003 Advances in Biochemical Engineering 3 (1974)

Prévia do material em texto

Advances 
in Biochemical 
Engineering 3 
Edited by 
T. K. Ghose, A. Fiechter, and 
N. Blakebrough 
With 119 Figures 
Springer -Verlag 
Berlin. Heidelberg. New York 1974 
T. K. GHOSE 
Dept. of Chemical Engineering, Indian Institute of Technology, New Delhi/India 
A . FIECHTER 
Mikrobiologisches Institut der Eidgen. Techn. Hochschule, Zfirich/Switzerland 
N . BLAKEBROUGH 
The University of Birmingham 
Dept. of Chemical Engineering, Birmingham 15/Great Britain 
ISBN 3-540-06546-6 Springer-Verlag Berlin • Heidelberg • New York 
ISBN 0-387-06546-6 Springer-Verlag New York. Heidelberg • Berlin 
This work is subject to copyright. All rights are reserved, whether the whole or part 
of the material is concerned, specifically those of translation, reprinting, re-use of 
illustrations, broadcasting, reproduction by photocopying machine or similar means, 
and storage in data banks. 
Under § 54 of the German Copyright Law where copies are made for other than 
private use, a fee is payable to the publisher, the amount of the fee to be determined 
by agreement with the publisher. 
© by Springer-Verlag Berlin . Heidelberg 1974. Library of Congress Catalog Card 
Number 72-152360. Printed in Germany. The use of registered names, trademarks, etc. 
in this publication does not imply, even in the absence of a specific statement, that 
such names are exempt from the relevant protective laws and regulations and therefore 
free for general use. 
Typesetting and offset printing: Zechnersche Buchdruckerei, Speyer. Bookbinding: 
Bri~hlsche Universit~itsdruckerei GieBen. 
Contents 
Ciba-Geigy/Lepetit 
Seminar on Topics of Fermentation 
Microbiology 
CHAPTER 1 
Genetic Problems of the Biosynthesis 
of Tetracycline Antibiotics 
Z. HOS'I"ALEK, M. BLUMAUEROVA, and Z. VAN~K, 
Praha (CSSR) 
With 22 Figures 
CHAPTER 2 
Some Aspects of Basic Genetic Research on Fungi 
and Their Practical Implications 
K. ESSER, Bochum (Federal Republic of Germany) 
With 5 Figures 
CHAPTER 3 
Microbial Oxidation of Methane and Methanol 
N. KOSARIC and J. E. ZAJIC, London, Ontario 
(Canada) 
With 7 Figures 
CHAPTER 4 
Modelling and Simulation in Biochemical Engineering 
H. W. BLANCH and I. J. DUNN, Zi,irich (Switzerland) 
With 26 Figures 
CHAPTER 5 
Transient and Oscillatory States of 
Continuous Culture 
D. E. F. HARRISON and H. H. TOPIWALA, 
Sittingbourne, Kent (Great Britain) 
With 24 Figures 
13 
69 
89 
127 
167 
CHAPTER 6 
The Significance of Microbial Film in Fermenters 
B. ATKINSON and H. W. FOWLER, 
Swansea (Great Britain) 
With 33 Figures 
221 
CHAPTER 7 
Present State and Perspectives of 
Biochemical Engineering 
I. MALEK, Praha (CSSR) 
With 2 Figures 
279 
Ciba-Geigy/Lepetit 
Seminar on Topics of 
Fermentation Microbiology 
June 19-23, 1972, Zermatt (Switzerland) 
The Microbiological Sections of the two pharmaceutical companies 
Ciba-Geigy Basel and Lepetit Milan held a joint Seminar on various 
microbiological topics in Zermatt, Switzerland, from June 19th to 23rd 
1972. 
With the exception of the invited speakers, the participation was re- 
stricted to members of both companies and a few scientists from 
University Institutes associated with them. Dr. Ch. Stoll, Ciba-Geigy 
Basel, was in charge of the administrative part of the meeting whereas 
Dr. J. N~iesch from the same company was responsible for the scientific 
programme. 
The aim of this Meeting was to transmit to the academically trained 
personnel of both companies some of the progress made in genetics and 
molecular biology. After the biosynthesis of industrially interesting 
antibiotics, tetracyclines, rifamycins and/~-lactam antibiotics was out- 
lined. Moreover the technique of continuous culture was delt with. 
This technique has various interesting applications for research and 
development, although it is relatively unknown in the field of anti- 
biotics. 
It was the intention of the organizers of the meeting to concentrate on 
fundamentals rather than applied aspects of the subject. Application in 
the industrial laboratories and plants was worked out during the dis- 
cussions. Considerable time was therefore reserved to analyze the various 
new aspects appearing in the course of the Seminar. This particular 
organisation proved to be very useful. 
2 Ciba-Geigy/Lepetit 
The scientific programme was organized as follows: 
1. Genetics 
1t. Introduction (G. Magni, Lepetit S.p.A., Milan, Italy) 
12. Genetics of fungi (K. Esser, Ruhr University Bochum, West 
Germany) 
13. Genetics of actinomycetes (D.A. Hopwood, John Innes Institute, 
Norwich, England) 
2. Regulatory aspects of enzymes 
21. Active and passive control of enzyme synthesis (R. Hiitter, ETH, 
Ziirich, Switzerland) 
22. Structure of allosteric proteins and their regulation properties 
(G. N. Cohen, Institute Pasteur, Paris, France) 
3. Continuous cultivation in application .for the study of biosynthesis and 
process development of antibiotics 
31. Continuous fermentation of filamentous organisms (S.J. Pirt, 
Queen Elizabeth College, London, England) 
4. Mechanisms of biosynthesis of antibiotics 
41. Biosynthesis of tetracyclines (Z. Vanek, Institute of Microbiology 
Czech., Acad. Sci., Prag, CSSR) 
42. Biosynthesis of rifamycins (G. Lancini, Lepetit, Milan, Italy) 
43. Biosynthesis of/Mactam antibiotics (J. Ntiesch, Ciba-Geigy, Basel, 
Switzerland) 
Some of the contributions were prepared for publication. Summary and 
reference of these articles are given here whilst two of them are re- 
ported in extenso in this volume (Chapters 1 and 2). 
HOPWOOD, D.A.: Genetics of Actinomycetes. G. Sykes (Ed.). Actino- 
mycetes. Symp. Soc. Appl. Bact., p. 9--31, 1973. 
Summary 
Genetic recombination has so far been discovered in members of four 
genera of actinomycetes: Streptomyces, Nocardia, Micromonospora and 
Thermoactinomyces. In the first three, there is reasonable evidence that 
some kind of "conjugation" process is responsible for gene transfer, 
whereas in Thermoactinomyces, transformation occurs. Transformation 
has not been unequivocally demonstrated in Streptomyces; results with 
Thermoactinomyces reveal one possible cause for this. 
By far the best known genetic system in the Actinomycetales is that of 
Streptomyces coelicolor A3(2), although studies of two other species, S. 
Seminar on Topics of Fermentation Microbiology 3 
rimosus and S. bikiniensis, have reached the stage of linkage analysis. 
Comparison of the linkage maps of these three species suggests that a 
common gene arrangement is (largely) conserved in the genus, even 
when strains have been subjected to enormous abuse by chemical and 
physical mutagens. 
Study of the fertility system of S. coelicolor A3(2) has reached the stage 
at which a donor (NF) and two classes of recipient strains (IF and 
UF) have been recognised. IF is the fertility of the wild-type, and NF 
arose from this by a chromosomal event (which may have been the 
integration of a plasmid); thus IF and NF segregate as "alleles" in a 
cross between these two fertility types. UF strains arise with a rather 
high frequency from IF strains by loss of plasmid SCPI, and can be re- 
infected with the plasmid by contact with an IF strain. The frequency 
of recombinant production in mixed cultures ranges from about 10-5 
in the least fertile combination (UF x UF) to 1 in the most fertile 
(NF x UF). 
Streptomyces genetics has reached the stage where it can aid indus- 
trial strain-improvement programmes in several ways. One has been 
the provision of more efficient procedures for chemical mutagenesis. A 
second concerns the predictive power of a common linkage map, which 
should allow the use of markers of known map location without the 
necessity of mapping the markers in a new strain. An area which is still 
untried, but is on the threshold of feasibility, is the introduction of 
specific characters from a donor strain into a recipient strain on sub- 
stituted plasmids, if not by transduction or transformation. 
Actinomyceteshave much to offer to the study of differentiation in being 
the most complex prokaryotes in which genetic analysis is yet feasible. 
Spore delimitation in S. coelicolor and the three-dimensional geometry 
of the Thermoactinomyces vulgaris endospore are two topics which are 
currently under investigation. 
HOTTER, R.: Passive and Active Control of Enzyme Synthesis. Regula- 
tion der Enzymsynthese bei Bacterien und Pilzen. Fortschr. Botan. 34, 
309--323 (1972). 
Summary 
The levels of gene products, be it RNA or protein, are controlled in 
diverse ways. An intensive effort to elucidate the mechanisms of reyuIa- 
tion has been made with the bacterium Escherichia coli. We wilt con- 
centrate on this organism and focus our attention on some selected 
examples. The result will necessarily be a very schematic and 9eneralized 
picture of the real situation. 
4 Ciba-Geigy/Lepetit 
PIRT, S.J.: Continuous Fermentation of Filamentous Organisms. A 
monograph on continuous cultivation of microorganisms is in prepara- 
tion. 
Summary 
1. Uses of continuous flow culture in fermentation studies 
Limitations of batch culture - - special functions of the chemostat - - 
difficulties in chemostat culture. 
Studies on effects of growth rate and environment on penicillin fermen- 
t a t i o n - the maintained state - - behaviour of microorganisms of growth 
rates. 
2. Production of penicillin by tysine auxotrophs of Peniciltium chrysogemm7 
Possible regulation of penicillin synthesis. Production and characterisa- 
tion of lysine auxotrophs. Effects of lysine and :~-amino-adipic acid on 
penicillin production by parent and auxotrophic mutants. 
LANCINI, G.: Biosynthesis of rifamycins. Lancini et al. Progr. Antimicrol. 
and Anticancer Res. 2, 1166 (1970); Lancini, G., White, R.J.: Proc. 
Biochem., p. 14, July 1973. 
N# ESCH, J.; Biosynthesis of/~-Lactam Antibiotics. 
Niiesch, J., Treichler, H.J., Liersch, M. (1970): Genet. Ind. Microorg. 
Van~k, Z., Hog~filek, Z., Cudlin, J. (Ed.), pp. 309--334. Prague: Academia 
1973. 
Lemke, P.A., Brannon, D.R. (1972): Cephalosporins and Penicillins. 
Flynn, E.F. (FA.), pp. 370--430. New York and London: Academic Press 
1972. 
Summary 
The /J-lactam antibiotics form a group of natural substances charac- 
terized by a bicyclic ring system: a four membered lactam ring fused 
with a five- or six-membered heterocycle. Although these compounds 
have many common features with regard to their structure, their bio- 
synthesis as well as their biological activities, they can be divided into 
two groups on the basis of certain aspects of their biosynthesis. 
The penicillium-type comprises/?-lactam antibiotics with 6-amino-peni- 
ciltanic acid (6-APA) as nucleus and various N-acyl side chains. Gen- 
erally of a nonpolar aliphatic or aromatic carboxylic acid type and 
L-a-amino acid. The synthesis of a specific penicillin is directly de- 
pendent on the N-acyl side chain precursor in the culture medium. In 
the absence of side chain precursor 6-APA may be accumulated. All 
producers in this groups are eucaryotic fungi. 
Seminar on Topics of Fermentation Microbiology 
a) Penicillium-type 
Nucleus 
( unvariable} 
C~H 3 CH 3 
s ' ~ C O O H 
R (N-Acyl side chain) 
{variable) 
( 6-AminopeniciIlanic acid } 
HOOC-CH {NH 2 } ICH213--CO-- 
(L-~--aminoadipic acid ] 
CH3- CH 2-- CH=CH-CH 2-co - 
(flsy-hexenoic acid ) 
CH3(CH 2 }3--CO-- 
loctanoic Ocld ) 
C6H5-CH2-CO- 
( phenylacetic acid) 
C6Hs-O-CH2-CO-- 
{phenoxyacetic acid ) 
Producers 
Penicillium notatum 
PeniciHium chrysogenum 
Other species of penicillia 
Dermatophytes 
Aspergil(us sp. 
Malbranchea pulchella 
b) Cephalosporium-type 
Nucleus R 1 (N-Acyl side chain} 
(variable) {unvariab{e) 
CH 3 CH 3 H- 
S , ,~CO01~ (6-aminopeniciilanic acid) 
\ N / HOOC-CH (NH2) ( CH 2)3- CO- 
F [ , (D-ez'--aminoadipic acid} 
R, HN~ % 
CH2-R 3 
coo. 
S \~ " HOOC-CH (NH~llCH.)~-CO- 
(D-(~-aminoadipic acid ) 
R1HN "O 
R 2 R 3 Producers 
Cephalosporium sp. 
Emericellopsis sp. 
Paeci|omyces persic i nus 
Streptomycetes 
H- -OCOCH 3 
H- -OH 
-OC~ I-OCOCH 3 Cephafosporium acremoniul 
H- -OCONH 2 Streptomycetes 
-OCH 3 -OCONH 2 
6 Ciba-Geigy/Lepetit 
The cephalosporium-type on the other hand is characterized by D-fi- 
aminoadipic acid as the unique N-awl side chain. In contrast to the 
penicillium-type two nuclei are found, namely 6-APA and 7-amino- 
cephalosporanic acid (7-ACA). Whereas in 6-APA the four membered 
lactam ring is fused with a five membered thiazolidine ring, 7-ACA is 
a bicyclic structure composed of the same lactam ring but fused with a 
six membered dihydrothiazine ring. Characteristic for the formation of 
this type offl-lactam antibiotics is its insensitivity to the addition of side 
chain precursors to the culture medium. Eucaryotic as well as proc- 
aryotic microorganisms are known as producers. 
Cysteinyl moiety and sulfur metabolism (after Lemke and Brannon, 1972) 
t 
HS-~~L-~Serine 
• S z 0 5 - 
J 
S05 - 
PAPS 
APS 
, Choline-SOj 
Choline 
SO2- (endo), 
I Sulfate permease 
SO2 (exo) 
L-CYSTEINE 
L-Cystathionine 
IT 
L-Homocysteine 
[ 
[ 
(endo) Dk-Methionine 
l Methionine permeases 
(exo) DL-Methionine 
Pen ic i l l i um C e p h a l o s p o r i u m 
B
io
sy
nt
he
si
s 
of
 t
he
 p
re
su
m
ab
le
 c
o
m
m
o
n
 p
re
cu
rs
or
 a
m
in
o
 a
ci
ds
 o
f 
th
e 
fl
-l
ac
ta
m
 a
nt
ib
io
ti
cs
 L
-2
-a
m
in
oa
di
py
l 
m
o
ie
ty
 
