-
7/26/2019 Biosintesis de Fosfomicina
1/12
A N T I M I C R O B I A L
A G E N T S AND
C H E M O T H E R A P Y , F e b . 1 9 7 4 , p .
1 2 1 - 1 3 2
C o p y r i g h t
0
1 9 7 4
A m e r i c a n S o c i e t y f o r
M i c r o b i o l o g y
V o l .
5 ,
N o .
2
P r i n t e d
i n
U . S A .
B i o s y n t h e s i s o f
F o s f o m y c i n
b y
S t r e p t o m y c e s
f r a d i a e
THOMAS 0 . ROGERS A N D
JEROME
BIRNBAUM
M e r c k
S h a r p
a n d
Dohme
R e s e a r c h
L a b o r a t o r i e s , R a h w a y , Ne w J e r s e y
0 7 0 6 5
R e c e i v e d f o r
p u b l i c a t i o n 2 3
O c t o b e r 1 9 7 3
T h e
a n t i b i o t i c
f o s f o m y c i n
w a s p r o d u c e d
a s a s e c o n d a r y
m e t a b o l i t e i n
a
g l u c o s e - a s p a r a g i n e
medium c o n t a i n i n g
c i t r a t e , L - m e t h i o n i n e , a n d
L - g l u t a m a t e .
T h e
c i t r a t e
r e q u i r e m e n t
f o r
a n t i b i o t i c
s y n t h e s i s w a s r e l a t e d t o
i t s r e q u i r e m e n t
f o r
g r o w t h . I n
c o n t r a s t ,
L - m e t h i o n i n e
a n d
L - g l u t a m a t e
c a u s e d
a
m a r k e d
s t i m u l a t i o n
o f
f o s f o m y c i n p r o d u c t i o n a n d
h a d
n o
e f f e c t
o n g r o w t h . L - M e t h i o n i n e h a d t o
b e
a d d e d
e a r l y
t o
e f f e c t m a x i m a l
a n t i b i o t i c
s y n t h e s i s
l a t e r
i n t h e
f e r m e n t a t i o n .
The
L - g l u t a m a t e r e q u i r e m e n t
w a s n o t
s p e c i f i c , s i n c e
s e v e r a l
t r i c a r b o x y l i c
a c i d
c y c l e
i n t e r m e d i a t e s
c o u l d
r e p l a c e t h i s a m i n o a c i d .
L - A s p a r a g i n e w a s
t h e m o s t e f f e c t i v e
n i t r o g e n s o u r c e
f o r
g r o w t h
a n d
p r o d u c t i o n
o f
f o s f o m y c i n . G l y c i n e ,
a n
a l t e r n a t e
n i t r o g e n
s o u r c e ,
s u p p o r t e d f o s f o m y c i n
s y n t h e s i s
o n l y w h e n a d d e d
i n
e x c e s s o f
t h a t n e e d e d f o r
g r o w t h . C o b a l t a n d
i n o r g a n i c
p h o s p h a t e
w e r e
r e q u i r e d
a l s o
f o r
a n t i b i o t i c
p r o d u c t i o n a t
c o nc e n t r a t i o n s e x c e e d i n g
t h o s e s u p p o r t i n g
m a x i m a l
g r o w t h .
R a d i o a c t i v e
i n c o r p o r a t i o n
s t u d i e s s h o w e d
t h a t t h e
m e t h y l c a r b o n
o f
m e t h i o n i n e w a s
t h e
p r e c u r s o r
o f t h e
m e t h y l
o f
f o s f o m y c i n .
C a r b o n 1 o f
f o s f o m y c i n
w a s
d e r i v e d f r o m
g l u c o s e c a r b o n s
1 a n d 6 ,
w h e r e a s
g l u c o s e - 2 -
1 4 C l a b e l e d
f o s f o m y c i n c a r b o n 2 .
R a d i o a c t i v i t y
f r o m
a c e t a t e - 2 - 1 4 C w a s
d i s t r i b u t e d
e q u a l l y
b e t w e e n
f o s f o m y c i n
c a r b o n s 1
a n d
2 .
No
i n c o r p o r a t i o n o f
a c e t a t e - l - 4 C ,
a s p a r a -
g i n e - U - 4 C ,
c i t r a t e - 1 ,
5 - 1 4 C ,
o r
g l u t a m a t e - U - 1 4 C
o c c u r r e d .
