How Many Genes Are Required for the Synthesis of Chlortetracycline? Z. VAN~K, J. CUDL:[N, M. BLUMAUEROV~ and Z. H O ~ - L E K Department of Biogenesis of Natural Substances. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague 4 Received August 1, 1970
When contemplating the applicability of the theory of polygenic heredity to the development of views on the genetic regulation of the biosynthesis of secondary metabolites one encounters two extreme concepts. The first assumes that with increasing knowledge of the molecular basis of the biosynthesis of these compounds the views of polygenic heredity (formulated at the beginning of this century) cannot fundamentally contribute to the interpretation of new experimental data and to the formulation of a new working hypothesis in accord with the present state of our knowledge. The second concept proceeds from the simple fact that there is no unified theory of genetics at the present time to account satisfactorily for the heredity of complicated quantitative characters, such as the number of eggs laid, milk production, body weight, the production of various pigments etc. Most geneticists studying the improvement of industrial strains producing antibiotics are of the opinion that an increase in the production of these substances represents a very complicated matter and, basically, possesses all the features of polygenic heredity. We selected the study of the production of chlortetracycline by Streptomyces aureofaciens (Blumauerovs et al., 1969) to answer some of the questions accumulating on the biosynthesis and
regulation of secondary metabolites as a quantitative character (Vanfik et al., 1969a). Biogenetically, chlortetracycline belongs among the oligoketides. It is formed by the condensation of 8 molecules of malonylcoenzyme A and 1 molecule malonamylcoenzyme A (McCormick, 1965, 1969). In very simplified form, the entire metabolic pathway from glucose to chlortetraeycline can be divided into three independent sections. The first section includes the conversion of glucose to acetylCoA, the second section comprises carboxylation ofacetylCoA to malonylCoA, condensation of the malonate units to a hypothetical nonaketide and its partial condensation to a tricyclic skeleton. The third section includes the secondary transformations of this skeleton leading to the formation of the chlortetracycline molecule.
(1) Glucose ~ acetylCoA The glycolytic pathway to acetylCoA includes a total of 10 chemical processes to which corresponding enzymes can be ascribed. On the assumption of the ratio of 1 enzyme per gene, we might take ten genes into consideration. However, the situation is more complex. It is known that some of the key enzyme reactions of this pathway are not single-step but are rather carried out by enzyme complexes. For example, the dehydro-
226
Z. VA_N]~K E T A L .
genase complex, oxidizing pyruvate to acetylCoA, may be expressed as a sequence of 7 different enzyme reactions. I t is assumed t h a t all the required components (three or more proteins, thiamine pyrophosphate, thioctic acid, flavine adenine dinueleotide, magnesium, and unidentified disulphide and other poorly
Vol. 16
From what has been said, 64 genes should be involved in the pathway, not counting the possible isoenzymes. I f it is taken into account that many of the enzymes mentioned possess their own regulatory and operator genes one may expect that more than 100 genes are directly or indirectly involved in the transformation of glucose into acetylCoA. NADH TPP When evaluating the importance of this pathway for the formation of acetylCoA as a fundamental structural unit for the tetracene skeleton, the viewpoint may be defended that the mutagenic treatment of this pathway will not i S-S S-S affect the production of chlortetraeycline. In other words, if sufficient glucose is TPP.-CO-R TPP supplied, the glycolytic pathway should ~a~uPfs~b not affect the production of chlortetracycline. This viewpoint is to a certain SHSH S-S degree supported by experiments where the source of carbon was repeatedly T,I~ TOP / supplied in the course of fermentation which, in some cases, resulted in increased production of secondary metabS-S ~ S-S o]ites. RCOSCoA CoASH This point of view is rather simplified. Fig. 1. Oxidative decarboxylation of 2-oxoacids. Individual strains of Streptomyces aureofaciens producing chlortetracycline differ defined components) are under physio- markedly in the rate of glucose (sucrose) logical conditions bound firmly in a huge consumption. This fact is clearly reflected complex with an integral structure. It in metabolic differentiation, during tranappears that the activity of the complex sition from the anabolic to the catabolic is regulated (inhibited) by nicotinamide phase, or rather during their suppression adenine dinueleotide in its reduced form, on passing to the chlortetracycline prothe true inhibitor being acetylCoA (Han- duction phase. It goes without saying that the glycosen & Henning, 1966). The accompanying