an
d 
L
-l
ys
in
e 
m
et
ab
ol
is
m
 
C
H
3-
-C
O
O
H
 
A
ce
ta
t+
 
~ 
C
H
--
C
O
O
H
 
C
H
--
C
O
O
H
j[
 
H
O
--
C
H
--
C
O
O
H
[ 
O
~-
--
C
--
C
O
O
H
 \ 
H
O
--
C
--
C
O
O
H
 
C
--
C
O
O
H
 
C
H
--
C
O
O
H
 
r 
\ 
C
H
2 
"J{
 ,
 
C
H
2 
, 
' 
C
H
2 
, 
' 
C
H
2 
r 
t 
I 
I 
C
H
2 
C
H
2 
C
H
: 
C
H
2 
I 
I 
I 
I 
C
O
O
H
 
C
O
O
H
 
C
O
O
H
 
C
O
O
H
 
O
=
=
C
--
C
O
O
H
 
~
=
C
--
C
O
O
H
 
I 
I 
C
H
--
C
O
O
H
 
C
H
2 
, 
...
. ' 
C
H
2 
, 
C
H
2 
,-
- 
I 
I 
C
H
2 
C
H
2 
d 
I 
C
O
O
H
 
C
O
O
H
 
2-
K
et
og
lu
ta
ra
te
 
H
om
oc
it
ra
te
 
H
om
oa
co
ni
ti
c 
H
om
oi
so
ci
tr
ic
 
O
xa
lo
gl
ut
ar
ic
 
2-
K
et
oa
di
pi
c 
ac
id
 
ac
id
 
ac
id
 
ac
id
 
H
2
N
--
C
H
--
C
O
O
H
 
H
2
N
--
C
H
--
C
O
O
H
 
[ 
[ 
C
H
2 
C
H
z 
J 
I 
C
H
2 
' 
C
H
2 
J 
I 
C
H
2 
C
H
2 
C
O
O
H
 
C
=
O
 
I 0 r 
L-
2-
Am
in
o-
 
O
=
=
P
--
O
- 
ad
ip
ic
 a
ci
d 
] 0 
A
de
no
si
ne
 
or
 N
N
 A
de
ny
l-a
m
in
oa
di
pi
c 
ac
id
 
H
2
N
-C
H
--
C
O
O
H
 
C
H
2 
' 
C
H
2 
........
 
C
H
2 
H
--
C
--
O
H
 
O
 
O
 I 
A
de
no
si
ne
 
A
de
ny
l-
am
in
oa
di
pi
c 
ac
id
 s
em
ia
ld
eh
yd
e 
H
2
N
--
C
H
--
C
O
O
H
 
H
2
N
--
C
H
--
C
O
O
H
 
C
H
2 
C
H
2 
I 
f 
, 
C
H
2 
;-
"-
-~
 
C
H
2 
, 
I 
I 
C
H
2 
H
O
O
C
 
C
H
2 
H
--
C
~
O
 
H
--
C
--
N
-H
 
+ 
C
H
2 
P 
H
2
N
--
C
H
--
C
O
O
H
 
C
H
2 
I 
[ 
C
H
2 
C
O
O
H
 
I CH
2 
Sa
cc
ha
ro
pi
ne
 
F CO
O
H
 
A
m
in
oa
di
pi
c 
ac
id
 
se
m
ia
ld
eh
yd
e 
+ 
L
-g
lu
ta
m
ic
 a
ci
d 
H
zN
--
C
H
--
C
O
O
H
 
[ CH
2 
' 
C
H
2 
I CH
2 
[ CH
2 
[ N
H
/ 
L
-L
ys
in
e + 
2-
K
et
og
lu
ta
ra
te
 