T h e
l a b e l i n g
p a t t e r n o f
f o s f o m y c i n
c a r b o n s
1
a n d 2
w a s
s i m i l a r
t o
t h a t f o u n d i n
2 - a m i n o e t h y l p h o s p h o -
n a t e
f r o m
T e t r a h y m e n a .
I n 1 9 6 9 ,
H e n d l i n e t
a l .
( 9 ) r e p o r t e d t h e
d i s c o v e r y
o f a
new c e l l
w a l l - a c t i v e
a n t i b i o t i c ,
p h o s p h o n o m y c i n .
T h e
c o m p o u n d
w a s
i d e n t i f i e d
a s
( - ) - ( l R ,
2 S )
- 1 ,
2 - e p o x y p r o p y l p h o s p h o n i c
a c i d
( 3 ) ,
a n d h a s
s i n c e
b e e n
r e n a m e d
f o s -
f o m y c i n .
T h e
a n t i b i o t i c h a s a
u n i q u e
s t r u c t u r e
( F i g .
1 )
c o n t a i n i n g
c h e m i c a l
g r o u p s
n o t f r e -
q u e n t l y
e n c o u n t e r e d
i n
a n t i m i c r o b i a l
s u b -
s t a n c e s ,
n a m e l y ,
e p o x i d e
a n d
p h o s p h o n a t e
g r o u p s . F o s f o m y c i n
i s
p r o d u c e d
b y
s e v e r a l
S t r e p t o m y c e s s p e c i e s
( 9 , 2 6 ) .
P h o s p h o n a t e s
h a v e b e e n
d e t e c t e d
i n
a
v a r i e t y
o f
t i s s u e s
( 2 1 ) ;
h o w e v e r ,
b e f o r e
t h e
d i s c o v e r y
o f
f o s f o m y c i n ,
t h e i r
e x i s t e n c e
i n
b a c t e r i a a n d r e l a t e d
m i c r o o r -
g a n i s m s h a d b e e n d o u b t f u l
( 2 1 ) .
J a c k s o n
a n d
S t a p l e y
( 1 4 )
i n v e s t i g a t e d
t h e
n u t r i t i o n
o f
S t r e p t o m y c e s
f r a d i a e
i n
c o m p l e x
a n d
s e m i s y n t h e t i c
m e d i a a n d d e f i n e d s e v e r a l
r e q u i r e m e n t s
f o r
f o s f o m y c i n p r o d u c t i o n .
T h e y
f o u n d
t h a t ,
i n t h e a b s e n c e
o f t h e
c r i t i c a l
n u t r i -
l i t e s ,
a
l o w
l e v e l o f
a n t i b i o t i c
was
p r o d u c e d .
T h i s
w a s
p r o b a b l y
d u e
t o t h e
p r e s e n c e ,
i n
t h e i r
m e d i a ,
o f l o w
c o n c e n t r a t i o n s
o f
c o m p o u n d s
e s s e n t i a l
f o r
f o s f o m y c i n s y n t h e s i s .
T h e r e f o r e ,
t h e s e
s t u d i e s
h a v e
n ow
b e e n
e x p a n d e d
b y u s i n g
a
c o m p l e t e l y
s y n t h e t i c
medium
a n d w a s h e d - c e l l
i n o c u l a . A
t e c h n i q u e
w a s
d e v e l o p e d
f o r
t h e
i s o l a t i o n
o f
f o s f o m y c i n
f r o m f e r m e n t a t i o n
b r o t h
w h i c h
e n a b l e d u s t o m e a s u r e
t h e
i n c o r p o r a t i o n
o f
r a d i o a c t i v e
c o m p o u n d s
i n t o
t h e a n t i b i o t i c .
T h e s e s t u d i e s
h a v e a l l o w e d u s
t o d e f i n e
t h e
p h y s i o l o g i c a l
r o l e
o f
s e v e r a l
c o m p o u n d s
i n
f o s -
f o m y c i n s y n t h e s i s .
MATERIALS AND
METHODS
O r g a n i s m
a n d
m e d i a . T h e
c u l t u r e
u s e d
w a s
M A - 2 9 1 3
(NRRL
B 3 3 6 0 )
w h i c h h a s b e e n
i d e n t i f i e d
a s
S .
f r a d i a e
( 2 6 ) .