figure illustrates the many possibilities lyric pathway is coupled with other of regulation, inhibition and activation metabolic pathways, e.g. with the hexose offered by this relatively simple enzyme monophosphate shunt (Boretti et al., system (Fig. 1). It may be useful to 1956; Ho~f~lek, 1964) which is a source realize that a further 28 enzymes can of NADPH. I f one starts with erythrosecompete for pyruvate as substrate, 7 en- 4-phosphate, one may count with 9 enzymes for phosphoenolpyruvate, 5 for zyme reactions plus some regulatory 3-phosphoglycerate, 7 for glyceraldehyde- genes. Experiments with radioactive glu3-phosphate, 3 for fructose-l,6-diphos- cose in Streptomyces aureofaciens showed phate, 11 for fructose-6-phosphate, and that a certain optimal ratio of these 6 for glucose-6-phosphate, to list the pathways is required for CTC promain enzyme reactions that have been duction. The activity of the glycolytic pathway described so far. +
FA~UPS2
1971
in Streptomyces aureofaciens is intimately linked with that of the tricarboxylic acid cycle (Hogf~lek et al., 1969; Jechovs et al., 1969; Tint~rovs et al., 1969). In its classical form, this cycle contains ten enzymes (and hence genes), all catalyzing simple reversible reactions (with the exception of decarboxylation of 2-oxoglutarate to succinylCoA). Two more genes can be ascribed to isocitrate lyase and malate synthase. One may assume that the whole cycle will have its own operator and regulatory system with a control and coordination function. The key reaction here is not only the condensation of acetylCoA with oxaloacetate and the formation of citric acid but also carboxylation of phosphoenolpyruvate to oxaloacetate (Vo~t~ek et al., 1969; 1970). It is particularly during protein synthesis, during the formation of aspartic and glutamie acids when 2-oxoglutamate and oxaloacetate are being removed from the cycle continuously that the rate of carboxylation significantly affects the running of the machine which provides energy for synthetic processes of the cell in the form of ATP (Janglov~ et al., 1969). An important role during the production of CTC is played by the coupling of the glycolytic pathway with the oxidative reactions. In a grossly simplified form (disregarding compartmentation which is frequently held responsible for unexplained findings) one may assume that oxidation-reduction enzymes participate in more than 100 reactions. NAD(P) alone take part in no less than 80 enzymic transfers of hydrogen, 10 to 20 reactions are catalyzed by the cytochromes, three reactions by FMN, one reaction by FAD. Ubiquinones could not be demonstrated in our strains of Streptomyces aureofaciens. However, the strains of Streptomyces aureofaciens used here (both production and nonpr0duction) differed subs~antially in their eytochrome equipment. In conclusion, one may say that some
GENES FOR CHLORTETRACYCLINE
227
200 enzymes (and genes) participate in the metabolic conversion of glucose to acetylCoA. Mutagenic interference with this very complex mechanism may fundamentally alter the quantitative course of the pathway, whether by cutting off some enzyme systems or damaging their control centres (operator and regulatory genes).
(2) AcetylCoA
--+ tricyclic nona/cetide
In the second section of the metabolic pathway, from acetylCoA to the anthracene derivative, one may count 6 enzyme reactions (Fig. 2). The heavy arrow in the figure shows the formation of malonamide from malonyl, attached to a protein carrier, by transamination from glutamie acid. This is the first reaction which is characteristic for the formation of the tetracene nucleus and which does not occur in other metabolites (an exception may be formed here by the glutar~mide ring or cyeloheximide and of ~elated antibiotics; Van~k et al., 1967; 1969b, Cudlfn et al., 1969). The figure also shows another hypothetical possibility where malonamide is formed by deamination of asparagine. Condensation with other malonyl molecules takes place on a protein template. The hypothetical nonaketide is then cyclized and aromatized, accompanied by a loss of hydroxyl in position 8. A mutagenic change leading to the enhancement of the production of CTC in reactions taking place on a protein template is not likely. One can conjecture, however, that during inhibition of the transaminase giving rise to malonamide the terminal group of a protein template can be replaced by another suitable precursor. 2-Acetyldecarboxamidotetracyeline represents a metabolite that can be derived from a decaketide. The terminal group during the biosynthesis of this compound was apparently formed by aeetoaeetylCoA which, by virtue of its size, may fully substitute
228
Z. VANI~]K E T A L .
pyruvate
= AcSCoA
Vol. 16
+C02 _ -
,/COS-E MaSCoA
~
CH2 ~C00H
glutamate--~ 2-oxoglqtarate-~---~ CO-COOH asparaqine-~.-,--
/COSCoA CH2 ~
I
/COS-E CH2
\C0NH 2
CH2-CONH 2
\CONH2
CH2- COS-E
i
C0-CHz-CONHz 0
Y 0
"If" y 0 0
C0NH2
~
0
~
0
Y
0
Y 0
"CONH2
Fig. 2. Pathway from acetylCoA to the tricyclic nonaketide.