8 Ciba-Geigy/Lepetit 
All/~-lactam antibiotics seem to derive from the same three amino acids, 
L-~-aminoadipic acid, L-cysteine and L-valine. 
Obviously these precursors form a tripeptide which undergoes internal 
cyclization to the final compounds. In both types the e-amino-adipic 
moiety derives from the lysine biosynthetic pathway. Whereas in the 
penicillium-type of antibiotics this amino acid is in general replaced in 
the final product by a variety of N-acyl side chains the same amino 
Valinyl moiety and L-valine metabolism 
TPP (CH3--CHO) 
CH~ 
£o 
H3C--C--OH 
COOH COOH 
NADH --H20 R--NH~ 
I 
CH3 CH3 / CH3 
I r 
H3C--C--OH H3C--C--H [ H3C--C--H 
[ , H / OH 1_~ / = O I tt --C--NH2I I I 
COOH COOH COOH 
Pyruvate 2-Acetolactate 2,3-dihydroxy- 2-Ketoisovalerate L-Valine 
isovalerate t 
| 
i 
I 
I 
L-Leucine 
acid is irreversibly bound as the D-isomer in the biosynthesis of the 
Cephalosporium-type. A further difference in biosynthesis between the 
two types is found in the formation of cysteine. In contrast to the 
Penicillium-type where inorganic sulfur is the optimal sulfur source for 
the cysteine which is subsequently incorporated into 6-APA, methionine 
is the optimal sulfur donor in the Cephalosporium-type. No funda- 
mental differences could yet be detected with regard to valine. 
The condensation of the three amino acids and their cyclization is still 
a matter of speculation. 
Formation of a common tripeptide and its cyclization to fl-lactam anti- 
biotics (Hypothesis) 
+H3N 9 9- +H3N ÷H3N "CH3 
L/xCHICH2)3 C-O-P-O- Adenine ~.CHCH2SH ¢CHC~ 
+H~N S..~ / 
÷H3N~c/S~--N,~CO o- 
-~× N~J-~o 
Penicillium Cepholosporium 
+ N 
m ~>..~A.coH_T__KS,h<: OOC~coH_~_KS., ~ 
- o o c o~...L_,~__Lcoo_ " , ~ , ~ o~J_,_~l.:coo - 
+ 
" 0 m >...,,v.~coHT__FSh _j~:~coo-~,~ " N 
f" CH2COO--ooc o~.j_~/...~CH3,~ ' 
COO- 
l 
A s 
+H N ~ v .CON..T. ~ h 
O.11 ~'- N --'-"~COO- 3 O~_L. ~ I(I,,~CH2OAc 
COO- 
After A. L. Demain: In J. Snell (Ed.): Biosynthesis of Antibiotics, Vol. 1, pp. 29 94 
(1966) and R.D.G. Cooper, S.L. Jos6: J. Amer. chem. Soc. 94, 1021 (1972). 
Although no in vitro systems for the formation of fl-lactam antibiotics 
are yet available a few enzymatic reactions have been found in connec- 
tion with the biosynthesis. 
10 Ciba-Geigy/Lepetit 
Known enzymatic reactions with real or hypothetical functions 
in the synthesis of fi-lactam antibiotics 
1. Peptide-symhetases 
1.1. 6-(L-~-aminoadipyl)-L-cysteine + DL@eC)-valine 
crude extract | ATP 
ofC. acremonium ~ PEP + PEP-kinase 
6-(L-~-aminoadipyl)-L-cysteinyl- ?-valine 
1.2. DL-c~-aminoadipic acid + L-cysteine + L-valine 
crude extract / ATP 
of P. chrysooenum ~ PEP + PEP-kinase 
6-(L-e-aminoadipyl)- L-cysteinyl-L-valine 
2. N-Acylases 
0 H 0 
I I / II 
R--C--N--6-PA ~ R- -C- -Of t + 6-APA 
3, 
3.1. 
3.2. 
AO,I- Transferases 
O H O H 
*R--C--N 6-PA + R--C--N--6-PA* ~ _ _ - , *R--C--N 6-PA* 
H 1 
O H + R--C N--6-PA 
II I 
O H 
R--C--N--6-PA* + 6-APA ~ R--C--N--6-PA + 6-APA* 
II i lI E 
O H O H 
References 
1. Reviews 
Abraham, E.P., Newton, G.G.: In: Antibiotics If. Gottlieb, D., Shaw, P.D. (Ed.), 
p. 1--16. Berlin - Heidelberg- New York: Springer 1967. 
Abraham, E. P.: In: Topics in Pharmaceutical Sciences. Perl-Man, D. (Ed.), p. 1 31. 
New York: Interscience Publisher 1968. 
Demain, A.L.: In: Biosynthesis of Antibiotics, Vol. 1. Snell, J. (Ed.), p. 29--94. 
New York: Academic Press 1966. 
Flynn, E.H.: Cephalosporins and Penicillins, 752 p. New York and London: 
Academic Press 1972. 
Seminar on Topics of Fermentation Microbiology 1 
2. Genetics 
Ball, C.: J. Gen. MicrobioL 66, 63 (1971). 
Elander, R. P.: Abhandl. Deut. Akad. Wiss. Berlin, K1. ffir Med. 2, 403 (1967). 
3. Biochemistry 
Arnstein, H.R.V., Morris, D.: Biochem. J. 76, 357 (1960). 
Benz, F , Liersch, M., Nfiesch, J., Treichler, H.J.: Eur. J. Biochem. 20, 81 (1971). 
Loder, P.B., Abraham, E. P.: Biochem. J. 123, 477 (1971). 
4. Regulation of Biosynthesis 
Goulden, S.A., Chattaway, F.W.: Biochem. J. 110, 55 (1968). 
Goulden, S.A., Chattaway, F.W.: J. Gen. Microbiol. 59, 111 (1969). 
Lemke, P.A., Nash, C.H.: Can. J. Microbiol. 18, 255 (1972). 
C H A P T E R I 
Genetic Problems of the Biosynthesis of 
Tetracycline Antibiotics 
Z. HOg~ALEK, MARGITA BLUMAUEROVA, and Z. VAN~K 
With 22 Figures 
Contents 
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 
1. Selection of ltigh-Production Strains of S. aureq[aciens and S. rimosus 15 
a) Mutagen Treatment . . . . . . . . . . . . . . . . . . . . . 15 
b) Selection in Mutant Populations . . . . . . . . . . . . . . . . 17 
c) Hybridization . . . . . . . . . . . . . . . . . . . . . . . . 18 
d) Transduetion . . . . . . . . . . . . . . . . . . . . . . . . 21 
e) Increase of Resistance to its Own Metabolite . . . . . . . . . . 2I 
2. Isolation and Characterization of Mutants Blocked in the Biosynthesis 
of Tetracycline Antibiotics . . . . . . . . . . . . . . . . . . . 22 
a) Mutant Metabolites of S. aureofaciens . . . . . . . . . . . . . 24 
b) Mutant Metabolites of S. rimosus . . . . . . . . . . . . . . . 31 
c) Interspecific Cosynthesis . . . . . . . . . . . . . . . . . . . . 35 
3. Genetic Recombination . . . . . . . . . . . . . . . . . . . . . 35 
a) The Linkage Map of S. rimosus . . . . . . . . . . . . . . . . 36 
b) Analysis of Loci Controlling Tetracycline Biosynthesis . . . . . . 38 
c) Interspecific Recombination . . . . . . . . . . . . . . . . . . 44 
4. Genetic Control of the Biosynthetic Process . . . . . . . . . . . . 44 
a) Genes Responsible for the Biosynthesis of Tetracyclines . . . . . . 44 
b) Quantitative Aspects of the Biosynthesis of Tetracyclines . . . . . 56 
5. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . 60 
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 
Introduction 
The g r o u p of te t racycl ine an t ib io t ics is compr i sed of ch lor te t racyc l ine 
(CTC) p r o d u c e d by S t r e p t o m y c e s aureo fac i ens (Duggar , 1948) and oxyte- 
t racycl ine (OTC) p r o d u c e d by S t r e p t o m y c e s r i m o s u s (F in lay et al., 1950). 
14 Z. HO~'IALEK et aI. 
The two antibiotics certainly represent the standard metabolites of these 
two species- in spite of their structural similarity and related biogenetic 
origin of the compounds one may discern the species specificity of 
several biosynthetic steps, namely the chlorination in position 7 of 
the tetracycline ring system of S. aureofaciens and the hydroxylation 
in position 5 ofS. rimosus. Another important antibiotic of the tetracycline 
series is tetracycline (TC) itself, produced by standard strains of S. 
aureofaciens or S. rimosus (Perlmann et al., 1960) as a minor metabolite, 
and by mutants of S. aureofaciens blocked in the chlorination step, 
as a major product (Doerschuk et al., 1959). The formation of TC 
has been described in a number of other producers but their taxonomy 
is an unresolved problem which will not be discussed here. The problems 
of genetic and metabolic regulation of the chlorination of the tetracycline 
nucleus were reviewed by Petty (1961). 
In addition to the above-named, both S. aureofaciens and S. rimosus 
produce a number of other compounds which occur under standard 
conditions as minor metabolites or are produced by biochemical mutants 
of the two species. A list of these compounds, some of known and 
some of unknown chemical structure, will be found in the second section 
of this chapter. Most of them are not active antibiotically but are 
worth investigating for a better understanding of the genetic control 
of the biosynthetic process. 
Immediately after their discovery, the tetracycline antibiotics became 
important fermentation products. This fact stimulated intense research 
into the process of biosynthesis of these compounds in a number of 
laboratories. More than twenty years of study have provided vast experi- 
mental material, including data on strain improvement, metabolism 
of the blocked mutants and on the genetic processes of the production 
strains. In the present paper we attempt to present an overall view 
of the genetic problems associated with the production of tetracycline 
antibiotics and, at the same time, to draw general conclusions on the 
genetic regulation of their biosynthesis. 
R 7 Me OH R 5 N Me 2 
OH 0 OH 0 
Fig. 1. Structures of 
tetracycline antibiotics 
R 5 R 7 
CTC H CI 
OTC OH H 
TC H H 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 15 
1. Selection of High-Production Strains of S. aureofaciens 
and S. rimosus 
a) Mu tagen Trea tmen t 
Extensive selection work focussingon increasing the production of 
tetracycline antibiotics has been done mostly in industrial laboratories 
and the results have not been published. However basic information 
on the possibilities of strain improvement can be obtained from several 
papers describing the effects of physical or chemical mutagens on S. 
aureofaciens (Van Dyck and De Somer, 1952; Katagiri, 1954; Niedz- 
wiecka-Trzaskowska and Stzencel, 1956; Wang, 1957; Alikhanian and 
Romanova, 1965; Alikhanian et al., 1957; 1959a; Goldat, 1958, 1961, 
1965; Goldat and Sokolova, 1964; Blumauerovfi et al., 1973a) and 
on S. rimosus (Borenztajn and Wolf, 1956; Mindlin and Alikhanian, 
1958; Alikhanian et al., 1959a; Htiber and Giinter, 1960; Goldat and 
Vladimirov, 1968). 
In spite of the fact that the work was carried out with different strains 
and under various experimental conditions, the results indicate that 
the most effective factor for increasing the productivity in both species 
is UV light, applied either alone or in combination with other mutagens. 
Thus, for example Blumauerovfi et al. (1973a) working with the produc- 
tion strain ofS. aureofaciens 84/25 in populations surviving after UV-irra- 
Table 1. Comparison of spontaneous and induced variability of CTC production 
in S. aureofaciens (Blumauerovfi et al., 1973a) 
Mutagen a 
Spread in productivity (% of controlp Frequency 
of superior 
Total range The most producers 
frequent class (%)~ 
None 40~110 80-100 5.3 
UV light 0---130 90--110 17.4 
X-rays 0-- 110 7 0 - 90 7.1 
y-radiation 0--I 10 0 - 10 1.8 
MNG 0~120 40-- 80 9.4 
N-mustard 0~110 80--100 5.8 
a The following doses of mutagens resulting in less than t % of surviving conidia 
were used: UV light, 50~sec; )'-radiation and X-rays, 100 kR; N-methyl-N'-nitro-N- 
nitrosoguanidine (MNG), 1 mg/ml, for 60 min (in 0.02 M phosphate buffer, 
pH 6.7): N-mustard, 0.01 M, for 30 min {in 0.15 M phosphate buffer, pH 8.0). 
b The productivity of the parent strain is taken as 100 per cent. 
c Expressed as the percentage of total number of isolates tested. 
16 Z. HO~1"ALEK et al. 
diation, obtained more than 17% variants with production exceeding 
the activity of the parent strain by 10--30%. Of the other tested mutagens 
the most effective one was N-methyl-N'-nitro-N-nitrosoguanidine while 
X-rays and nitrogen mustard were relatively ineffective. On the other 
hand, 7-radiation induced the highest number of inactive variants (Table 
1). Goldat (1961) applied an eight-step selection procedure with UV 
light in combination with photoreactivation or with preceding treatment 
with sublethal doses of X-rays or ethylenimine, to achieve a five-fold 
increase of CTC production, in comparison with the activity of the 
parent low-production strain of S. aureofaciens 77 (Fig. 2). Working 
I 
X 
2O5 (1700) 
77 (60O) 
1 
u.v 
i 
536(t000) 
I 
UV-~PR4.UV 
112 (1200) 
[ 
El -~V 
" 15(14001 
2185 (3000) 
] 
UV 
546(1000) 
1 
XTUV 
134 (1700) 
I 
XTUV 
16 (2200) 
I 
XTUV 
542 (2260) 
I 
El 
E4f2-2 (2460) 
I 
X-~ El -... UV 
t 
f 
2201 (,:3500) 
Fig. 2. Scheme of selection of new active variants in S. aureojaciens (after Goldat, 
1961). Key: UV, UV light; PR, photoreactivation; EI, ethyleueimine; X, X-rays. 
Numbers in parenthesis represent the production level in ~tg CTC/ml 
with S. rimosus, Mindlin and Alikhanian (1958) applied repeatedly UV 
light to the parent production strain 8229 to obtain the variant LS-T 
118 with production activity by 67% higher. Further UV-irradiation 
of S. rimosus LS-T 118 produced variant LS-T 293 which displayed 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics t7 
only a little greater production activity than the parent strain but was 
more resistant to higher levels of inorganic phosphate in the fermentation 
media (Alikhanian et al., 1959b). 
Both species showed considerable morphological variability after treat- 
ment with mutagenic factors (alterations in size, shape, structure and 
pigmentation of colonies and in sporulation ability), most of the morpho- 
logical mutants having a decreased antibiotic activity. On the other 
hand, most variants with greater biosynthetic activity did not differ 
morphologically from the standard type (Mindlin and Alikhanian, 1958). 
It thus appears that mutagens and doses bringing about maximum 
frequency of morphological mutants produce also the highest number 
of (--) variants and are not suited for selection work. 
S. aureofaciens is frequently reported as a typical example of a highly 
variable species of Streptomyces (Backus et al., 1954; Duggar et al., 
1954; Kutzner, 1967). Its high spontaneous variability may frequently 
cause the instability of the production strains obtained through selection 
(Horvfith, 1954). Likewise, some strains of S. rimosus were observed 
to segregate spontaneously into morphological and nonproduction var- 
iants (Alikhanian et al., 1959a). Using multiple-step selection of producers 
of tetracycline antibiotics we found it useful to subject the isolates 
to natural selection before applying further mutagenic treatment. In 
this way we obtained not only a general stabilization of the given 
isolate but also frequently a further increase of its biosynthetic activity, 
as well as an increase of mutagen efficiency in the subsequent selection 
step. 
b) Selection in Mutant Populations 
To increase the efficiency of the selection procedure of S. viridifaciens 
and S. rimosus a method was developed, based on a selection of high-pro- 
duction variants from different types of mutant populations. The highest 
number of these variants was obtained among revertants of nonproduc- 
tion mutants (i.e. mutants in which a previous mutagenic treatment 
has already caused mutation in loci controlling tetracycline biosynthesis). 
Some superior producers were also obtained by an indirect selection 
among auxotrophic mutants growing in the presence of high concentra- 
tions of the required growth-factor and among prototrophic revertants 
of induced auxotrophs (Dulaney and Dutaney, 1967; Dulaney, 1969). 
However, yield improvement through auxotrophy does not seem to 
be the way of choice with S. aureofaciens where most nutritional mutants 
show a very much decreased or completely lost antibiotic activity even 
in media with increased amounts of the given factor (Blumauerovfi 
et aI., 1973b). 
18 Z. HO~ALEK et al. 
c) Hybridization 
After the discovery of genetic recombination in S. rimosus (Alikhanian 
and Mindlin, 1957) and in S. aureofaciens (Alikhanian and Borisova, 
! 961) the possibility of practical application of the recombination process 
was investigated as a new method for breeding the production strains 
in both species (J~irai, 1961; Borisova et al., 1962a;'Alikhanian et aI., 
1959a; Mindlin et al., 1961a, 1961c; Vladimirov and Mindlin, 1967). 
The starting material for these experiments consisted of biochemical 
mutants (single auxotrophs or double mutants carrying at the same 
time markers of nutritional deficiency and resistance to streptomycin) 
derived after mutagenic treatment from various strains differing among 
themselves in their production capacity and some other characteristics. 
The nutritional mutations exerted usually a negative effect on antibiotic 
activity which, in most auxotrophs of S. aureofaciens and S. rimosus, 
attained only ~ - I 0 % or even less of the production level of the parent 
prototrophs. 
When estimating the biosynthetic activity of prototrophic recombinants 
selected from mixed cultures of pairs of biochemical mutants of S. 
aureofaciens different authors obtained different results. Recombinants 
obtained by Alikhanian and Borisova (1961) from crossing mutants 
derived from strains LS-536 and Bd (producing 850--1000 pg CTC/ml) 
generally showed a higher activity than the parent monoauxotrophs 
but mostly did not exceed the level produced by the prototrophic parents. 
Only the cross of arg-3 × ilv (as the only one of 11 tested arg × ilv 
crosses) gaverise to recombinants with greater activity. Similarly, Bori- 
sova et al. (1962a) obtained most recombinants with activity lower 
than or identical with that of the parent strains. An exception here 
were some recombinants from the arg x ala crosses, exceeding the activity 
of the production prototrophic parent LS-B 2201 (3,300 pg CTC/ml) 
by about 6% (Table 2). On the other hand, auxotrophic mutants isolated 
by Jfirai (1961) from six strains of S. aureofaciens (derived from LS-536 
and LE-8234 and producing 1100--1690 pg CTC/ml) gave rise to three 
different types of recombinants: group I (11.5% of the total number 
of recombinants tested) produced 40~60% more of the antibiotic than 
the prototrophic parents, group II (31.7% recombinants) showed the 
same activity as the prototrophic strains and group III (56.8% recom- 
binants) had an activity as low as the auxotrophic parent strains. The 
most active recombinants were obtained from the arg x met crosses. 
Hybridization experiments with S. rimosus were carried out with mutants 
derived from four production strains (101, 8229, LS-T 293 and BS-21). 
The recombinants obtained were usually more active than the parent 
auxotrophic mutants and frequently attained the productivity of the 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 19 
Table 2. Productive activity of the original strains of S. aureofaciens, their biochemical mutants 
and prototrophic recombinants (after Borisova et al., 1962) 
Strain 
Activity Biochemical mutants 
of original 
strain Code Activity 
(gg/ml) 0ag/ml) 
LS-B 2201 3300 
5,8.10 
13, 26 arg 50-- 70 
1 thr 250~ 370 
14 ala 60(0--1000 
536 900 6 ilv traces 
Bd 900 4 his traces 
Prototr0phic recombinants 
Strains C ros sed Activity 
mutants (gg/ml) 
220t x 2201 
1 x 13 6(X)---3300 
lx 8 10(O2700 
5 x 14 560--3500 
10 x 14 25(~-2500 
26 x 14 40(02000 
2201x 536 14× 6 500~-2300 
1 x 6 1000--2700 
2201xBd 14× 4 180~ 880 
1 x 4 40(02500 
original prototrophs; the most active recombinants (isolated only from 
the his x ilv cross) then produced 10.5--13.5% more OTC than the 
prototrophic parents (Alikhanian et al., 195%; Mindlin et al., 1961c). 
One of these most active strains, the LS-T Hybrid (derived from LS-T 
293 and BS-21 which are parent strains of remote origin) was character- 
ized by lower foam formation during submerged cultivation in rich 
media (Mindlin et al., 1961a). 
It appears that the results of hybridization of S. aureofaciens and S. 
rimosus are affected by a number of factors, the most important of 
these being the selective markers of auxotrophic partners used for cross- 
ing, and the production capacity and mutual relationships (related or 
remote) of the prototrophic parents of the auxotrophs (Jfirai, 1961; 
Vladimirov and Mindlin, 1967; Mindlin, 1969). If the original proto- 
trophic strains are closely related and have similar characteristics it 
cannot be expected that crossing of their mutants will give rise to 
recombinants with markedly different properties. On the other hand, 
a crossing of strains originating from different parents and selected 
independently in different laboratories gives some hope of obtaining 
recombinants with higher production (Table 3). This assumption was 
supported by reciprocal crosses of active hybrids with one of the parent 
strains (Vladimirov, 1968; Mindlin, 1969). 
It should be noted here that certain differences in the properties of 
recombinants ofS. aureofaciens and S. rirnosus were observed, as concerns 
morphological properties and stability. With S. rimosus, recombinants 
selected from different combinations of crossed mutants as well as those 
T
ab
le
 3
. 
P
ro
du
ct
iv
e 
ac
ti
vi
ty
 o
f 
th
e 
or
ig
in
al
 s
tr
ai
ns
 o
f 
S.
 r
im
os
us
, 
th
ei
r 
bi
oc
he
m
ic
al
 m
ut
an
ts
 a
nd
 p
ro
to
tr
op
hi
c 
re
co
m
bi
na
nt
s 
(a
ft
er
 
