inven~ion. All su~h ~o~m~ are ~ ont~mpla~d a~
equi~ralent~ ~or us~3 in th~ ~ invention.
Ac¢ording ~o thi lJ~ ntic)n, anaïogou~ ~o th~3
goregoin~, ~liaitor dexived ~rom ~ teria~ a.g.,
inactivat13d elicitor~ aining hac:te~ia, ~ra~fffl~nt~
thereof or e~n~etion~ elic:itc~r-cont~ining l~aa~sria,
aan ~lso be employed for lnc~ ing en~ iviti~:
~nd synthe~i~ performana~3 in tis~u~, tis6~ cultur~s,
c~ ultures , e'cc ., derlv~d fr~ his~her organi~m~ , ~uc:~
a~ plant~ imal~, e.g., n~ unals, in~;luding h~man~,
e~p~ci~lly pl~nt~. A~ h~ b~n mentioned ~bove, u~e c~
enz~me ~I~e~ eliaitor-a~n~ining ma1~erial o~ b2~t~x:ia
will inar~as~ the performanc~e o~ th~ ~ynthe~i~; o~
produat~ ~y higher pl~nt~ a~ ha~ b~en ~on~$~ by
experim~n~ hi~ will ~e oi~ importanc: e ~or th~
utiliza'c~on o~ ~reg~able c:ell ~ul~urç~ fo~ th~
3 o prepa~a~ n o:~ active medic~inal ~gents ~M . H . Zenk in:
i'Pharma2i~ heute" [Today ' ~ Pharmac:y~ rol 3 : 131-
13~ 2). The ~la~its:~r-c~n~ining materlai o~
bac~erie~ will lik~wi~ rve per ~hi~ in~rention ~or
inc:rea~ing ~he pl3rformanc:e o~ ~he ~ynthe~l~ oP
ingr~dients in cultu~e~ of anir~al ti~ Ç~ or a~
inaludin~ human and ot~er mammalian ti~uex or
~ 3 ~
cells, and will be useful in therapy -- for example, in
treatment of wounds by increasing the rate at which ~ells
produce wound-treating components.
While this application primarily discusses the
enhancemsnt 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,
inerferon, 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 whatsoevex.
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.
--1'1 --
' `':
Example 1
Stimulation of Synthesis of Colored Ingredients
(Actinorhodin, Prodigiosin) in Skreptomyces lividans (ATCC
19844) by Cell Wall Preparations
___________..._________________________________________________.
80 ml of a nutrient medium consisting of
103 g sucrose
1010 g glucose
10.12 g magnesium chloride hexahydrate
0.25 g potassium sulfate
0.1 g Casaminoacids (Difco Labs, Detro.it~USA)
800 ml distilled water
is sterilized (20 minutes, 120C) 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
30200 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
35tetrahydrate
0.5 ml IN sodium hydroxide solution
- 15 -
Respectively 1.8 ml of this nutrient solution is
introduced under sterile conditions into the 24 chamb-ers of a
polystyrene multidish with a volume of respectively 3 ml
(Multidish, Nunc, 6200 Wiesbaden 12). Respectively 2 mg of
the cell wall preparations to be tested for elicitor content
is sterilized in twice-distilled water for 20 minutes at
120C, 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 conditions with twice-distilled water.
Each chamber is inoculated under sterile conditions
identically with 5 ~l of a spore suspension of Streptomyces
lividans (~TCC 19844). The incubation of the test batch
takes place aerobically ("Tablar" shaker; 100 rpm) at 26C.
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 supernatant
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 weighiny the absorption peaks of the
automatically recorded spectra.
Table 3 below shows the results obtained in the t~st
series of Example 1.
- 16 -
,~
, ~, ,.
~3~2~7
TABLE 3
Bacterial Cell Walls Dry Cell Yield Color Content
Tested of (mg) Rel. Absorption
Units
Without (Control) 22
B. ammoniagenes (ATCC 6872) 25 38
B. glutamingenes (ATCC 13747) 32 24
B. pumilus (ATCC 7061) 34 2
B. linens (ATCC 19391) 23
C. diphtheriae (Mass. 8) 24 34
C. melassecola (ATCC 17965) 26 34
C. glutamicum (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
B = Brevibacterium
C = Corynebacterium
Ce = Cellulomonas
L = Lactobacillus
S = Staphylococcus
- 17 -
p
,
~72~7
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 120C, 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
tActinorhodin, 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.