for malonamyl on the protein template as the starting unit. AcetylCoA may be metabolized under catalysis of at least 28 different enzymes. We assume that sufficient supply of acetylCoA for "anthracene synthetase" is one of the principal conditions for the satisfactory production of CTC. The biosynthesis of oligoketides is frequently related to the synthesis of fatty acids. However, "anthracene synthetase" differs fundamentally from f a t t y ~cid synthetase. First of all, it may be expected that it will have a much lower molecular weight. The carbonyl groups of condensing units of malonylCoA are not reduced, hence the cycle does not require NADPH, the dehydratase which forms a double bond, and FMNH2 for the reduction of this double bond t h a t would give rise to a saturated chain of the acetyl-protein carrier. In our experiments dealing with the acetylCoA carboxylase activity of the production and low-production strains of Streptomyces aureofaciens we observed
(B6hal & Van6k, 1970), B6hal & Van6k unpublished results) that the enzyme activity was twice as high in the production strain. The maximum activity of acetylCoA carboxylase is found during the anabolic phase of cultivation. At the period of intense formation of CTC, its activity is considerably lower. This finding led to the view that the formation of malonylCoA via acetylCoA carboxylase need not be the only way this precursor is formed. One may also take into consideration other reactions, such as the decarboxylation of oxaloacetate as was described for Phaseolus vulgaris and
Penicillium islandicum. One may deduce from the results obtained in the study of lipid synthesis by Streptomyces aureofaciens (B~hal et al., 1969a, 1969b) that fatty acid synthesis does not compete with CTC synthesis. Streptomyces aureofaciens does not accumulate lipids as storage material. Using incorporation of labelled acetate it was shown that lipid synthesis reaches its maximum at about 12 hours of
1971
GENES FOR CHLORTETRACYCLINE
derivative formed from the nonaketide by a three-fold dehydration. The scheme proceeds from the presently known metabolitcs isolated from the cultivation medium of Streptomyces aureofaciens. It cannot be excluded t h a t other metabolites will be described among other metabolic series, such as derivatives of the nonaketide with a hydroxyl group in position 8. The first and third branches include derivatives of tetracene, the first intermediate to be described being methylpretetramide. The second and fourth branches comprise metabolites with an open fourth ring (deoxoprotetrone). I t follows from the figure that at an early stage of biosynthesis methylation may take place in position C6 (paths Bl and B2). Metabolic paths B3 and B4 then result in the demethyltetracycline series (B3), or in protetrone (B4).* It follows from Fig. Bl that the tetracycline series may be split into two metabolic paths in a subsequent stage of biosynthesis. The decisive step here
cultivation. The rate of incorporation of acetate into lipids was higher in the production strain of Streptomyces aureofaciens. F a t t y acids in the lipid fraction were mostly branched which indicates t h a t their terminal group originates in or has a relation to the synthesis of branched amino acids. Although one may conclude from the experimental data that fatty acid biosynthesis does not compete with the biosynthesis of tetracycline-type compounds, the biosynthesis of oligoketides of types different from nonaketides with a ~erminal malonamide group is certain to interfere. Inhibition of the biosynthesis of these compounds, particularly when dealing with secondary metabolites, might lead to increased levels of compounds of the tetracycline series.
(3) Tricyclic nonaketide --> chlortetracycline Fig. A shows two possibilities of transformation of the hypothetical tricyclic Me_C 6
|
229
A-ring
+2H
cyclizotion
- H20
Me to t82
S
OH OH OH OH
meth~lpretelromid
ov-dT-C os/
~,.,.~.~.
y
o o o o
BI
OH OH OH
Me
/
~
-CONH2
Me
-
rOH
~
to eantl~rones ~'
~
B2
~CONHz
OH OH OH 0
o o o o
~ H
/'~y.-coN,~ OH OH
~
OH
to demethyltetmcycli~_~ B3
CONHz
OH
pretetromid
OcOH -
~0 proTBtfonO
~ B 4
ONH2
* Formulae with aromatic ring B and C are somewhat misleading (one could not envisage a hydroxylation in position 6); one must always keep in mind the keto-enol(aromate)-tautomerism of these tetracyclic polyhydroxy- (or polyoxo-) compounds.
230
Z. V A N E K E T A L .
V o l . 16
| OH-C 4
-2H
H20-C4a, IZo
Cl
Me
Me
~
o
i~
OH
H
~"~
.-
OH 0
OH 0
0
- ~---::~
6-d.o.~,.,,o.,,dq....
~
~
O
H
""~YY'CONH
-........... ~ - ' -
" " : ~
4-hydroxy-6-mithylpretetromid
,
z
~ ~.
.
.
.
