Table Of ContentBIOGENESIS OF ANTIBIOTIC
SUBSTANCES
Edited by: ZDENKO VANEK
Institute of Microbiology
Czechoslovak Academy of Sciences
Prague
and ZDENEK HOStÄLEK
Research Institute of Antibiotics
Roztoky near Prague
0
Publishing House Academic Press
of the Czechoslovak Academy New York and
of Sciences, Prague London
1965
Academic Press Inc., Ill Fifth Avenue, New York, New York 10003
Library of Congress Catalogue Card Number 64-66186
(Q) Nakladatelstvi Ceskoslovenske akademie ved 1965
All rights reserved
No part of this book may be reproduced in any form, by photostat, microfilm,
or any other means without written permission from the publishers
Printed in Czechoslovakia
Preface
The period of stormy development of antibiotic substances seems to be over.
During this period the principal effort was focused on finding new antibiotics and
even with small financial expenditure some success could be expected. Nowadays,
however, with more than 1000 antibiotic substances already discovered and only some
15 of them in broad clinical use, it has become apparent that for the discovery of
a major, so far unknown substance, large teams of highly specialized and experienced
laboratory workers are necessary, who must screen increasingly huge numbers of
microorganisms among which producers of a new substance might be concealed. It
may take years before such a search brings results, and sometimes the results are
never brought to practical use and all effort appears wasted.
It is therefore logical that attempts are being made to utilize the tremendous
amount of work hidden in such antibiotic substances which — because of their
toxicity or other untoward properties — remain unused, and also that ways are
sought to obtain modified substances with a more favourable chemotherapeutic index.
Results obtained in the group of new penicillins and tetracyclines show that this ap-
proach is quite realistic, but a prerequisite for success is a thorough knowledge of the
biochemical mechanisms by which these substances are formed.
Knowledge of basic units, of enzymatic systems participating in the biosynthesis
and of mechanisms which govern the biosynthesis and determine the amounts of
substances produced under given conditions — all this is of imminent practical im-
portance, and not only for directing the fermentation process: in fact it becomes
impossible to imagine any progress in the genetics of producing microorganisms
without profound understanding of biosynthetic pathways leading to the production
of the required secondary metabolite.
The study of mechanisms regulating the biosynthesis of primary (essential) meta-
bolites has in recent years met with such success that it is imperative to follow this
line also with secondary metabolites, such as antibiotic substances. We must learn
more about the relationship between secondary metabolite production (usually
culminating after the logarithmic phase of microbial growth) and synthesis of cell
matter or cell wall. We must learn why some metaboUc cycles cease working and new
ones are formed. Does the biosynthesis of some secondary metabolites have an in-
ductive character? Is their synthesis governed by mechanisms analogical to repression
and feedback inhibition? Those are the questions to which the answers are urgently
needed.
8
I have been happy to see how earnestly all participants in the panel discussion
"Basic research and practical aspects of antibiotic production" held during the Anti-
biotic Congress in Prague* tackled these problems and I wish to thank them all for
the high scientific standard of their contributions which made the basis for this book.
Milos Herold
Roztoky near Prague
* "Antibiotica Congressus cum Participatione Internationali", held under the patronage of the
Government of the Czechoslovak Socialist Republic in Prague, June 15—19, 1964.
Contributors
E. P. ABRAHAM, Sir William Dunn School of Pathology, University of Oxford, England.
S. I. ALIKHANIYAN, Kurchatov Institute of Atomic Energy, Moscow, USSR.
R. BENTLEY, Department of Biochemistry and Nutrition, Graduate School of Public Health,
University of Pittsburgh, USA.
J. D. BU'LOCK, University of Manchester, England.
W. D. CELMER, Medical Research Laboratories, Chas. Pfizer & Co., Inc., Groton, USA.
J. W. CORCORAN, Department of Biochemistry, Western Reserve University, Cleveland, USA.
R. DONOVICK, Squibb Institute for Medical Research, New Brunswick, USA.
S. GATENBECK, Institute of Biochemistry, University of Lund, Sweden.
D. GOTTLIEB, University of Illinois, Urbana, USA.
C. H. HASSALL, Chemistry Department, University College of Swansea, Great Britain.
J. J. H. HASTINGS, Lilly Industries Ltd., London, England.
M. HEROLD, Research Institute of Antibiotics, Roztoky near Prague, Czechoslovakia.
E. KATZ, Georgetown University, School of Medicine and Dentistry, Washington, USA.
H. A. LECHEVALIER, Institute of Microbiology, Rutgers University, New Brunswick, USA.
I. MÄLEK, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czecho-
slovakia.