A
li
kh
an
ia
n 
et
 a
l.,
 1
95
9a
) 
A
. 
C
ro
ss
es
 o
f 
lo
w
-a
ct
iv
it
y 
st
ra
in
s 
A
ct
iv
it
y 
B
io
ch
em
ic
al
 m
ut
an
ts
 
S
tr
ai
n 
of
 o
ri
gi
na
l 
st
ra
in
 
C
od
e 
A
ct
iv
it
y 
(~
tg
/m
l)
 
(t
.tg
/m
l)
 
82
29
 
14
29
 
13
61
 t
hr
 
26
2 
31
0 
ilv
 
97
 
P
ro
to
tr
op
hi
c 
re
co
m
bi
na
nt
s 
} 
C
ro
ss
ed
 
, 
m
ut
an
ts
 
j 
i 
31
0x
87
0 
I II
 
II
I 
I 
13
61
 x
 8
70
 
lI
 
Il
l 
10
1(
2a
) 
12
34
 
87
0 
hi
s 
28
 
R
ec
om
bi
na
nt
 
A
ct
iv
it
y 
ty
pe
 
(g
g/
m
l)
 
70
5 
16
22
 
! 5
55
 
66
3 
28
1 
12
69
 
b~
 
A
ct
iv
it
y 
S
tr
ai
n 
of
 o
ri
gi
na
l 
st
ra
in
 
(p
g/
m
l)
 
L
S
-T
 2
93
 
26
50
 
B
 S
 - 2
1 
19
(1
0 
B
. 
C
ro
ss
es
 o
f 
hi
gh
-a
ct
iv
it
y 
st
ra
in
s 
i 
...
...
 
B
io
ch
em
ic
al
 m
ut
an
ts
 
P
ro
to
tr
op
hi
c 
C
od
e 
A
ct
iv
it
y 
i 
C
ro
ss
ed
 
(g
g/
m
l)
 
| 
m
ut
an
ts
 
1 
ilv
 
7 
l 
x 
49
 
49
 h
is
 
11
5 
24
 a
rg
 
0 
1 
x 
24
 
re
co
m
bi
na
nt
s 
R
ec
om
bi
na
nt
 
A
ct
iv
it
y 
ty
pe
 
(~
tg
/m
l)
 