C4
/ - ........ ~ ~ o . . , ---
~-
--"
OH 0
:
O~H6-deoxychloraureovocidin
"" - C5
A CI Me
met hylr hlorpreletromid
-- r
@ NH 2
2xMe
OH- C--6
_-_ ~
B1
Cl Me OH O H
CONH2 OH 0 OH OH met hylhydroxychlorpretetremid
Me OH BI
CONH 2 OH 0 OH OH methylhydroxypretetramid
1971
GENES FOR CHLORTETRACYCLINE
is the course of hydroxylation of ring A in position 4. After hydroxylation and another step (it may be assumed to involve either the addition of a molecule of water (C4a,t2a) or a hydroxylation (C12a) with subsequent reduction of the double bond) 4-oxoanhydrotetracycline is formed, which is chlorinated in position 7, giving rise to 4-oxoanhydrochlortetracycline (path CI). The series nonhydroxylated in position 4 results in methylhydroxypretetramid and, after chlorination, in the hitherto undescribedmethylhydroxychloropretetramid. If no addition of water occurs, tetramid-green and three other, so far unisolated compounds, are formed (paths s and {4). During inhibition of oxidation of ring A to qninone, one would expect the formation of chloraureovocidin and, in the absence of the chlorinating system,
231
that of aureovocidin (which is an aglycone of aureovocin; Vokoun et al.,
1970). In another step of the biosynthesis (Fig. s the derivatives of the tetracycline series may branch depending on whether the oxygen in ring A is replaced by an amino group. I n the next step 4-aminoanhydrochlortetracycline undergoes a two-fold methylation giving rise to anhydrochlortetracyeline. The last two steps of the biosynthesis are represented by hydroxylation in position 6 giving rise to dehydroch[ort~tracycline and by the final reduction giving rise to chlortetracycline. The branch in which no amination takes place could theoretically give rise to 4-oxoehlortetracyeline. Fig. s shows the metabolites formed after blocking of chlorination in position 7. The amination of these deriva-
@ NH 2
2xMe
OH -(~
1 Me OH
OH
cO~NH2
BI
OH O OH OH chloraureovocidin
Me OH
9H
BI
OH O OH aureovocidin
OH
Z. VAN]~K E T A L .
~,32
Vol. 16
@ 2xMe
NH 2
C(
M4
NH2
CI
4 - aminolnh~drochlor tetlo cycline
OH-C 6
Me
NMe2
2H
C( Me OH
onhydr
NMe2
C{ M# OH
dehydlochlocte,tra.cyr
NMo~
chlorlltracycline
....
BI
O
CI Me OH
C! Me OH
O
CONHz
4 -oxodehydror
CONHz
4,- oxochlortetr
cyclinlt
@ NH z
2 x Me
Me
NH2
/
/
OH-C6
Me
o. oH o
4"ominoa~ydrotetroc)lclJne
Nl~e2
o
anhydroIetroCyr
2H
Mo OH
o. o
o
,NMe2
o
dehydrotefrocycl~ne
~ Y
T~Tc~
tetracycline
B!
Me OH
OH 0
0
0
0
4 - oxodehydroletmcycline
I ~ OH
OH O
0
OH"0
4 - OXOtelvocyeline
CONHz
GENES
]971
FOR CHLORTETRACu
233
@ NHz
2 x Me
/~
OH
OH-C6
C[ Me
CONH2
OH O
NMe,20H~
H
OH OH
CHO
CH OH"CONH2
methylchloftetromid- blue
Bt
Cl MeOH
0 ONH 2
OH O
OH 0
ch~ortetramid - green
@ NH z
2 x Me
Me
# iY OH O
NH2
OH OH
OH - C 6
_
Mie
N~Me2
Y Y ~ ~'CONH z OH O OH OH
Me OH
N.NI",ZOH
ONH2
OH O
OH OH methyltetramid - blue
B1
Me OH
,0
OH
O H ~ ~ C O N H tetramid --green
z
234
Z. V A N ~ K E T A L .
Vol.
tires in the case that now methylation and hydroxylation have taken place gives rise to tetracycline. If no amination occurs the last metabolite of this path would be 4-oxotetracycline. Figs. C3 and C4 represent tctracene derivatives in which no addition of water in position C4a, 12~ took place. The aminated derivative after methylation and hydroxylation on Ca gives rise to methylchiortetramid blue. During inhibition of exchange of oxygen for an amino group the metabolite is the hitherto, undescribed chlorotetramide green (Fig. (:3). Fig s shows the metabolites in which neither the addition of water at Cda, Cz2a nor chlorination took place. After methylation and hydroxylation one would expect the formatfion of methyltetramid blue. During inhibition of transamination the metabolite is the previously described tetramid green. Fig. B3 contains the beginning of the biosynthetic pathway in which methyl-
16
ation in position Ca is inhibited. The first step is the described 4-hydroxypretetramid. The following sequence of reactions gives rise to 4-oxoanhydrodemethyltetracycline and 4-oxoanhydrodemethylchlortetracycline. From the hypothetical 6-dcmethyl-6-deoxytetramid green one may derive both chlorotetramide blue and chloro A C diquinone (Fig. E9), and the nonchlorinated analogues, tetramid-blue and A-C-diquinone (Fig. El{}). When the oxidation of ring A to quinone is inhibited one may derive two more monoquinone derivatives (Fig. s (:12). Fig. (:7 shows the last steps of the biosynthetic pathway leading up to demethylchlortetracycline. In this pathway, the individual intermediates have been isolated and described. In the absence of the enzymic ~ransamination system one may visualize the formation of the hypothetical 4-oxoanalogues.