J. R. D. MCCORMICK, Lederle Laboratories, Pearl River, USA.
W. OBERZILL, Institute for Biochemical Technology and Microbiology, University of Technology,
Vienna, Austria.
J. A. ROPER, Department of Genetics, The University, Sheffield, England.
G. SERMONTI, Istituto Superiore di Sanitä, Rome, Italy.
C. SPALLA, Laboratori di Ricerche Microbiologiche e Chemioterapiche, Farmitalia Milano,
Italy.
J. F. STAUFFER, The University of Wisconsin, Madison, USA.
S. W. TANENBAUM, College of Physicians and Surgeons, Columbia University, New York, USA.
R. THOMAS, Department of Biochemistry, Imperial College of Science and Technology, University
of London, England.
H. THRUM, Institute of Microbiology and Exp. Therapy, Jena, German Democratic Republic.
CHAPTER 1
About Mechanisms Regulating the Biosynthesis
of Secondary Metabolites
I. MÄLEK
Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia
The conception of the metabolic regulation of the biosynthetic pathways of primary
metabolism is today generally understood as the regulation of enzyme synthesis,
especially repression and induction and the regulation of the enzymatic activity of the
final product (feedback-allosteric inhibition). In many processes leading to the syn-
thesis of a final product, the activity of the enzyme catalyzing the initial reaction is
inhibited. The final product is then usually defined as the last small molecule in a
series of consecutive reactions (amino acids, nucleotides) before incorporation into
the macromolecule.
Most work on the mechanism of repression and allosteric inhibition has until now
been limited to studying the regulation and control of the biosynthesis of the so-called
essential metabolites, such as amino acids and nucleotides, which are essential for the
growth of microorganisms. The results obtained in these studies are most stimulating
and necessitate similar considerations in the study of the regulation of the biosyn-
thesis of secondary metabolites (antibiotics, pigments, etc.), whose molecules are
much more complicated. If it were shown that the biosynthesis of secondary meta-
bolites is controlled by mechanisms similar to repression and feedback, it could be
assumed that some techniques which permit the attaining of the state of derepression
and insensitivity to allosteric inhibition, which were used successfully in studying the
biosynthesis of essential metabolites, could also be used for increasing the production
of the required secondary metaboHte. Here, however, we run against the difficulty
that the exact definition of secondary metabolites formed by microbial cells is very
difficult. We consider them to be substances formed in static bacterial cultures up to
the end of the exponential phase, and often in large amounts {e.g. the dry weight of
Penicillium islandicum contains up to 30% of pigments). Their formation is not dir-
ectly connected with the basic biosynthetic process of biomass formation. Secondary
metabolites appear to be quite without significance for the actual course of the pro-
liferative phase of the microorganisms. Many of these substances have a very com-
12 I. MÄLEK
plicated structure which can be understood as the result of several quite different bio-
synthetic pathways.
The great progress in knowledge of the mechanisms regulating the biosynthesis of
primary metabolites has led quite logically to detailed study of the basic metabolic
pathways of primary metabolism, the enzymes of the different reactions and the
kinetics of these reactions. On the other hand, the metabolic pathways leading to the
biosynthesis of most secondary metabolites are known only approximately and in
broad outline and the enzymatic systems taking part in their synthesis have not yet
been studied in detail.
A further important difference results from the economic evaluation of the import-
ance of allosteric inhibition and inhibition of repression for bacterial cells. The intra-
cellular concentration of primary metabolites is very low since their outflow is neces-
sary for the proliferation of bacteria. Therefore cells react to an increase in their con-
centration with surprising sensitivity; the synthesis or the activity of the enzyme,
which led to excessive production of the primary metabolite, immediately stops. As
opposed to this, secondary metabolites are often formed in large quantities and it is
difficult to form a rational conception of the teleological significance of this. We do
not know why secondary metabolites are formed in concentrations far in excess of
their solubility in the cultivation medium, we do not know the principles of their
physiological or ecological significance. Any kind of generalization must necessarily
be unsuccessful.
