Biosynthesis of Antibiotics

238 Pages·1966·3.5 MB·English

by  J. Snell (Eds.)

CONTRIBUTORS TO THIS VOLUME
J. D. BU'LOCK
MALCOLM CHICK
JOHN W. CORCORAN
ARNOLD L. DEMAIN
JOSEPH MENDICINO
D. PERLMAN
J. M. PICKEN
J. F. SNELL
ROY H. TURLEY
BIOSYNTHESIS
OF ΑΝΉΒΙΟΉΟβ
EDITED BY
J. F. SNELL
DEPARTMENT OF BIOCHEMISTRY
OHIO STATE UNIVERSITY
COLUMBUS, OHIO
Volume I
ACADEMIC PRESS New York · London 1966
COPYRIGHT © 1966, BY ACADEMIC PRESS INC.
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.
ACADEMIC PRESS INC.
Ill Fifth Avenue, New York, New York 10003
United Kingdom Edition published by
ACADEMIC PRESS INC. (LONDON) LTD.
Berkeley Square House, London W.l
LIBRARY OF CONGRESS CATALOG CARD NUMBER: 66—29994
PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions
begin.
J. D. Bu'Lock (141), Department of Chemistry, University of Man­
chester, Manchester, England
Malcolm Chick (159), Department of Biochemistry, School of
Medicine, Western Reserve University, Cleveland, Ohio
John W. Corcoran (159), Department of Biochemistry, School of
Medicine, Western Reserve University, Cleveland, Ohio
Arnold L. Demain (29), Merck Sharp and Dohme Research Labora­
tories, Division of Merck and Company, Inc., Rahway, New
Jersey
Joseph Mendicino (121), Department of Biochemistry, Ohio State
University, Columbus, Ohio
D. Perlman (1), Squibb Institute for Medical Research, New Bruns­
wick, New Jersey
J. M. Picken (121), Department of Biochemistry, Ohio State Uni­
versity, Columbus, Ohio*
J. F. Snell (95), Department of Biochemistry, Ohio State Univer­
sity, Columbus, Ohio.
Roy H. Turley (95), Department of Chemistry, Otterbein College,
Westerville, Ohio
* Present address: Department of Biochemistry, Creighton University,
Omaha, Nebraska.
ν
Preface
This volume is devoted to the biosynthesis of antibiotics. Reviews
cover the groups of antibiotics that have been studied for sufficient
time to gain some tentatively satisfactory picture of a biosynthetic
route.
The arrangement of the chapters in this volume (following an
introductory survey chapter on radioactive antibotics) is roughly
chronological with respect to the advent of the antibiotics on the
research scene. It also happens to be roughly at the moment of
writing, the order of completeness of our present knowledge of
their biosynthesis. No better criterion of arrangement was apparent
inasmuch as the antibotics are inherently a heterogeneous group of
compounds held together tenuously only by certain apparent gross
similarities in one aspect of their biological activity.
The classification of compounds according to therapeutic effect
or biological activity has been common in medical and pharmaco­
logical circles since antiquity. Antibiosis is the most striking and
readily identifiable biological activity discovered for these com­
pounds at the present moment. All of these compounds have other
biological activities (sometimes termed "side effects"!). Since there
is woeful lack of information regarding the factors behind the
genesis of the ability of a microorganism to produce antibiotics
(whether induced as a "deliberate" response to predators or simply
some fortuitous genetic or environmental condition) one must not
speculate too far regarding the teleology behind these biosynthetic
processes. However, the grouping together of information on the
antibiotics will hopefully lead alert students to make their own
survey of the comparative data presented here; if there are to be
important deductions in the comparative biochemistry of the biosyn­
thetic processes, we hope that they will be stimulated and ac­
celerated by the juxtaposition of these reviews.
This volume will be useful to academic and industrial research
workers, graduate students, and faculties of biological sciences.
vii
viii PREFACE
My sincere appreciation is hereby expressed to the contributors
to this volume. Each has been more than patient in enduring the
editorial and production phases of its birth. I must also acknow­
ledge with gratitude many persons whose interest and cooperation
have been of great value to me, several of whom have also served
as typists, proofreaders, or bibliographic assistants: C. Andrews,
G. Becher, D. Buchanan, M. Graner, C. Inglesby, J. Last, W. Rich­
ards, D. Sypowicz, P. Thomas. I should especially like to thank the
staff of Academic Press for sympathetic cooperation and patience.
J. F. SNELL
December, 1966
1
Microbial Processes for the Preparation
of Radioactive Antibiotics
D. PERLMAN
I. Introduction 1
II. Microbial Methods Used in the Preparation of Radioactive
Antibiotics 2
A. Methods of Addition of Precursor to Microbial Systems 2
B. Recovery of Labeled Compounds 6
C. Determination of the Purity of Labeled Compounds . . 6
D. Stability of Radioactive Compounds 7
III. Some Radioactive Antibiotics Prepared by Microbial
Processes 9
A. Penicillin and Cephalosporins 9
B. Streptomycin, Neomycin, and Paromomycin 20
C. Tetracyclines 22
D. Polyene Antifungal Agents 22
E. Polypeptide Antibiotics 23
F. Other Antibiotics 23
IV. Summary 24
References 24
I. INTRODUCTION
Organic compounds labeled with radioactive isotopes have found
widespread use in research in biochemistry, pharmacology, toxicol­
ogy, and medicine. In many laboratories they long ago lost their
novelty and have become conventional research tools. However,
their preparation has become a new branch of practical organic
chemistry, and in some instances the organic chemist and the bio­
chemist have made use of microbial systems to prepare the desired
labeled compounds.
This use of microbial methods is often necessary in the prepara­