@ o~-c~
-all
a~o-cd,,c~2o
CL Cl
~~.~o. z0 ~.J.,,~ .,~%..J.,~ .,~ o,
u. u u. ,,
,~M
/'
~
C
O
/ N
H
2
coN. z . . . .
c~'
4 - oxoClnhydro d e m e f l l y ~ r o c y c l i n 0
4-oxo~h~dtodeff'Mlth)'ltetrocycli~l
% ~
0
CI
"0
- C~.
~._----rC'~T TC'~T I"
C~
G- ~ , ~ I
ClO
- 6 - deoly -7-r
CII
Cl 0H 0NH 2
CI2
9~ C l Z
1971
GENES F(}K CHLORTETRACYCLINE
235
@ 2 x Me
NH2
OH-C 6 -2H CI
OH
i Y ~ OH O
~ ~'C~H 2 OH OH
~
NMeZH._~
~
CI
~
"111~
OH O
0
~
O
NMez
O
H
~-CONH2
OH OH
CONH2 OH O OH OH chlortetramid-blue
B3
O
C!
0
CONH2 OH 0 OH C) chloro -A-C-diquinon, e
@ 2 x Me
NH 2
OH-C 6 -ZH
NH 2
NMe2
CONH2 OH 0
OH OH
Q
NMe2
NHz OH 0
ONH2
OH OH
OH 0 OH OH tetramid-blue
B5
0 = O
H
0 ~
~
A - C - diquinone
OH
CONH2
236 Z. VAN~K E T A L .
Vol. 16
@ NHz
2xMe
OH-C6 -2H CI
0
OH
OH 0
OH OH
0
OH
B5
B3 CONH2
@ NH2
2xMe
OH-C6 -2H Cl
B3
~
0
O
H CONH2
OH 0
~
0 O
OH OH
H
B3 CONH2 OH 0 OH OH naphthacenequinone derivative
1971
GENES FOR CHLORTETRACYCLINE
If a metabolic block involves chlorination the final product will be demethyltetracycline or rather the hypothetical 4-oxodemethyltetraeycline (Fig. ES). If the fourth ring is not olosed (Fig. A) in the hypothetical anthracene precursor
237
the list of the possible intermediates and final metabolites is heavily reduced. Enzymes modifying the A ring in tetracyclic derivatives apparently cannot play a role here. The enzyme hydroxylating tetracenes in position 6 appears to be
@ NH2
2 x Me
Ci
NH2
CI
/ ~ o ~ "c~ /
OH-C6
4- aminoonh:fdrodemethyl chlortetracycline
2H
NMe-
~o~J~oC~
~o~o'PT'o~O,.~ ;.'~:~T'o oo,.~
Onh)tdrodemethylchlor tetracycline
dlhydrodemethylchlor tetrm:ycline
demethylchlodltrocycliF4
B5
c,o.
,L ~
.
"-~ , ~
o
.