- ~8 -
,.
~ 3 ~ 7
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 for~ation (supposedly likewise actinorhodin) is
achieved also in case of this bacterium.
Example 5
Stimulation of Synthesis of Colored Ingredients
in Streptomyces griseoruber tDSM 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.
- 19 -
. ~
~3~72~7
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 40082), a significant increase
in pigment production is obtained also with this bacterium.
Example 9
Stimulation of the Formation of ~-Lactam Antibiotics
(Cephalosporins, Penicillin N) by Streptomyces clavuligerus
(ATCC 27064)
___________________________________________ _________________
g 3 (N-morpholino)propanesulfonic acid tMQPS)
3.5 g dipotassium hydrogen phosphate
0.6 g magnesium-sulfate heptahydrate
2 g L-asparagine
g 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 steriliæed
(20 minukes; 120C).
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 wall
preparations to be tested for elicitor activity, or
- 20 -
~72~'~
lo mg of the cells to be tested are added as homogeneous
suspensions sterilized in double distilled water (20 -and,
respectively, 45 minutes at 120C). 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 ~1 of a spore
suspension of Streptomyces clavuligerus (~TCC 27064).
Incubation of the test series is conducted under aerobic
conditions ("Tablar" shaker; 160 rpm) at 26C.
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 (106cells per
ml). On standard filter plates (0.9 cm diameter),
respectively 2~ ~1 of centrifuged (48,OGO x g) culture broth
from the test chambers are applied. After a diffusion period
of 4 hours at ~C, the biotest is incubated for 24 hours at
~OC.
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 ~-lactam
antibiotics ~penicillin N, cephalosporins) by Streptomyces
clavuligerus.
~3~72~7
Example 10
Stimulation of Production of Alkaloids ~Sanguinarine,-
Chelirubin, Marcarpine and Chelerythrine) by Cultures of
Eschscholtzia californica
________________________________________--.____________________
Under the conditions described to be optimal by J.
Berlin et al. (Z Naturforsch. [Journal of Natural Science.s]
~8c : 346-352, 1983), tissue cultures of Eschscholtzia
californica are grown individually in 24 1 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/l of heat-extracted and
ethanolprecipitated yeast elicitor (prepared according to
Kocourek, J., and Ballou, C.E., J. Bacterial. 100 :
1175-1181, 1969); the remaining chambers each receive 266
mg/l of cell wall preparation of the bacteria listed in Table
3. Then the cultures are incubated for 72 hours at 240C 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.
22 -
13 ~72~-~ 7
TABLE 4
Bacterial Cell Walls% Elicitor Activity
Tested
Brevibacterium Butanicum 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 lilium ATCC 15990108
Corynebacterium striatum ATCC 694017
Corynebacterium petrophilum ATCC 19080 0
Corynebacterium xerosis ATCC 373102
Corynebacterium diphtheriae Strain Mass. 8 137
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
- 23 -
,
,. .-
~3~
ExamPle 11
Stimulation of the Production of Indolc Alkaloids
tVallesiacotamine) in Cultures of Rauvolfia serpen~ina
A suspension culture of Rauvolfia serpentina (Stsckigt,
J., A. Pfitzner and J. Firl : Plant Cell Rep. ï ~ 36-39 ,
1981) iS cultivated in Linsmaier and Skoog tLS) medium
~Physiol. Plantarum 18 : 100-127, 1965) on rotary shakers
(100 rpm) at 23C and constant light ~600 lux). For
eliciting, 200 g of cell fresh weight per liter of LS medium
is used for inoculation. Cell wall preparations of the
microoryanisms listed in Table 4 are utilized as elicitor-
containing fragments of microorganisms, in a concentrati~n o~
130 mg/l of medium.
After an incubation of 5 days, the cellul~r 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 contain only 1.16 mg/l
of medium, the yield in the elicited cultures is maximally 58
ml/l. This corresponds to an increase of 50 times by the
elicitor.