T h e
o r g a n i s m
w a s
p r e s e r v e d
i n
t h e
l y o p h i l i z e d
s t a t e
a n d
m a i n t a i n e d o n
c o r n s t a r c h -
a s p a r a g i n e
a g a r
s l a n t s
( 1 4 ) .
F o s f o m y c i n
p r o d u c t i o n
m e d i u m
c o n t a i n e d
( p e r l i t e r ) :
g l u c o s e
( 2 0
g ) ;
L -
a s p a r a g i n e ( 5 g ;
D i f c o ) ;
m o n o s o d i u m
g l u t a m a t e ( 1
g ;
M e r c k ) ;
L - m e t h i o n i n e
( 0 . 1 5 g ) ;
s o d i u m
c i t r a t e
d i h y -
d r a t e
( 4
g ) ;
K,HPO4
( 1
g ) ;
C a C l 2 . 2 H 2 0
( 0 . 5
g ) ;
M g S O 4 * H 2 0
( 0 . 2 g ) ; C o C l 2
. 6 H 2 0 ( 0 . 1
g ) ;
F e S O 4
. 7 H 2 O
( 1 0
m g ) ;
M n S O 4 - 1 H 2 O
( 1 0
m g ) ;
C u C l 2
2 H 2 0
( 2 . 5
M g ) ;
H S B O S
( 5 . 6
M g ) ;
Z n S O 4
7 H 2 O
( 2
M g ) ;
a n d
( N H 4 ) , M o 7 0 2 4 - 4 H 2 0
( 1 . 9
g g ) .
T h e
pH
w a s
7 . 0 .
G l u -
c o s e
w a s s t e r i l i z e d
s e p a r a t e l y .
S e e d medium
h a d t h e
s a m e
c o m p o s i t i o n ,
e x c e p t
t h a t C l i n t o n
c o r n s t a r c h
r e p l a c e d
g l u c o s e .
F o s f o m y c i n
p r o d u c t i o n .
F e r m e n t a t i o n
s t u d i e s
w e r e
c a r r i e d
o u t
i n 2 5 0 - m l
E r l e n m e y e r
f l a s k s
i n c u -
1 2 1
onA
pril11
,2
01
6b
yguest
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ac.a
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http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/ -
7/26/2019 Biosintesis de Fosfomicina
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1 2 2
ROGERS AND
BIRNBAUM
H H
CH3 f
P 0 3 H 2
F I G .
1 . S t r u c t u r e o f
f o s f o m y c i n .
b a t e d a t
2 8 C
o n a
r e c i p r o c a t i n g
s h a k e r
( 2 5 0
r p m ) .
B a f f l e d f l a s k s ,
c o n t a i n i n g
4 0 m l o f
s e e d
m e d i u m ,
were
i n o c u l a t e d f r o m
agar
s l a n t s a n d
i n c u b a t e d
f o r
4 8 t o
7 2
h . C e l l s w e r e
h a r v e s t e d
b y
c e n t r i f u g a t i o n
( 1 2 , 0 0 0
x
g ) ,
w a s h e d t h r e e t i m e s w i t h
s t e r i l e
D a v i s
s a l t s
( 6 ) ,
a n d
s u s p e n d e d
i n D a v i s
s a l t s t o
t h e
o r i g i n a l
h a r v e s t
v o l u m e .
U n l e s s o t h e r w i s e
i n d i c a t e d ,
u n b a f f l e d
f l a s k s
c o n t a i n i n g
3 0 m l o f
p r o d u c t i o n
m e d i u m
w ere
i n o c u -
l a t e d
w i t h
1
m l
o f
t h e w a s h e d - c e l l
s u s p e n s i o n
a n d
i n c u b a t e d a t
2 8
C . A t
a p p r o p r i a t e
t i m e s ,
c e l l s
were
h a r v e s t e d b y
c e n t r i f u g a t i o n
a n d
t h e
s u p e r n a t a n t
f l u i d
was
a s s a y e d
f o r
f o s f o m y c i n .
P r e p a r a t i o n
o f
p h o s p h a t e - s t a r v e d
i n o c u l u m .