I 
20
25
 
II
 
29
25
 
II
I 
17
00
 
I 
15
00
 
II
 
63
0 
II
I 
18
00
 
.N
 
.at
 
7~
 
e~
 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 21 
originating from the same cross differed in their morphology; moreover, 
each of the mutant combinations usually gave rise to at least 2--3 
morphological types of recombinants (Alikhanian et al., 1959a; Mindlin 
et al., 1961c). On the other hand, prototrophic colonies selected from 
S. aureofaciens usually belonged to a single morphological type, similar 
to the parent strains. The offspring of these recombinants exhibited 
a segregation into 3~S different types of colonies resembling in pheno- 
type one or the other auxotrophic parent, or the prototrophic parent 
strains, or showing some features of both. This segregation process 
continued even in other subcultures of the recombinants for a number 
of generations (Alikhanian and Borisova, 1961; Mindlin et al., 1961c). 
In our recombination studies with S. aureofaciens we obtained similar 
results. With S. rimosus such segregation patterns were observed only 
rarely, most of the recombinants being relatively stable (Alikhanian 
et al., 1959a; Mindlin et al., 1961c). 
The stability of the production strains is one of the prerequisites for 
their successful application in the industry. Jfirai (1961) or the other 
authors who obtained high-production hybrid forms of S. aureofaciens 
do not mention the stability of this improved production capacity. 
In view of the observed segregation of morphological variants from 
the recombination cultures one can assume, however, that the biosynthe- 
tic activity also underwent subsequent spontaneous changes and most 
likely gradually decreased. 
d) Transduction 
Alikhanian and Ilyina (1957) observed in experiments with Streptomyces 
olivaceus that the actinophage brings about a great increase of morpho- 
logical variability. Mutagenic effects of the actinophage were also demon- 
strated in two production strains of S. aureofaciens; treatment with 
different phage types led to an increased variability in the production 
of the antibiotic-as compared with the untreated control by 6 0 - 200%, 
depending on the phage type and on the activity of the strain from 
which the phage had been isolated. The best results were obtained 
with mutant phages obtained after exposure to mutagens (Fedorova 
and Alikhanian, 1965). 
e) Increase of Resistance to its Own Metaboli te 
Using S. aureofaciens, a relation between the CTC production and 
the interaction of the antibiotic with the protein-synthesizing system 
was demonstrated (Mikulik et aL, 1971a, 1971b). The results obtained 
with some other antibiotic producers indicate that the final concentration 
22 Z. HOgt~,LEK et al. 
of produced metabolite may be limited by a mechanism resembling 
feedback control (Dole~ilowi et al., 1966; Legator and Gottlieb, 1953, 
Malik and Vining, 1970;Gordee and Day, 1972). Differences in the 
biosynthetic activity of various strains of S. aureofaciens and S. rimosus 
might thus be affected by the genetically determined degree of their 
resistance to their own antibiotic. The possibility of increasing this 
resistance either by physiological adaption to gradually increasing con- 
centrations of the antibiotic in the medium or by a mutation process 
was also checked as one of the ways toward improving the biosynthetic 
capacity of the production strains. 
Katagiri (1954) attained a 4-fold increase of productivity of S. aureofaciens 
by repeated transfers of the standard strain in a medium containing 
200 400 lag CTC/ml. The resulting high-production variant R-9-70 
was not stable and its biosynthetic activity gradually dropped to the 
original level. Veselova (1967) investigated the variability of different 
strains of S. aureofaciens and S. rimosus growing in media with an 
addition of CTC or OTC (200--1000 lag/ml) and compared it with 
the variability induced by mutagenic factors. The sensitivity of each 
strain to increasing concentrations of the antibiotic was in direct propor- 
tion to its production capacity. It was found that at a survival rate 
which in mutagen-treated populations led to increased frequency of 
low-production variants, tetracyclines resulted in an increased number 
of active forms. These results are explained by a lethal effect of the 
antibiotic which selectively deprives the natural population of low-active, 
sensitive forms. The method might thus be most effective during the 
initial stages of strain improvement or in cases when a population 
of a high-production strain was to be purified (e.g. during preparation 
of inocula for industrial-scale fermentations). 
2. I so la t ion a n d C h a r a c t e r i z a t i o n of M u t a n t s B locked in 
the Biosynthes is of Te t r acyc l ine Ant ib io t ics 
A prerequisite for the study of tetracycline biosynthesis and its genetic 
control is the preparation of mutants of S. aureofaciens and S. rimosus 
with point mutations at different steps of the biosynthetic pathway. 
Alterations in the morphology and pigmentation of colonies are 
obviously used as the first criterion in the selection of these mutants 
in populations surviving after mutagen treatment (Alikhanian et at., 
1961; McCormick et al., 1961; McCormick, 1969; Mindlin et al., 1968; 
Blumauerovfi et al., 1969b; Delid et al., 1969). For a qualitative analysis 
of the spectrum of metabolites produced by submerged cultures of 
mutants it was found most expedient to use paper chromatography 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 23 
and subsequent detection of spots under UV light (Erokhina, 1965; 
Mindlin et aI., 1968; Blumauerovfi et al., 1969b). Basic information 
on the character and probable sites of genetic blocks in the biosynthetic 
pathway can be obtained by studying metabolic complementation (cosyn- 
thesis) in mixed cultures of pairs of blocked mutants, either under 
submerged conditions (Mindlin et al., 1966, 1968; McCormick et al., 
1961; Blumauerovfi et al., 1969a) or by the agar method (Deli6, 1969; 
Deli6 et al., 1969; Pigac, 1969). 
The phenomenon of cosynthesis is explained by a mechanism of intercel- 
lular transmission of the intermediate or enzyme (coenzyme) essential 
for the biosynthetic pathway leading to tetracyclines (McCormick, 1966). 
It is an advantage of the agar method (Fig. 3) that one can determine 
which of the two participating mutants is the donor (secretor) and 
which is the recipient (converter) of cosynthetic activity. However, sub- 
merged cultivation and subsequent chromatographic analysis permit 
detection also of cosynthesis of antibiotically inactive substances (Blu- 
mauerovh et al., 1969a). 
Inact ive mutant A A 
B. subt i l is 
I 
Inact ive mutant B B 
Mutant A X , " Y ~ Z 
Mutant B X :: - Y ~' Z ant ib io t ic 
Fig. 3. Detection of cosynthesis of tetracycline antibiotics by the agar method 
(Deli6, t969). Two inactive mutants were streaked on opposite halves of a plate 
containing agarized production medium (each mutant covering one half of the 
plate) about 1--2 mm apart. The plate was incubated 5--7 days at 28°C. A 
strip of agar was cut from the dish and placed on the surface of an agar plate 
containing the test organism Bacillus subtilis. After overnight incubation, any 
antibiotic activity was revealed as an inhibition halo near the middle of the 
strip. The mutant surrounded by the halo was evidently the one which converted 
to tetracycline a compound secreted by the other mutant 
24 Z. HO~;~,~LEK et al. 
A definitive characterization of mutants is possible only on the basis 
of understanding the structure of the corresponding mutant metabolites. 
Isolation and study of the blocked mutants of producers of tetracycline 
antibiotics were recently taken up in several laboratories; the results 
obtained are at different stages of development. 
a) Mutant Metabolites of S. aureofaciens 
Standard strains of S. aureofaciens are characterized by the production 
of CTC (in mixture with about 5% TC) and of the antibiotically inactive 
glucoside aureovocin (Vokoun, 1970; Podojil et al., 1970). The position 
of its aglycon aureovocidin in the biosynthetic sequence is shown in 
the Fig. 16. Aureovocidin originates probably as a product of the overflow 
metabolism of 4-hydroxy-6-methylpretetramid, oxidation of ring A being 
apparently the rate limiting reaction of CTC biosynthesis under standard 
conditions (Fig. ll). Its glucosidation to aureovocin seems to be a 
detoxicating mechanism. The substrate specificity of enzyme system 
responsible for glucosidation and its general properties have been de- 
scribed by Hovorkov'~ et al. (1974) and Mat~jfi et al. (1973). 
Mutations leading to qualitative changes in the spectrum of standard 
products can be induced both in low- and in high-production strains 
rather easily (with a frequency of 5--30% of the surviving population, 
depending on the parent and mutagen used). Most frequently, however, 
only the ability to form aureovocin is lost, which is accompanied by 
a decreased production of tetracycline antibiotics. On the other hand, 
mutants with completely blocked synthesis of CTC and TC, or mutants 
producing novel substances different from the metabolites of standard 
strains, occur only at a low frequency (Blumauerovfi et al., 1971a, 1972). 
Of mutational changes resulting in the formation of qualitatively different 
metabolites, the most frequent is the block in the methylation at carbon-6 
of the tetracycline skeleton (Fig. 10) (Blumauerovfi et al., 1971a, 1972). 
In many cases, however, the transmethylation activity is not completely 
lost: such leaky mutants then produce demethyltetracyclines as well 
as trace amounts of CTC, TC, or even aureovocin (Blumauerovfi et 
al., 1969b). 
The series of blocked mutants used by McCormick and co-workers 
for studying the biosynthesis of tetracyclines was obtained from a wild 
strain of S. aureofaciens A 377 (NRRL 2209 or ATCC 10.762) or from 
its derivatives. For induction of mutants, various types of radiation, 
chemical mutagens and physical manipulation (grinding) were used. 
UV light, X-rays and nitrogen mustard have been of particular usefulness 
as mutagenic agents (McCormick et at., 1961). 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 25 
When cultivated on agar media, the wild parent strain A 377 forms light- 
yellow substrate mycelium (the reverse side of the colonies is yellow to 
yellow-orange or yellow-brown), white or light-grey aerial mycelium, 
light-grey spores and light-yellow to orange pigment (Duggar, 1948; 
McCormick et al., 1961); under submerged cultivation conditions the 
production may reach 20--300 lag CTC/ml. The nonproduction blocked 
mutants were either completely colourless or formed pigments different 
from those of the parent strain (dark-brown, dark-green, copper-red,etc.), or they differed in other characteristics, e.g., in the size or surface 
character of the colonies, formation of fluorescent diffuse zones (McCor- 
mick et al., 1961). Most mutant pigments belonged to tetracycline deriva- 
tives. 
Mutants blocked in the first step of biosynthesis, i.e. in the synthesis 
ofmatonamylCoA, form, in the place of tetracyclines, 2-acetyl-2-decarb- 
oxamidotetracyclines, possessing in position 2 an acetyl group (Miller 
and Hochstein, 1962; McCormick, 1965). The presence of the acetyl 
group deriving from acetoacetylCoA apparently has no influence on 
the further course of biosynthesis which is analogous to that during 
formation of the tetracyclines (McCormick, 1966). 
2-acetyl analogues of tetracyclines were found also in cultures of produc- 
tion strains of S. psammoticus (Lancini and Sensi, 1964) and S. rimosus 
(Hochstein et al., 1960; Orlova and Smolenskaya, 1965, Frolova et 
al., 1971) as side products during the biosynthesis of TC or OTC; 
a strain of S. rimosus yields mutants producing increased amounts of 
2-acetyl-2-decarboxamidooxytetracycline and only very low concentra- 
tions of OTC (Hochstein et al., 1960). The formation of the 2-acetyl 
analogue of OTC can be suppressed by an addition of amides (Orlova, 
1968). 2-acetyl-2-decarboxamidotetracyclines exhibit a similar antibac- 
terial spectrum as the corresponding tetracyclines but they are 5 to 
50 times less effective (Hochstein et al., 1960; Miller and Hochstein, 
1962; Orlova and Smolenskaya, 1965). 
Genetic block in methylation at carbon-6 results in the formation of 
demethyltetracyclines (McCormick et at., 1957), and in combination 
with another block(s) -- in the accumulation of non methylated com- 
pounds, homologues of the methylated series (Fig. 18). 
Using mutants (e.g. ED 1639) blocked in the cyclization step of biosyn- 
thesis (Fig. 10) two biologically inactive compounds of the anthraquinoid 
type were isolated; protetrone (McCormick and Jensen, 1968) and its 
6-methyl analogue (McCormick et al., 1968b) formed probably as by-pro- 
ducts of oxidation of a hypothetical anthrone precursor of tetracyclines 
(Fig. 19). Protetrone is identical with the dark red-brown pigment of 
mutants which, in submerged cultures, is bound mostly in the mycelium 
(McCormick and Jensen, 1968). 
26 Z. HOg'I'ALEK et al. 
Mutant V 655 blocked before the 12a-hydroxylation and 7-chlorination 
of the tetracycline ring system produces 4-hydroxy-6-methytpretetramid 
(Fig. 11) at a concentration of up to 500 lag/ml (McCormick et al., 
1965; McCormick and Jensen, 1965). The green pigment of this mutant 
was identified as the oxidation product of 4-hydroxy-6-methylpretetra- 
mid, the tetramid green (Fig. 15) (McCormick, 1965). 
The T 219 mutant and other strains blocked in the synthesis of 5a,6- 
anhydrotetracycline accumulate metatetrene originating, probably spon- 
taneously, from anhydrotetrene (McCormick 1965, 1966). The position 
of anhydrotetrene in the biosynthetic pathway is not very clear. 
So far no mutant has been prepared that would have a blocked capacity 
for hydroxylation in position 6 and that would thus accumulate large 
amounts of 5a,6-anhydrochlorotetracycline (McCormick, 1966). This 
is attributed to the high toxicity of anhydrochlorotetracycline (Goodman 
et al., 1955) and to its lethal effects on the producer. However, mutant 
1 E 1407 was described that produce 4-amino-6-demethylanhydrochlor- 
tetracycline (McCormick et al., 1968a), and mutant 1 E 6113, producing 
6-demethylanhydrochlortetracycline (McCormick and Jensen, 1969). The 
formation of N-demethylanhydrotetracyclines by S. aureofaciens BC-41 
in the presence of L-ethionine was described by Miller et al. (1964). 
A block in the last step of biosynthesis connected with the loss of 
a hydrogenating enzyme or cofactor results in accumulation of dehydro- 
tetracyclines (Fig. 12) (McCormick et al., 1958). 
Another group of mutants derived from strains producing demethyltetra- 
cyclines and blocked also in the last stage of biosynthesis is characterized 
by the production of pigments of naphthacenequinone type (McCormick 
and Gardner, 1963; McCormick, 1965). The colour of naphthacene- 
quinones changes in dependence on pH from blue in the alkaline medium 
to red in the acid medium. A typical representative of these compounds 
is tetramid blue isolated from the E 504 mutant. 
From cultures of the pigment-free mutant W 5 the so-called cosynthetic 
factor I has been isolated. In a mixed culture of this mutant with 
strains producing dehydrochlortetracycline, it stimulated the synthesis 
of CTC; a similar effect was produced by adding the filtrate of the 
fermentation liquor of W 5 or of a pure preparation of this cofactor 
to cultures of complementary mutants (Miller et al., 1960; McCormick 
et al., 1960, 1961). The fluorescence, colour, solubility, composition 
and some other properties, e.g. the ability of reversible oxidation and 
reduction, indicate that the cosynthetic factor is probably related to 
flavines or pteridines. 
The results of the extensive work of McCormick's group, dealing with 
the characterization of mutants and identification of biosynthetic 
sequences using either cosynthetic experiments or transformation of 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 27 
natural and synthetized intermediates were published in several excellent 
reviews (McCormick, 1965, 1966, 1969). 
Beside this there exist isolated publications on the preparation and 
properties of S. a u r e o f a c i e n s mutants blocked in the biosynthesis of 
tetracyclines. One of these mutants (obtained from the production strain 
LSB-2201 FU-57 after treatment with nitrosomethylurea) produced 
demethyltetracyclines and differed from the parental type in its morpho- 
logical properties and in the production of a dark brown-red pigment 
(Makarevich et al. , 1966). On applying nitrosomethylurea, the same 
parent strain yielded six mutants producing three new, hitherto unidenti- 
fied, compounds with yellow-green fluorescence in UV light which can 
be distinguished chromatographically from the tetracycline antibiotics; 
one of the compounds was apparently antibiotically active (Gutnikova, 
1966). 
Deli6 e t al. (1969) isolated blocked mutants from S. a u r e o f a c i e n s A-4 
(wild type isolated from soil) after treatment with UV light, nitrous 
acid, ethyleneimine and 1,3-diepoxybutane. The most effective for their 
induction was UV light (0.1--1% surviving cells). Many of the mutants 
lost their ability to sporulate, and differed from the parent type in 
changes in pigmentation but some retained traces of antibiotic activity. 
The metabolites of mutants were not characterized further, nevertheless 
their mutual cosynthetic ability was studied. 
The results of these tests made it possible to divide the mutants into 
four complementation groups suggesting the probable sequence of ge- 
Table 4. Grouping of inactive mutants of S. aureofaciens A 4 (Deli6 et al., 1969) 
Attribution of mutants to 
complementation groups Cosynthesis between groups a 
Mutants Group Groups A A C D 
c tc - l ,2 ,4 A A - - B C A 
ctc-3 B B - - C - - 
ctc-5 C C - - 
ctc-6, 7 D D 
ctc-8, 9,10 Doubtful 
Complementation patternb: C B A 
D 
a The sign (--) indicates no cosynthesis. Each letter indicates cosynthesis with 
a halo on the side of the strain of the corresponding group. 
b Non-overlapping segments correspond to complementing groups. 
28 Z. HO~/~LEK et al. 
netic blocks in the biosynthetic pathway (Table 4). Groups ctc B and 
ctc C (and possibly also ctc A) included mutants which were mutually 
complementary during the biosynthesis of the antibiotic and functioned 
in different combinations either as secretors or as converters. These 
mutants are probably blocked in their structural genes, controlling 
the main metabolic pathway during biosynthesis of CTC. On the other 
hand, the ctc D (and possibly ctc A) groups included mutants withno mutual complementarity with those of the preceding groups, acting 
always as secretors. These mutants are probably not formed by multiple- 
point mutations covering several structural genes because they occur 
at relatively high frequency in comparison with the usually observed 
frequencies of single-point mutations of structural genes. The authors 
suggest that these mutants are blocked rather in the regulatory genes 
(or quite generally in genes controlling the onset of secondary metabo- 
lism, i.e. the shift of normal metabolic channels toward the antibiotic 
pathway) rather than in the structural genes of the secondary metabolic 
pathway itself. 
Another series of S. aureofaciens mutants was isolated from two standard 
strains of S. aureofaciens (84/25 and Bg) differing in their production 
capacity, after treatment with UV light, X-rays and 7-radiation, N- 
methyt-N'-nitro-N-nitrosoguanidine, nitrosomethylurea, nitrous acid, 
hydroxylamine and nitrogen mustard (Blumauerovfi et at., 1969b). 
According to the differences in biosynthetic activity, the mutants were 
divided into twelve metabolic groups (Table 5). In eight of these, chroma- 
tography showed the production of novel compounds differing from 
the metabolites of the standard strains; in nine groups atypical pigments 
were also produced. The identification of these metabolites is still in 
progress, our preliminary results suggest that most of them belong 
to the tetracycline series. 
Comparison of the frequency of occurrence of the individual metabolic 
types in treated and untreated populations of both parent strains indicates 
a relative specificity of the mutagens used. Qualitative changes in metabo- 
lism resulting in the production of new compounds were induced with 
UV light, N-methyt-N'-nitro-N-nitrosoguanidine and nitrous acid. On 
the other hand, ?- and X-radiation induced only quantitative changes 
in the production of standard metabolites or a loss of their synthesis. 
Very specific was the inhibitory effect of the two types of radiation 
on the production ofaureovocin, accompanied by a decreased prod uction 
of tetracycline antibiotics induced at a 2 5 times higher frequency 
than after application of other mutagens (Blumauerovfi et al., 1971 a). 
Cosynthetic experiments (Blumauerovfi et al., 1969a) revealed that 
mutants can be complementary not only during biosynthesis of tetracy- 
cline antibiotics but also during production of other metabolites 
T
ab
le
 