tion of labeled antibiotics since in most instances an efficient chem­
ical synthesis of the antibiotics has not been reported. The purpose
1
2 D. PERLMAN
of this discussion is to present in a relatively brief form some of the
available literature concerned with the microbial preparation of
radioactive antibiotics. Since other chapters of this book will be
concerned with the biosynthetic mechanisms involved, relatively
little attention will be given to discussion of the possible explana­
tions of mechanisms involved in incorporating the various precur­
sors into the antibiotics. Instead, emphasis will be placed on the
microbiological aspects of these processes, and technical problems
likely to be encountered will be mentioned. However, no detailed
procedures will be presented, and the reader is advised to consult
the original literature for this information. Only radioactive isotopes
will be included, and studies with the stable nonradioactive isotopes
15N, 13C, 1 80, and deuterium, often used to prepare labeled com­
pounds, will not be stressed.
II. MICROBIAL METHODS USED IN THE PREPARATION
OF RADIOACTIVE ANTIBIOTICS
Although a wide variety of experimental equipment and pro­
cedures have been described as being useful in preparing labeled
antibiotics by microbial processes, there seems to have been little
recognition of the advantages, requirements, and limitations of
these processes and techniques. We have reviewed a number of
the papers that discuss the preparation of various labeled com­
pounds and have determined the similarities and differences in
the processes described. If allowances are made for problems
peculiar to preparation of certain types of chemically unstable com­
pounds, it can be seen that only a few methods have found wide
acceptance. Since the advantages and disadvantages of these
methods are of concern to the laboratory investigator preparing
labeled compounds by microbial processes, these will be stressed
in the following discussion.
A. METHODS OF ADDITION OF PRECURSOR TO MICROBIAL SYSTEMS
Two methods have been used in the preparation of most labeled
antibiotics by microbial processes:
1. Growth of the microorganism in a medium containing "nor­
mal" medium constituents (and the labeled material) under condi­
tions where appreciable amounts of the labeled substrate are ab­
sorbed by the culture and incorporated into the desired antibiotic.
1. PREPARATION OF RADIOACTIVE ANTIBIOTICS 3
2. Growth of the microorganism in a "normal" medium, collec­
tion of the microbial cells, and resuspension of the cells in a solu­
tion containing the labeled precursor. Incubation is continued until
a "high" yield of the desired metabolite is obtained.
In some instances cell-free enzymes have been used in convert­
ing labeled compounds to desired products. While cell-free en­
zymes may have application on a preparative scale, there are very
few reports of successful scale-up of these processes from a micro-
scale to a preparative level.
The advantages of using growing cultures in preparing labeled
compounds are derived in part from the ease of operation. The
selected microorganism is grown in conventional media under con­
ditions where useful concentrations of the desired metabolite are
usually obtained. The radioactive precursor is added to the grow­
ing culture, and incubation is continued until maximum incorpora­
tion is achieved or some other end point is reached. The fermenta­
tion is harvested, and the desired metabolite (labeled) is recovered
by conventional procedures. All these operations can be carried
out and standardized without the use of the labeled materials, and
the addition of the labeled precursor does not make an apprecia­
ble difference in either the fermentation operations or the type of
equipment used.
When 14C-labeled precursors are used and appreciable amounts
14
of metabolic CC>2 may be produced, precautions against contam­
ination of the incubation chamber atmosphere are usually taken.
Hunter et al. (1954), Craig (1960), Snell (1960), and Rinehart
(1964) are a few of those who have described modified equipment
of shaken flask size designed to take care of these problems. A
diagram of the equipment used by Numerof et al. (1954) is shown
in Fig. 1. The fermentation flasks are arranged in a "closed" system
so that air is swept through the flask and the metabolic C0 is
2
trapped in alkali. The flow rate of the air through the flask must
be carefully controlled. When it is not, the rate of metabolism of
the organism is often affected. An alternative system (patterned
after Craig, 1960) is shown in Fig. 2. Here, only the air outside
1 4
the flask is swept through alkali (to trap the evolved C0 ), and
2
the aeration of the shaken fermentation is not noticeably affected.
There may be a need to be concerned about the efficiency of
conversion of the precursor to the desired metabolite, especially
if a costly precursor is used. In these situations addition of the pre-
4 D. PERLMAN
Flow meter
FIG. 1. "Closed" system apparatus for 1C4-supplemented fermentation. From
Numerof et al (1954).
cursor may be delayed until the growth phase of the fermentation
has been finished, or the precursor may be added repeatedly to
the growing culture during the incubation period. Under these lat­
ter circumstances the apparatus shown in Fig. 1 is more difficult
to handle than that shown in Fig. 2, since the control of air-flows
in the former is often a very critical matter.
ROTARY
SHAKER
FIG. 2. "Open" system apparatus for 1C4-supplemented fermentation. From
Perlman and Semar (1965) after Craig (1960).