C e l l s
g r ow n
f o r
7 2 h
i n
s e e d m e d i u m
were
h a r v e s t e d
b y
c e n t r i f u g a t i o n ,
w a s h e d
t h r e e t i m e s w i t h
s t e r i l e
0 . 8 %
N a C l ,
a n d
s u s p e n d e d
i n
4 0 ml o f
p h o s p h a t e - f r e e
p r o d u c t i o n
m e d i u m . T h e s e c e l l s
w ere
s h a k e n
f o r
1 6
h
a t
2 8
C ,
h a r v e s t e d ,
washed
t h r e e
t i m e s
w i t h
0 . 8 %
N a C l , a n d
s u s p e n d e d
i n
4 0 ml o f
0 . 8 % N a C l .
T h i s
s u s p e n s i o n
was
u s e d
as i n o c u l u m
i n
s t u d i e s
o f
t h e
e f f e c t
o f
p h o s p h a t e
o n
g r o w t h
and
f o s f o m y c i n s y n t h e -
s i s .
C e l l
d r y
w e i g h t
d e t e r m i n a t i o n s .
C e l l s
( f r o m
1 t o
5
m l
o f
b r o t h )
w e r e
c o l l e c t e d
on
t a r e d membrane
f i l t e r s
( 0 . 4 5 jm
pore
s i z e ;
M i l l i p o r e
C o r p . ) ,
washed
w i t h
D a v i s
s a l t s ,
d r i e d
o v e r n i g h t
a t 9 0
C ,
and
w e i g h e d .
G r o w t h
was
e x p r e s s e d
as
m i l l i g r a m s
o f
d r y
c e l l s
p e r
m i l l i t e r
o f
b r o t h .
A s s a y s .
F o s f o m y c i n
was
q u a n t i t a t e d
m i c r o b i o l o g i -
c a l l y
a s
d e s c r i b e d
b y
S t a p l e y
e t
a l .
( 2 6 ) .
G l u c o s e w a s
d e t e r m i n e d
b y
t h e
G l u c o s t a t method
( W o r t h i n g t o n
B i o c h e m i c a l
C o r p . ,
F r e e h o l d ,
N . J . ) .
R a d i o a c t i v i t y
measurements.
R a d i o a c t i v i t y
w a s
m e a s u r e d
i n
a
P a c k a r d T r i - C a r b
s c i n t i l l a t i o n
s p e c t r o -
p h o t o m e t e r . The s c i n t i l l a t i o n
f l u i d
c o n t a i n e d
( p e r
l i t e r ) :
1 ,
4 - b i s - 2 - ( 5 - p h e n y l o x a z o l y l ) - b e n z e n e
( 0 . 3 7 5
g ) ;
2 , 5 - d i p h e n y l o x a z o l e
( 5 g ) ;
t o l u e n e
( 6 8 8
m l ) ;
a n d
T r i t o n
X - 1 0 0
( 3 1 2
m l ) . A q u e o u s
s o l u t i o n s o f
1 . 0 ml
or
l e s s
were
a d d e d t o s t a n d a r d
c o u n t i n g
v i a l s
f o l l o w e d
b y
1 0 m l
o f
s c i n t i l l a t i o n
s o l u t i o n . When l e s s
t h a n 1 . 0 m l
o f
s o l u t i o n was t o
b e
c o u n t e d ,
s u f f i c i e n t
w a t e r was
a d d e d t o
t h e
v i a l t o
g i v e
a
f i n a l
aqueo u s
v o l u m e
o f
1 . 0
m l . I n
c h e m i c a l
d e g r a d a t i o n
s t u d i e s ,
t h e
r a d i o a c t i v i t y
o f
CO2
was
d e t e r m i n e d i n
a
s c i n t i l l a t i o n
f l u i d c o n s i s t -
i n g
o f
t o l u e n e :
m e t h y l
c e l l o s o l v e : e t h a n o l
( 1 1 0 : 8 8 :
1 3 )
c o n t a i n i n g
5 . 5
g
o f
2 , 5 - d i p h e n y l o x a z o l e
per
l i t e r
o f
s o l u t i o n
( 1 6 ) .
I s o l a t i o n
o f
r a d i o a c t i v e
f o s f o m y c i n b y
co-crys-
t a l l i z a t i o n .