5.
 
C
ha
ra
ct
er
is
ti
cs
 
of
 s
ta
n
d
ar
d
 
st
ra
in
s 
o
f 
S.
 
au
re
of
ac
ie
ns
 
an
d
 
th
ei
r 
b
lo
ck
ed
 
m
u
ta
n
ts
 
(B
lu
m
au
er
ov
fi
 
et
 
al
., 
19
69
b,
 
19
71
a)
 ~ 
S
p
ec
tr
u
m
 o
f 
p
ro
d
u
ce
d
 m
et
ab
o
li
te
s 
M
et
ab
o
li
c 
T
et
ra
- 
ty
pe
 
cy
cl
in
es
 
C
T
T
C
 
U
n
k
n
o
w
n
 s
u
b
st
an
ce
s 
P
ig
m
en
t 
(l
ag
/m
l)
 
T
C
 
D
T
C
 
A
V
C
 
B
 
C
 
O
th
er
 
N
u
m
b
er
 o
f 
Pz
 
w
 
m
u
ta
n
ts
 
te
st
ed
 
~
=
 
B
g 
40
0 
+ 
+ 
+ 
+ 
~ 
84
/2
5 
20
00
 
+ 
+ 
+ 
+ 
"U
 
b
ro
w
n
-o
ra
n
g
e 
to
 b
ro
w
n
 
--
 
~-
 
b
ro
w
n
-o
ra
n
g
e 
to
 b
ro
w
n
 
--
- 
,<
 
I 
50
--
15
00
 
+ 
" 
+ 
+ 
+ 
--
 
li
gh
t 
b
ro
w
n
-o
ra
n
g
e 
to
 b
ro
w
n
 
34
3 
~"
 
II
 
0 
--
 
--
 
+ 
+ 
+ 
--
 
d
ar
k
 g
re
en
 
5 
II
I 
0 
--
 
--
 
+ 
+ 
+ 
D
, 
E
 
ye
ll
ow
 
20
 
IV
 
50
--
- 
10
0 
÷ 
--
 
--
 
+ 
--
 
cr
ea
m
 o
r 
b
ro
w
n
is
h
-c
re
am
 
14
7 
p~
 
V
 
0 
--
 
--
 
+ 
--
 
n
o
n
e 
54
 
V
I 
10
0 
--
 
+ 
--
 
--
 
+ 
F
, 
G
 
re
d-
vi
ol
et
 
5 
V
II
 
15
0 
+ 
+ 
+ 
--
 
+ 
F
, 
G
 
re
d
-b
ro
w
n
 
2 
V
II
I 
15
0 
+ 
+ 
+ 
--
 
+ 
G
, 
H
 
re
d
-o
ra
n
g
e 
5 
:~
 
IX
 
25
0 
+ 
--
 
+ 
K
, 
J 
b
ro
w
n
-g
re
en
 t
o 
b
ro
w
n
-b
la
ck
 
6 
9.
 
X
 
20
 
--
 
+ 
--
 
-~
 
+ 
F
, 
L
, 
M
 
b
ro
w
n
-v
io
le
t 
2 
o-~
" 
X
I 
0 
--
 
--
 
--
 
+ 
L
, 
M
 
gr
ey
-v
io
le
t 
2 
g-
 
X
II
 
0 
.
.
.
.
.
.
.
.
.
.
.
.
 