Examplè 12
Stimulation of the 17-Keto Steroid Reductase Activity
of Rhodotorula glutinis IFO 0389
_________ ________.,____________________...____________
~a) A 2-liter Erlenmeyer flask with 500 ml of stsrile
nutrient medium containing
5 % glucose monohydrate
2 % corn steep li~uor
- set at pH 6.5 -
- 24 -
~ ~,r~2D~7
is inoculated with a smear of an agar slant of Rhodotorula
glutinis IFO 0389 and incubated for 40 hours at 30C -with 190
rpm.
~ b) A 500 ml Erlenmeyer flask with 100 ml of sterile
nutrient medium containing
1 % corn steep liquor
% "Nurupan " (manufacturer: Nurupan Gm~H, 4000
Dusseldorf 1, Germany)
1 ~ "Metarin ~i (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 7hours at 30C and 180 rpm.
Thereafter, the culture is combined with 10 mg of 3-
hydroxy-1,3,5(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-1,3,5(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.
Trademark
- 25 -
~7~
Example 13
Stimulation of Steroid ~ 1-Dehydrase Activity of ~ -
Bacillus lentus (ATCC 13805)
__________________________________________________
(a) A 2-liter Erlenmeyer flask with 500 ml of a steril~
nutrient solution containing
0.5 ~ corn steep liquor
0.05 ~ glucose monohydrate
0.1 ~ yeast extract
- 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 30C.
(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
- setat pH 7.3 -
is inoculated with 10 ml of the Bacillus lentus subculture
and shaken at 180 rpm for 7 hours at 30C. Then a sterile-
filtered so].ution of 40 mg of 6~,9~-difluoro~ ,17~-
dihydroxy-16~~methyl-4-pregnene,-3,20-dione in 4 ml of
dimethylformamide is added to the culture and the latter
incubated for another 41 hoursO
Then the culture is extracted with methyl isobutyl
k~tone, the extract is concentrated under vacuum, and the
residue is purified by chromatography over a silica gel
column, thus obtaining 16 mg of 6~,9~-difluoro~ ,17~-
dihydroxy-16~-methyl-1,4-pregnadiene-3,20-dione (= 40% of
theory).
:
~3~'72~7
(c~ Under the conditions of Example 14tb), 40 mg of
6~,9~-difluoro~ ,17a-dihydroxy-16~-methyl-4-preynen~-3,20-
dione is fermented with a culture o~ 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 6~,9~-difluoro-
11~,17~-dihydroxy-16~-methyl-1,4-pregnadiene-3,20-dione is
obtained (= 52.5% of theory).
Example 14
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 -70C, of
Claviceps paspali (ATCC 13895) and shaken for 5 days at 24C
with 240 rpm.
~ 3~2~
(b) A 50~ ml Erlenmeyer flask with 50 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 24C.
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 isobutyl
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/l 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 walI 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/l of culture).
- 28 -
~72~
Example 15
Stimulation of 15~-Hydroxylase Activity of Penicilli~m
raistrickii (ATCC 10490)
__________.._________________________________._____________
~a) A 2-liter Erlenmeyer flask with 500 ml of a sterile
nutrient medium containing
3 % glucose monohydrate
l % 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 30C
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 phosphata
0.2 % dipotassium hydrogen phosphate
0.05 % magnesium sulfate heptahydrate
- set at pH 6.0
is inoculated with lO ml of the Penicillium subculture
produced in accordance with (a).
Thereafter, the culture is combined with 300 mg of 13-
ethyl-4-gonene-3rl7-dione, and fermented for 120 hours at
30C with 180 rpmr
- 29 -
Then the culture is extracted with methyl isobutyl
ketone, the extract is concentrated, and the resultan~t crude
product is purified by chromatography over a silica gel
column, thus obtaining 180 mg of 13-ethyl-15~-hydroxy-4-
gonene-3,17-dione.
(c) Under the conditions of (b), 300 mg of 18-
mathylnorandrostenedione is fermented with a culkure 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
MassO 8) in water. After the culture has been worked up, 210
mg of 13-ethyl-15~-hydroxy-4-gonene-3,17-dione is obtainedO
- 30 -
- 31 - 13172~
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 e~amples.
From the foregoing description, one ski,lled 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.