~
0
"~ o Y ~ o
CONH2
OH
OH'~'*~O
CONH2
4 - oxodemothylchlorte~acychM
@ NH 2
2xMe
NH2
OH-C 6
NMe2
2H
9H
N,Me2
OH
NMe2
-c-, /
4 - ominoonhydrodemethyttetracycline
anhydrodemethyltetracyr
dehydrodemethylte/ra cycline
demethyltetrQcycline
'B3
\\
OH
G
OH CONH2
4 - oxodehy~odemethyltetracycline
0 COflH2
4 *oxodlmethyltltcacyr
238
Z. VANI~K E T A L .
Vol. 16
nonspecific, hydroxylating also the two hypothetical precursors to metabolites t h a t have already been isolated, i.e. anthrone and protetrone (Fig. B2, ]14). The chlorinated derivatives have not y e t been isolated. A general s u m m a r y of ~ll the enzyme reactions considered here, including all the intermediates and final metabolites, is shown in the scheme in which the metabolites are shown as full circles (the ones already described) and as e m p t y circles (the ones not yet isolated or simply hypothetical) (Fig. 3). The scheme contains a total of 72 compounds, 27 of which have been isolated, 45 hypothetical. These compounds m a y be derived from a hypothetical tricyclic derivative b y combination of a relatively small number (11) of enzyme reactions. They include eyclization of the A ring,
removal of the oxo group at Cs, methylation at C6, hydroxylation at C4, oxidation of the A ring, hydration at C4a, Clo.a, chlorination at 07, transamination, double :N-methylation, hydroxylation at C6 (and possibly oxidation to an oxo group) and the final reduction. In the scheme, the metabolites corresponding to the individual metabolic reactions are shown in vertical columns. The maximum of metabolites are formed after hydroxylation at C6 which would suggest considerable unonspecificity of the corresponding enzyme. Metabolic blocks (mutations) at the level of these enzyme reactions lead to the corresponding metabolites which might accumulate in the cultivation medium. In this way, one m a y prepare e.g. tetracyclines, demethyltetracyclines or aureovocidin, the accumulation of
|174 OH-C4 -2H
Ci-C 7 NHz
H20"C4aZl,a
2xMe OH-C6
~OOH
A
"anthrone"
OH-Cs -2H 0 /L
~
C
OH ONHz
protetrone
1971
GENES FOR C H L O R T E T R A C Y C L I N E
which in the cultivation medium results from a block in the oxidation of the A ring to a quinone. In other words, when considering the a m o u n t of a given metabolite of the whole branched family of secondary . . . . . . . . . . . . . . . . . . . . . . . . , ~. . . . . . . .~. . . . . . . . . . . . . . .
1
" <,
|
,
4
,s_. . . . . . . . . . . . . . . :c~_-__6_. . . . . . . . . .
1/\ i
chlortetraeycline, is the last member of the metabolic sequence (in the production of which all the 11 enzyme systems take part), a mutagenic t r e a t m e n t of this area cannot bring a b o u t its higher production. This consideration requires an operon ctc-cluster of structural genes controlled b y a single operator gene. Our production strains of Streptomyces aureofaciens produced increased amounts not only of chlortetraeycline but also of other pigments. This means t h a t the formation of the nonaketide proceeded at such a rate that it could not be transformed all to chlortetracycline b u t overflowed into lateral metabolic paths so t h a t compounds usually described as minor were also produced in increased amounts. In conclusion of this consideration of the genetic regulation of chlortetracycline synthesis one should, at least in general terms, observe the fact t h a t after the production phase has terminated secondary metabolites (and hence also ehlortetracycline) are degraded or transformed enzymically to other metabolites. This enzymic activity is probably also under a genetic control so that the overall picture of the genes t h a t m a y affect quantitatively the result of the biosynthesis of a given metabolite must be supplemented with these mechanisms. ]n our work where the ability of the ribosomes (isolated from low-production and production strains of Streptomyces aureofaciens) to bind chlortetracycline was investigated, we observed (Mikulik, unpublished results) t h a t the ribosomes of low-production strains are more sensitive to chlortetracycline and readily from nondissociable aggregates. A considerable difference was displayed b y the pattern of structural proteins from the ribosomes of production and nonproduction mutants. The sensitivity of the microorganisms toward their own metabolites apparently represents another imp o r t a n t regulatory mechanism. I t is to be regretted that so far it has not been extensively studied. One might easily
/ -- ~ ...................... i___~ ! V\
LB__3.............. ~- .
.
.
.
.
.
.
.
.
.
.
.
.
i
. . .Lcy_-_12____ ............... ! .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
I
A
~
I
4
9
Fig. 3. Summary of biosynthetic steps involved in the biosynthesis of tetracycline type compounds.
metabolites based on the nonaketide, one (;an imagine t h a t m u t a n t s derived from Streptorayces aureofaciens strains producing considerable quantities of CTC will also produce an increased amount of secondary metabolites corresponding to the enzymic block. F r o m the point of view of the rate of flow of acetylCoA or malonylCoA into this metabolic branch no quantitative change has taken place. I f the metabolite investigated here,
;
239
240
Z. V A N ~ K E T AL.
VoL 16
envisage other possibilities of regulation but the strength of the ice on which we seem to be moving, i.e. the strength of our arguments, would be weaker still. The views t h a t we have developed in this communication may certainly be extended to secondary metabolJtes other than oligoketides. This would represent
push ahead our knowledge in a field which, in spite of its extraordinary importance in practical application, remains little elaborated in its theoretical aspects.
a very useful confrontation which could
No. 845[RB.