N
, 
O
, 
P
, 
R
 
y
el
lo
w
-b
ro
w
n
 
1 
S
,T
,U
 
a T
h
e 
p
ro
d
u
ct
io
n
 o
f s
ec
o
n
d
ar
y
 m
et
ab
o
li
te
s 
w
as
 d
et
er
m
in
ed
 b
y 
p
ap
er
 c
h
ro
m
at
o
g
ra
p
h
y
 i
n 
th
e 
sy
st
em
 c
h
lo
ro
fo
rm
-b
u
ta
n
o
l-
M
cl
lv
ai
n
 b
uf
fe
r 
(p
H
 4
.5
)-
-4
:1
: 
5,
 a
n
d
 b
y 
sp
ec
tr
o
p
h
o
to
m
et
ri
c 
as
sa
y 
at
 4
40
 n
m
. 
A
b
b
re
v
ia
ti
o
n
s 
us
ed
: 
D
T
C
, 
d
em
et
h
y
lt
et
ra
cy
cl
in
es
: 
A
V
C
, 
au
re
o
v
o
ci
n
; 
B
-U
, 
su
bs
ta
nc
es
, 
th
e 
st
ru
ct
ur
e 
o
f 
w
h
ic
h
 h
as
 n
o
t 
be
en
 y
et
 d
ef
in
it
el
y 
es
ta
bl
is
he
d.
 
30 Z. HO~TALEK et al. 
Type 
~4utants 
RFI'° ¢~E <1') 
0.9- 
0.81_ 
0.7_ @ B @ CT- 
0.6- 
0. 5 - TC" 
_9"Q° i 0.3 - ~) 0.2 - EP 
0.1- 
Fluoreseention in UV-light: 
O yellow 
Q yellow green 
t~ Tetracyctlnes ~r~ Substance A 
UV45 8-96 Mixture B-t03 ~801/26Mixture 
)r ()CT() 
'° i 
)T( 
A 
E~ Substances V,X,Y [~Z. Substances H, Z 
Bq03 0202Mixture Bq03 HA-V/382M~xture 
( ) x tll~z - 
L t~ 
4 ) 
tnt~ 
(]]) yeltow orange t~ brown Q red 
(~ orange O blue, blueviolet O violet 
Fig. 4. Chromatographic analysis of substances produced by pure and mixed 
cultures of blocked mutants of S. aureofaciens {Blumauerovfi et al., 1969a). Key: 
CT, chlortetracycline; TC, tetracycline: EP, epimers: DT, demethyltetracycline; 
A, aureovocin; B-Z, substances, the structure of which has not yet been definitely 
established. Paper chromatography in the system chloroform-butanol-Mcllvain 
buffer pH 4.5 (4: 1:5) was used for the analysis 
aureovocin and some novel compounds, probably intermediates or by- 
products of the tetracycline pathway (Fig. 4). These results indicate 
that multiple blocks in this pathway are possible. In contrast with 
the results described by McCormick et at. (1960) all the types of cosyn- 
thesis, observed by Blumauerovfi et at. (1969a) took place only during 
mixed cultivation of both complementary mutants. The presence of 
any of the partners could not be replaced either by filtrates of its 
culture or by metabolites isolated from the mycelium. A similar finding 
was made by Shen and Shan (1957) in S. aureofaciens where an increased 
production of CTC in mixed cultures was accompanied by a substantial 
improvement of growth. 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 31 
From the point of view of the present knowledge on the biogenesis 
of tetracyclines and its control (Van~k et al., 1971 ; Ho~filek and Van~k, 
1973) it appears that the phenotypic expression of many mutants seem- 
ingly blocked in the secondary metabolic pathway can be actually due 
to the genetic changes in primary metabolism responsible for the supply 
of a specific precursor (acetylCoA) to secondary biosynthesis. This 
assumption is in agreement with the conclusions expressed by Deli6 
et al. (1969) on the basis of cosynthetic experiments. Likewise, the 
concomitant characteristics of mutants, e.g. changes in morphology, 
growth rate, respiratory activity, ability to utilize carbohydrates, sensiti- 
vity to their own metabolites (Blumauerovfi, 1969) and in cell ultrastruc- 
ture (Ludvik et al., 1971) suggest the possibility of multiple-point muta- 
tions, affecting simultaneously a greater number of biochemical functions 
of the organism. Subsequent changes in the biosynthetic activity of 
unstable mutants never led to complete quantitative reversal to the 
standard parental type and they thus appear to reflect spontaneous 
suppressions affecting the overall metabolic balance rather than back 
mutations of the originally altered genes (Blumauerov~ et al., 1972). 
b) Mutant Metabolites of S. rimosus 
The preparation, properties and the results of genetic studies of blocked 
mutants of S. rimosus were described by Alikhanianand co-workers 
in 1956~1970. The mutants were obtained from the production strain 
LS-T 118 with the aid of various mutagens, the most efficient being 
UV light. Good results were also achieved by using fast neutrons, 
diethyl sulphate and combined action of ethyleneimine and UV light. 
The mutants differed from the parent strain by a substantially reduced 
production of OTC, by morphological properties, changes in pigmenta- 
tion of submerged cultures and by the formation of new compounds 
with characteristic fluorescence in UV light (Table 6). The structure 
of these compounds and their physico-chemical properties have not 
been described. In mixed cultures most mutants showed the capacity 
of mutual complementation during OTC biosynthesis; it is hence 
assumed that these mutants are genetically blocked in different loci 
controlling the biosynthesis of the antibiotic (Alikhanian et aI., 1961; 
Erokhina, 1965; Erokhina and Alikhanian, 1966; Mindlin et aI., 1966, 
1968; Zaytseva et al., 1961; Boronin and Mindlin, 1970). 
The nonpigmented "white'" mutants of group 6 were cosynthetically 
active only in combination with the "black" mutants of group 3. The 
"white" mutants were found to produce the so-called X-factor. This 
compound which was found in a number of other actinomycete species 
probably has the function of a coenzyme; a stimulatory effect on the 
T
ab
le
 6
. 
C
h
ar
ac
te
ri
st
ic
s 
o
f 
b
lo
ck
ed
 m
u
ta
n
ts
 o
f 
S,
 r
im
os
us
 d
es
cr
ib
ed
 b
y
 M
in
d
li
n
 e
t 
al
. 
(1
96
8)
 a
n
d
 M
in
d
li
n
 (
19
69
) a
 
M
u
ta
n
t 
N
u
m
b
er
 
C
o
lo
u
r 
o
f 
A
n
ti
b
io
ti
c 
g
ro
u
p
 
o
f 
m
u
- 
cu
lt
u
re
 
ac
ti
v
it
y
 
ra
n
ts
 
fl
ui
d 
(g
g
/m
l)
 
M
u
ta
n
t 
m
et
ab
o
li
te
s 
fl
u
o
re
sc
en
t 
in
 U
V
 l
ig
h
t b
 
b
lu
e 
b
lu
e-
 
vi
o-
 
y
el
lo
w
 
vi
o-
 
li
gh
t 
y
el
lo
w
 
o
ra
n
g
e 
g
re
en
 
le
t 
le
t 
b
lu
e 
g
re
en
 
C
o
sy
n
th
es
is
 
C
o
m
p
le
- 
m
en
ta
ry
 
O
T
C
 
g
ro
u
p
 
la
g/
m
l 
ta
O
 
1 
10
 
O
ra
n
g
e 
1
--
2
0
 
+
 
+
 
.
.
.
.
.
.
.
.
.
.
.
.
 
+
 
2 
1.
5 
3 
1
0
(O
 
40
0 
4 
14
0-
--
 3
00
 
5 
I
~
 
45
0 
6 
0
.5
--
 
30
 
3 
10
0 
60
0 
2 
7 
D
ar
k
 
0.
5-
--
1.
0 
+
 
+
 
--
 
+
 
--
 
+
 
--
- 
- 
4 
2
0
(0
 
40
0 
cr
im
so
n
 
5 
1
0
0
~
 
35
0 
6 
1
--
 
4 
D
ar
k
 
4 
2
0
0
--
 1
00
0 
3 ~
 
3 
b
ro
w
n
 
1
5
--
3
0
 
+
 
--
 
+
 
--
- 
+
 
..
..
..
. 
5 
4
0
(0
 
80
0 
to
 b
la
ck
 
6 
7
~
1
2
0
0
 
4 
5 
D
ar
k
 
10
--
--
80
 
+
 
--
 
+
 
--
 
+
 
--
- 
+
 
--
 
5 
1
5
0
~
 
20
0 
b
ro
w
n
 
6 
1
0
--
 
80
 
5 
3 
G
re
en
- 
0.
3-
--
6.
0 
+
 
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
 
6 
0
.5
--
 
6.
0 
b
ro
w
n
 
6 d
 
11
 
N
o
n
e 
0.
1-
~.
 1
.0
 
+
 
+
 
.
.
.
.
.
.
.
 
N
 
a 
M
u
ta
n
ts
 
w
er
e 
d
er
iv
ed
 f
ro
m
 
S.
 
ri
m
os
us
 
L
S
-T
 
11
8 
p
ro
d
u
ci
n
g
 
ab
o
u
t 
30
00
 
la
g 
O
T
C
/m
l.
 
T
h
is
 
st
ra
in
 
fo
rm
ed
 
a 
b
ro
w
n
 
p
ig
m
en
t 
u
n
d
er
 s
u
b
m
er
g
ed
 c
o
n
d
it
io
n
s.
 
b 
P
ro
d
u
ct
io
n
 (
+
) 
o
r 
ab
se
n
ce
 (
--
) 
o
f 
m
et
ab
o
li
te
s 
w
as
 d
et
ec
te
d
 b
y
 p
ap
er
 c
h
ro
m
at
o
g
ra
p
h
y
 i
n 
sy
st
em
 c
h
lo
ro
fo
rm
-n
it
ro
m
et
h
an
e-
p
y
ri
d
in
 
(1
0:
20
:3
) 
o
r 
b
u
ta
n
o
l-
ac
et
ic
 a
ci
d
-H
2
0
 (
16
:4
: 
5)
. 
"B
la
ck
" 
m
u
ta
n
ts
. 
d 
"W
h
it
e"
 m
u
ta
n
ts
. 
..a
 t 
t-
 