Despite the above differences between primary and secondary metabolites we are
of the opinion that, in work on the biosynthesis of antibiotic substances we could
find many examples explainable in line with our present knowledge by mechanisms
similar to allosteric inhibition and repression. For example, LEGATOR and GOTTLIEB1,
when investigating the dynamics of the biosynthesis of chloramphenicol by the actino-
mycete, S. venezuelae, found that in this strain under the given conditions, there was
a limiting concentration of antibiotic substance which was normally produced by the
organism. When different amounts of antibiotic (smaller, the same and larger) were
added to the fermentation medium at the beginning of fermentation, the final con-
centration was always the same. With initial concentrations higher than the limiting
one, this meant that the antibiotic was actively broken down into a nonactive sub-
stance. Since this regulating mechanism was apparent during the whole course of
fermentation it could be concluded that it is evidence of the regulation of the bio-
synthesis of secondary metabolites by allosteric inhibition. As already mentioned,
one of the characteristics of the biosynthesis of secondary metabolites is their exces-
sive production up to the end of the logarithmic phase of the growth of microorgan-
isms. It is certain that profound changes take place in the organism at this stage of
development. There is a decrease in the importance of mechanisms up to then con-
trolling the main stream of basic structural units for building up cell mass and cell
walls of the rapidly proliferating cells (see Scheme 1).
REGULATION MECHANISMS 13
11 metabolism η
pyrimidines r-
cell wall ^^_ -^ purines ~i j
I . <'
pyruvate
~l I 1
1 | |
proteins aminoacids
*
* t i ♦
1! ,
secondary
acetate
J metabolites
lipids fatty acids A A
1' 1 1
| |
Krebs' cycle J 1
■· 1
SCHEME 1
Subsidiary metabolic pathways then become the main metabolic stream. DAVIS2 con-
siders that antibiotic substances are the coproducts of disorganized control mechan-
isms rather than the co-products of biosynthetic pathways created so that the
microorganism can better hold its own in life. However, the use of the term dis-
organization is probably not justified. It is more probable that in cells which cease to
grow and begin to divide, there is a dissolution of the organization which controlled
the building up of cell proteins and cell walls and that the stream of building sub-
stances (amino acids, sugars, etc.) whose biosynthesis is controlled by other centres
is now directed to the building up of secondary metabolites. It can also be assumed
that some links in the dissolving organization could be utilized in systems taking over
the leading role. It is similar to what occurs, for example, on cultivating Escherichia
coli under limiting conditions of nitrogen deficiency. The stream of metabolites is
then directed towards glycogen formation and by this is transposed from the state
necessary for active metabolism to the state of slower metabolism.
The centre of interest of much work at the present time is the relation between the
biosynthesis of protein to the biosynthesis of secondary metabolites of a polypeptide
character. It is known that at a certain stage of development bacterial cells produce
considerable amounts of polypeptide substances (with very different molecular
weights). The mechanism of biosynthesis of these substances, however, is not known.
In general, three modes of biosynthesis of polypeptides can be envisaged. They could
be synthetized like proteins along a ribosome matrix, they could arise from the de-
gradation of larger protein molecules, or finally, they could be synthetized without
a RNA-matrix by the progressive condensation of amino acids.
It is impossible to exclude the existence of a quite specific transfer in which the
function of RNA is limited to the synthesis of polypeptides. Simple mechanisms,
14 I. MÄLEK
however, could also be considered, specific enzymes, perhaps also multienzyme com-
plexes (as in the biosynthesis of fatty acids), which could ensure structural specificity
for both the still not completed peptide and for further amino acids.
The fact that some bacteria form polypeptide antibiotics which contain the same
D-amino acids as their cell walls suggests that these antibiotics could be side-products
in the synthesis of cell walls. This very urgent problem of the relation of secondary
metabolites to the biosynthesis of polypeptides will be dealt with much more thorough-
ly in our discussions by E. P. ABRAHAM.