This work is supported by the International Atomic Energy Agency under research contract
References
B6hal, V., ProchAzkov~, V., Van6k, Z.: Re@-ulation of biosynthe,~is of secondary metaboHt~s. I[. SyntheM,s of fatty acids and chlortetracycline in S. aureofaciens. Fol. microbial. 14 : 112, 1969a. B6hal, V., Cudlin, J., Van~k, Z.: Regulation of biosynthesis of secondary metabolites. H I . Incorporation of 1-14C-AcOH to fatty acids and chlortetracycline in Streptomyce,s auteofaciens. ~'ol. microbial. 14 : 117, 1969b. B~hal, V., VanAk, Z.: Regulation of blosynthssi~ of
secondary metabolites. X I I~ Acetyl CoA carboxylase in Streptomyces aureofaciens. Fol. microbiol. 15 : 354, 1970. Blumauerovgt, M., Mra6ek, M., Vondr&6kov~, J., Podojil, M., How Z., Van6k, Z.: Regulation of
biosynthesis of secondary metabolites. IX. The biosynthetic activity of blocked mutants of Streptomyces aureofaciens. Fol. microbial. 14 : 215, 1969. Boretti, G., DiMarco, A., Julita, P., Ragg, F., Bardi, N.: Presenza de4jli enzimi della via eso8omonofosfam ossidativa n6lla S~reptomyces aureafaciens. Giorn. Microbial. 1 : 4 0 6 , 1956. Cudlin, J., Pfi~a, M., Yanfik, Z., RickarcL% R. W.: Biogenesis of streptimidone. Fol. microbial. 14 : : 406, 1969. Hansen, R. G., Henning, W.: Regulation of pyruvate
dehydrogenase activity in Escherichia colt K lg. Biochim. biophys. A c t s 122 : 355, 1966. How Z.: Relationship between the carbohydrate
metabolism of Streptomyces aureofaciens and the biosynthesis of chlartetracycline. III. The effect of benzyl thiocyanate on carbohydrate metabolism of Streptomyees aureafaciens. Fol. microbiol. 9 : 96, 1964. How Z., Tinti~rov~, M., Jechov/*, V., Blumauerov~, M., Such~r, J., Van6k, Z.: RegulatioT,
of biosynthesis of secondary metabolites. I. Biosynthesis of chlartetracycline and tricarboxylic acid cycle activity. Biotechnol. Bioeng. 11 : 539, 1969. Janglov~, Z., Such:~, J., Van6k, Z.: Regulation of biosynthesis of secondary metabolites. VII. Intracelhdar adenosine-5'-triphosphate concel~tration in Streptomyces aureofaciens. Fol. microbial. 14 : 208, 1969. Jechovgt, V., How
Z., Van{tk, Z.: Regulation of biosynthesis of secondary metabolites. V. Malate
dehydrogenase (decarboxylating) in Streptomyces aureofaeiens. Fol. microbial. 14 : 128, 1969. McCormick, J . R . D . : Biosynthesis of the te~ra. cyelines, p. 73 in Van~k Z., Ho~f~lek Z. (Eds.): Biogenesis of Antibiotic Substances. Academic Press, New York -- London, 1965. McCormick, $. R. D.: Point-blocked mutants and the biogene~ of tetracyclines, p. 163 in G. Sermonti, M. Ala6evid (Eds.): Genetics and Breeding of Streptomyces. Yug. Acad. Sci. and Arts, Za4greb 1969. TintArovA, M., I-Io~tfAlek, Z., Van~k, Z.: Regulation
of biosynthesis of secondary metabolites. VI. Characteristic.~ of isoenzymes of malate dehydrogenase in Streptomyces aureofaciens. Fol. microbiol. 14 : 135, 1969. Van6k, Z., Cudlin, J., Vondr~6ek, M.: Cyeloheximide and other glutarimide antibiotics, p. 222 in Gottlieb D., Shaw P. D. (Eds.): Antibiotics, Vol. II. Biosynthesis. Springer-Verlag, Heidelberg, 1967. Van6k, Z., Cudlfn, J., Mikullk, K,: Biogenesis and
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W. M. STARK E T A L .
B I O S Y N T H E S I S OF NEBI:tAMYC~N
o
o
.................i~84~
o~
-~.~ %~
%~
/
r..)
W . M. S T A R K E T A L .
BIOSYNTHESIS
P l a t e 5. I n t e r c o n n e c t i n g myeeliaI bridge: b e t w e e n clones of s t r a i n 12 g r o w i n g on a g a r m e d i u m 2.
P l a t e 7. Microscopic view of two clones of s t r a i n 13 g r o w i n g on a g a r m e d i u m 1 w i t h h e t e r o k a r y o n - t y p e g r o w t h b e t w e e n them.
OF NEBRAMYCIN
W . M. S T A R K E T A L .
B I O S Y N T H E S I S OF N E B R A M Y C I N
Plate 6. S~ra!n 13 growing on a g a r m e d i u m 1 after first t r a n s f e r to slants.
W . M. S T A R K E T A.L.
BIOSYNTHESIS
OF N E B I ~ A M Y C ] I ~
P l a t e 8. Multiple clones of s t r a i n 22 growing on a g a r m e d i u m 1,
P l a t e 9. Clones of s t r a i n 23 g r o w i n g on a g a r m e d i u m 2 e x h i b i t i n t e r c o n n e c t i n g m y e e l i a b u t no h e t e r o k a r y o t i c type growth.
W . M. STARK E T A L .