7~
 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 33 
biosynthesis of OTC was found also in partly purified preparations 
of the X-factor added to cultures of "black" mutants at the beginning 
of cultivation (Orlova et al., 1961, 1964). 
Cultures of "black" mutants were found to contain the antibiotically 
inactive compound Y. Its biosynthesis, just as that of OTC, was unfavour- 
ably affected by excess inorganic phosphate and by powerful aeration. 
Underoptimumconditions, compound Ywas produced at concentrations 
of up to 550 ~tg/ml (together with 15--30 ~tg OTC/ml), the maximum 
production having been reached after about 72 hours of cultivation. 
After adding a catalytic amount of the X-factor the production of OTC 
rose considerably with simultaneous disappearance of compound Y. 
Complementation took place even after transfer of the washed mycelium 
of the "black" mutant to the filtrate of a culture of a "white" mutant 
containing the X-factor. It is assumed that compound Y is either a 
precursor or a by-product during biosynthesis of OTC (Zaitseva and 
Orlova, 1962). 
Mutants of groups 3 and 6 thus could mutually complement each 
other during the formation of the antibiotic in a way similar to that 
described by McCormick (1966) for S. aureofaciens. It appears, however, 
that in other systems where different combinations of mutants of group 
1 to 5 were tested, a different mechanism played a role during complemen- 
tation. In all cases, the cosynthesis of OTC required direct contact 
between the two complementary types of mycelium. After plating the 
mixed cultures, as much as 10% of mosaic colonies were obtained, 
with mycelial sectors of both starting forms. The mosaic colonies were 
antibiotically active, in contrast to the colonies of pure mutant cultures. 
These results indicate that heterokaryons were formed in the mixed 
cultures. However, since the mixed cultures contained always besides 
heterokaryotic cells also the original homokaryotic mycelium of both 
blocked mutants, no full restoration of production comparable with 
that of the parent strain has been achieved (Mindlin et al., 1968). 
Data on the complementation activity of mutants obtained in the above- 
mentioned cosynthetic experiments indicate the existence of at least 
two separate groups of loci controlling the biosynthesis of OTC (Mindlin 
et al., 1968). Using newly prepared series of inactive mutants (Boronin, 
1970) the preliminary results were further supplemented by Boronin 
and Mindlin (1970): according to complementation patterns found 
with the agar method, the mutants were divided into several complemen- 
tation groups, three of which have not been previously described. 
A new scheme of blocks in the sequence of OTC biosynthesis 
was advanced. 
Mutants of S. rimosus studied by Deli6 et al. (1969) and Pigac (1969) 
were isolated from the standard strain R-6 under the same conditions 
34 z. HO~;J'ALEK et al. 
as the above mutants of S. aureofaciens (p. 27). The cosynthetic ability 
of mutants was tested by the agar method. On the basis of the results 
the mutants were divided (according to their complementation pattern) 
into eight groups (Table 7). In analogy with the mutants of S. aureofaciens, 
Table 7. Grouping of inactive mutants of S. rimosus R-6 (Deli6 et al., 1969) 
Attribution of mutants 
to complementation groups 
Mutants Group 
Cosynthesis between groups" 
GroupsE C B A F G H D 
otc-4, 5, 13,65, 123 A E 
otc-17 B C 
otc-15 C B 
otc-90, 98, t04, 118 D A 
otc-2 E F 
otc-10,91,94, II 1,112, 113, 114, 117 F G 
otc-8, 64, 105 G H 
otc-95, 96, 119, 120 H D 
Complementation patternb: 
- - E E E E E 
C C C - - C - - 
B 
E C B A 
F 
G 
H 
D 
a,b See notes to Table 4. 
two different mutant classes were described. One of them (groups otc 
B, otc C, otc E, and perhaps otc A) included mutually complementary 
mutants (and hence apparently blocked in the secondary metabolic 
pathway) whereas the other class (groups otc D, otc F, otc G, otc 
H and possibly also otc A) included mutants of unknown character. 
This second class contained also up to 90% of the isolated mutants; 
it thus appears that only a small number of mutants are really blocked 
in the proper biosynthetic pathway. 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 35 
c) Interspecific CosynthesisIn cosynthetic experiments designed by McCormick et al. ( 1961) a certain 
possibility ofinterspecific complementation was found; thus, for example, 
formation of OTC was observed in mixed cultures of S. aureojaciens 
and S. rimosus, and formation of CTC during combined cultivation 
of S. aureofaciens with mutants of S. albus and S. platensis. 
The cosynthetic activity was also apparent in combination of inactive 
mutants of S. rimosus and S. aureofaciens, tested by Deli6 et al. (1969). 
Two mutants of S. rimosus, belonging to the groups otc E and otc 
B, yielded in combination with the mutant of S. aureofaciens of group 
ctc A a zone of antibiotic activity at the side of S. rimosus mutants 
which thus acted as converters. This result was predictable since the 
cte A gene is a late gene in CTC biosynthesis while the otc B and 
otc E are earlier genes in OTC biosynthesis. A cosynthesis between 
several wild strains of streptomycetes and inactive mutants of S. aureofa- 
ciens or S. rimosus was observed. In these experiments usually the 
highest cosynthetic activity ever observed was found, the wild-type strain 
acting as the secretor (similarly to the above-discussed class 2 of mutants 
of S. aureofaciens and S. rimosus). 
3. Gene t i c R e c o m b i n a t i o n 
The existing data on genetic recombination of producers of tetracycline 
antibiotics may be divided into several principal groups. Preliminary 
results obtained by the application of the recombination technique 
for strain improvement have already been discussed in the first section. 
Another group includes papers on the genome topology, i.e. on the 
construction of the linkage map of the chromosome on the basis of 
analysis of loci controlling the biosynthesis of essential metabolites 
(Ala~evi6, 1969a, 1973; Ala~evi6 et al., 1972; Friend and Hopwood, 
1971). The third group of papers is focussed on the genetic analysis 
of loci controlling the secondary biosynthesis (Mindlin et aI., 1961b, 
1966, 1968; Vladimirov and Mindlin, 1967; Boronin and Mindlin, 1971; 
Blumauerovfi et al., 1971b, 1972). Most of these investigations were 
conducted on the model of S. rimosus while data on the genome of 
S. aureofaciens are still rather scarce (Ala~evi6, 1969a; Blumauerov~i 
et al., 1971b, 1972). Interspecies recombination between S. rimosus and 
S. aureofaciens has also been described Ala~evi6, 1963, 1965a, 1965b, 
1969b; Ala~evi6 et al., 1966; Polsinelli and Beretta, 1966; Blumauerov~i 
et al., 1971b). 
36 Z. HO~I",~LEK et al. 
a) The Linkage Map of S. rimosus 
By adapting methods developed for S. coelicolor A 3/2/ (Hopwood 
and Sermonti, 1962; Hopwood, 1967; Sermonti, t 969), basic information 
on the genetic map of S. rimosus was obtained independently in two 
laboratories. 
Ala~evi6 (1969a, 1973) worked with auxotrophic mutants of a tow-pro- 
duction standard strain, S. rimosus R 7 (ATCC 10.970). Most of them 
had a decreased or practically nonexistent antibiotic activity but their 
products were not characterized in detail. A selective analysis of haploid 
recombinants obtained by four-point crosses showed a variety of recom- 
binant phenotypes. In most cases an excess of prototrophs was obtained 
which distorted the results for the estimation of linkage relationships 
between genes. 
The main criterion used for the mapping was therefore based on data 
from heteroclone analysis. In the first stage of the work (Ala~evi6, 
1969a) 10 markers were localized on the chromosome map by analyzing 
trios of markers from many heteroclones. For further more refined 
analysis, four-, five- or six-point crosses of multiple mutants were used 
(Ala~evi6, 1973). From the segregation data obtained, the arrangement 
of markers was deduced by checking all the possible permutations 
of the circular arrangement of the markers involved and by choosing 
the arrangement from which the segregants were formed by the minimum 
number of crosses. Although in some cases contradictory results were 
obtained, a preliminary map of the chromosome containing 24 markers 
his.7~ 
~.'~//his-12 4,6 2124 \\ ,// ,., .,v,_._ ', arg-130 44 
":// / • rib-4 pro-l/ \ 
(met-108) ~ / / 
pao-1 /J,'--" 
(rib-5) /~- 
Fig. 5. Linkage map of Streptomyces rimo,sus R-7 (Alabevi6, 1973) 
Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 37 
could be constructed. A clustering of some genes controlling the biosyn- 
thesis of histidine was established, six mutations appeared to be closely 
linked, similarly to the situation described previously by Piperno et 
al. (1966) in S. coelicolor. Likewise, three of the genes controlling the 
biosynthesis of arginine appeared to be in close linkage on the map 
of S. rirnosus (Fig. 5). This map is consistent with that constructed 
in another strain, S. rimosus R 6 (Ala~evi6 et aL, 1972). 
Friend and Hopwood (1971) used for their mapping studies auxotrophic 
mutants derived from the industrial production strain of S. rimosus. 
During crossing of these mutants they observed frequently variable 
numbers of heterokaryons which could be eliminated by a suitable 
choice of selected marker combinations. A map showing the linkage 
relationship of 24 marker loci (Fig. 6) was constructed on the basis 
gtu B 
gLuA i ~ - - ~ . 
/ \ 
/ hisA \ / met A ser A 
// p a n A ~ ' ~ a r g A 
,/ leuA / ~- ribB'~ 
/proA / X~ ur.B\~ 
[ athA -~ J-cysD J 
thiB~ , A~ II ~ /serC 
\aae, X / l e u ~ / 
met B . x'b.....~ j . X / " itvB 
nicC his D~hrA lysB 
guaA 
Fig. 6. Linkage map of Streptom),ces rimosus (Friend and Hopwood, 197 I). The 
order of the bracketed loci is unknown. Certain loci have not been ordered 
relative to loci covered by broken lines. Loci are arbitrarily spaced at equal 
intervals 
of results of selective analysis of haploid recombinants obtained both 
in four-point crosses of mutants and in multi-factor crosses between 
mutant and recombinant strains; heteroclones were not selected in these 
experiments. 
Mutants used in the two laboratories for genetic analysis were derived 
from different strains and in many cases displayed different growth 
requirements. From an incomplete characterization of the auxotrophic 
38 Z. HO~A.LEK et al. 
markers (defined usually only on the basis of the required final metabo- 
lites) it cannot be concluded with certainty whether in mutants with 
the same growth requirement an identical locus has been mapped, 
viz. the one controlling the same enzyme step of biosynthesis of the 
given primary metabolite. In spite of these facts, the identical sequence 
of at least nine markers suggests the similarity of the two genetic maps 
presented. Ala~evi6 (1973) as well as Friend and Hopwood (1971) 
observed a similarity between their map of S. rimosus and the linkage 
map of S. coelicolor A 3/2/(Hopwood, 1967). A similar arrangement 
of the markers was described by Coats and Roeser (1971) for Streptomyces 
bikiniensis var. zorbonensis UC 2989 (NRRL 3684), the only other indus- 
trial streptomycete where a tentative chromosome map has been con- 
structed. These findings suggest a remarkable evolutionary stability 
of gene arrangements on prokaryote genomes (Friend and Hopwood, 
1971). 
Ala~evid (I969a) reported also the results of some heteroclone analysis 
of S. aureofaciens. The pattern of segregation from the heteroclones 
seems to be comparable with that observed in S. rimosus. For any 
definitive conclusions, however, more data should be gathered, following 
the same markers introduced in crosses in different coupling arrange- 
ments. 
b) Analysis of Loci Controlling Tetracycline Biosynthesis 
To analyze the production of secondary metabolites, Blumauerovfi et 
at. (1972) suggested the following working procedure (Fig. 7). Mutants 
point-blocked at different steps of the biosynthetic pathway must be 
labelled in the next mutation step by supplementary nutritional or 
drug-resistance markers, essential for the detection of genetic recombina- 
tion. For linkage mapping it is desirable to obtain