We consider that some secondary metabolites, whose molecule may be partly or
completely derived from the metabohc pathways transforming sugars, have a primary
relationship to the biosynthesis of cell walls and polysaccharides of which the walls
are composed. It was observed in S. griseus that streptomycin is bound to the myce-
lium (in some cases more than half its total amount). This streptomycin can be
liberated if the cell is treated with acids, alkalies or ionizable salts. The same was de-
monstrated for streptothricin, neomycin, etc. MAJER and coworkers3 reported this year
that after the completion of the growth of S. griseus, L-rhamnose, which is an essential
component of the cell wall, is used for the biosynthesis of streptose which is a part
of the streptomycin molecule. REHÄCEK4 assumes that there is a relationship between
the sugar components of the cell membrane of S. erythreus and the biosynthesis of
erythromycin.
Acetic acid can be considered to be a key metabolite of a number of cell components
and a large group of secondary metabolites. It was recently shown that the production
of fatty acids is regulated by acylCoA of the long chains of the fatty acids which
inhibit the carboxylase of acetylCoA. It seems that the carboxylation of acetylCoA
to malonylCoA is the critical site which controls the synthesis of long chains of fatty
acids. The carboxylation of acetylCoA can be considered as a point from which
further metabohc pathways branch off, e.g. the biosynthesis of aromatic substances
formed by the condensation of "acetate units". By analogy with the regulation of the
branched biosynthetic pathways of primary metabolism, it can also be assumed in
this case that there are several carboxylases of acetylCoA which differ in sensitivity
to the final products of the separate metabohc pathways.
In producing some antibiotics which can be derived from the condensation of
"acetate units" it proved of value to add fats to the fermentation medium since they in-
creased the production of the required antibiotic substance. It is worth considering
whether in this case fats were used only as a source of carbon or whether there was
a suppression of the synthesis of fatty acids. Acetate units would then be reorientated
to the biosynthesis of other secondary metabolites, in this caSe, antibiotic substances.
A further case of a change in the equilibrium of metabohc pathways using "acetate
units" as building material was described by DOLEZILOVÄ and coworkers5 in S. noursei
in the biosynthesis of substances of the cycloheximide series and fungicidine (see
Scheme 2):
REGULATION MECHANISMS 15
melanins
t ■
tyrosine
t
phenylalanine <+— shikimic acid ·*— hexose —»* · mycosamine —»- fungicidin
1 1 1 A
dioxopiperazines pyruvic ^ propionic
acid acid
1
acetic malonic
•1
acid acia
1 i
tricarboxylic fats
acid cycle 1
1 — >K
1
dehydrocyclo- , cycloheximide streptovitacins
heximide I If
i 1
act if> henol
anhydrocycloheximide
SCHEME 2
The so-called high-production mutants, producing only fungicidine did not produce
cycloheximide and biogenetically similar substances. It can be assumed that in these
mutants the stream of acetate units was mainly directed to the biosynthesis of fungi-
cidine. Since, however, mutants have been described which, while not forming sub-
stances of the cycloheximide series, did not produce increased amounts of fungicidine,
it must be assumed that still a further change (block) occurs in high-production
mutants, e.g. in sensitivity to aliosteric inhibition. If this were the case it would pro-
vide a rational explanation for the numerous cases in which the actual antibiotic sub-
stance was used with success in improving the high-production strains. More details
of these problems which are so interesting for scientific workers, are given in the
paper of MCCORMICK on the biosynthesis of tetracycline antibiotics.
If biochemists speculate on mechanisms leading to the increased production of
certain substances, they usually start from the chemical structure of the given sub-
stance and from experimental data, often very incomplete, on the biosynthetic path-
ways leading to the substance studied. The fact that the microorganism produces
many other substances, some of which are known, but most of which are not, that
the biosynthesis of these substances is interrelated and that their formation and pro-
duction level is genetically regulated, is pushed into the background. It is not only
a question of the mechanical increase in the level of building units (precursors) or the
saving of building material for the substances whose production we wish to increase,
but of the establishing of a new metabolic equilibrium between mutually competing