BIOSYNTHESIS OF NEBRAMYCIN
Plate 10. Clones of strain 23 growing on agar medium 1 demonstrate both heterokaryotic-type growth and interconnecting mycelia.
W . M. S T A R K
ET AL.
BIOSYNTHESIS
Plate II
OF NEBRAMYCIN
Plate 12
: P l a t e 11. T h i n l a y e r c h r o m a t o g r a p h o f n e b r a m y c i n f a c t o r s p r o d u c e d b y S. tenebrarius a t d a i l y i n t e r v a l s i n f e r m e n t a t i o n m e d i u m w i t h c r u d e s u b s t r a t e s . L a n e 1 - - f a c t o r 2; l a n e 2 - - f a c t o r 4; l a n e 3 - - f a c t o r s 5 a n d 5'; lane 4 -- 1-day sample; lane 5 -- 2-day sample; lane 6 -- 3-day sample; lane 7 -- 4-day sample. : P l a t e 12, C h a r a c t e r i s t i c t h i n l a y e r c h r o m a t o g r a p h o f n e b r a m y c i n f a c t o r s p r o d u c e d i n f e r m e n t a t i o n m e d i u m w i t h c r u d e s u b s t r a t e s b y s t r a i n s o f S, tenebrarius, L a n e 1 - - p a r e n t ; l a n e 2 - - s t r a i n 12; l a n e 3 - - s t r a i n 1 3 ; l a n e 4 - - f a c t o r 2; l a n e 5 - - f a c t o r 4; l a n e 6 - - f a c t o r s 5 a n d 5 ' ; l a n e 7 - - s t r a i n 22; l a n e 8 - - s t r a i n 23.
!i
z
Plate 13
3
4
5
6
:7
o Plate 14
P l a t e 13. C h r o m a t o g r a p h y bioautograph of nebramycin f a c t o r s p r o d u c e d b y S. tenebrarius s t r a i n s i n a chemically-defined medium. The solvent system was methylethylketone : t-butylalcohol :methanol : : 6.5 ~ - N I - I 4 O t t (16 : 3 : 1 : 6). L a n e 1 - - p a r e n t ; l a n e 2 - - s t r a i n 12; l a n e 3 - - s t r a i n 13; l a n e 4 - - f a c t o r s 2 a n d 5 ' ; l a n e 5 - - f a c t o r 4; l a n e 6 - - s t r a i n 16; l a n e 7 - - s t r a i n 22; l a n e 8 - - s t r a i n 23. P l a t e 14. C h r o m a t o g r a p h y b i o a u t o g r a p h o f n e b r a m y e i n f a c t o r s p r o d u c e d b y s t r a i n 23 i n b a s a l , c h e m i c a l l y -defined medium with variation in added substrates. The solvent system was methylethylketone : t-butyla l c o h o l : m e t h a n o l : 6 . 5 ~-NI-I4OI-I (16 : 3 : 1 : 6). L a n e 1 - - b a s a l m e d i u m ; l a n e 2 - - p l u s x y l o s e ; l a n e 3 - p l u s g l u c o s e ; l a n e 4 - - f a c t o r s 2 a n d 5 ' l a n e 5 - - f a c t o r 4; l a n e 6 - - p l u s g a l a e t o s e ; l a n e 7 - - p l u s f r u o t o s e ; lane 8 -- plus mannose.
T. I C H I K A W A E T A L .
I M P R O V E M E N T OF KASUGAMYCIN P R O D U C T I O N
Plate 1. Field test of k a s u g a m y e i n ,
Plate 2. The agar pi~ees placed in a P e t r i dish
1~. V. V I G F U S S O N E T A L .
STERILITY
M U T A N T S OF N E U R O S P O R A
CRAS,SA
2
P l a t e 1. P h o t o g r a p h s h o w i n g t h e effect of t h e e x t r a c t of cross E m A x E m a on t h e cross E m ,4 X E m a (1 anc~ 2)~ w h e n c o m p a r e d w i t h t h e u n t r e a t e d c o n t r o l s (3 a n d 4).
N . V. V / G F U S S O N E T A L .
S T E R I L I T Y MUTANTS OF N E U R O S P O R A
CRASSA
Plate 2. Photogcaphs s h o w i n g the effect of e x t r a c t s of single strains E m A (1) or E m a (2) on the cross 5366-A (sterile) • E m a w h e n c o m p a r e d with u n t r e a t e d control (3).
:N. V. V I G F U S S O N E T A L .
S T E I ~ I L I T Y M U T A N T S OF N E U R O S P O R A CRA,~,..qA=
Plate 3" P h o t o g r a p h s h o w i n g the effect of t h e e x t r a c t of the cross E m A • E r n a on the cross 9312-A (1 a n d 2) w h e n c o m p a r e d w i t h the u n t r e a t e d control (3 and 4).