C e l l s
g r ow n
i n
t h e
presence
o f
r a d i o a c t i v e
c o m p o u n d s w e r e
h a r v e s t e d f r o m
f e r m e n t a t i o n
b r o t h
b y c e n t r i f u g a t i o n
( 1 2 , 0 0 0
x
g )
o r
b y
f i l t r a t i o n
t h r o u g h
a
N a l g e n e
f i l t e r
( 0 . 2 jm p o r e
s i z e ) . T h e
f o s f o m y c i n
c o n t e n t o f
t h e
c e l l - f r e e b r o t h
w a s
d e t e r m i n e d
b y
b i o a s s a y .
T h r e e
m i l l i l i t e r s
o f
b r o t h
w a s
m i x e d
w i t h
1 2
m l
o f
i c e - c o l d
2 - p r o p a n o l
a n d
p l a c e d
i n i c e f o r
3 0
m i n .
I n s o l u b l e
m a t e r i a l
w a s r e m o v e d
b y
f i l t r a t i o n
t h r o u g h
a
m e m b r a n e
f i l t e r
( 0 . 4 5
gm
p o r e s i z e ;
M i l l i p o r e
C o r p . ) .
T e n
m i l l i l i t e r s o f
f i l t r a t e
w a s
t r a n s f e r r e d t o
a
C o r e x
c e n t r i f u g e
t u b e
( C o m i n g
G l a s s
C o r p . ) ,
a n d
6 0 0
mg
o f
+ - a - p h e n e t h y l a m m o n i u m
f o s f o m y c i n
m o n o h y -
d r a t e
( P E A - f o s f o m y c i n )
w a s
a d d e d .
T h e
s o l u t i o n
w a s
h e a t e d
a t
7 0
C
t o
d i s s o l v e
t h e c r y s t a l s ,
c o o l e d t o
r o o m
t e m p e r a t u r e ,
a n d
p l a c e d
i n
i c e f o r 1
h .
T h e
r e s u l t i n g
c r y s t a l s
w e r e
h a r v e s t e d
b y c e n t r i f u g a t i o n
a t
4
C ,
w a s h e d
o n c e w i t h
i c e - c o l d
9 0 %
2 - p r o p a n o l ,
a n d
s u s -
p e n d e d
i n 8 0 %
2 - p r o p a n o l .
Two
a d d i t i o n a l
r e c r y s t a l l i -
z a t i o n s
w e r e
d o n e
s i m i l a r l y .
F i n a l l y ,
t h e c r y s t a l s
w e r e
c o l l e c t e d
o n a
m e m b r a n e
f i l t e r
( 0 . 4 5
u i m p o r e
s i z e ;
M i l l i p o r e
C o r p . ) ,
w a s h e d w i t h
i c e - c o l d
8 0 %
2 - p r o p a -
n o l ,
a n d
d r i e d f o r
1 6 h
a t
r o o m
t e m p e r a t u r e
i n
a
vacuum
d e s i c c a t o r .
T h e r a d i o a c t i v i t y
o f
t h e
c r y s t a l s
w a s
u s e d
t o
c a l c u l a t e
t h e s p e c i f i c
a c t i v i t y
o f
t h e
f o s f o m y c i n
s y n t h e s i z e d i n
f e r m e n t a t i o n
m e d i u m .
A f t e r
t h r e e
r e c r y s t a l l i z a t i o n s ,
t h e i s o l a t e d
f o s f o m y c i n
w a s
a s s u m e d t o
b e r a d i o c h e m i c a l l y
p u r e , s i n c e
t h e
s p e c i f i c r a d i o a c t i v i t y
o f
t h e
c r y s t a l s
r e m a i n e d
c o n -
s t a n t
u p o n
a d d i t i o n a l
r e c r y s t a l l i z a t i o n .
T h i n - l a y e r
c h r o m a t o g r a p h y
( 2 3 )
o f
t h e
i s o l a t e d
f o s f o m y c i n
r e -
v e a l e d n o
c o n t a m i n a t i o n
w i t h
s t r u c t u r a l l y
r e l a t e d
p h o s p h o n a t e s
o r
o r g a n i c
p h o s p h a t e s .
When
g l u c o s e -
U - 4 C
o r
m e t h y l - l a b e l e d
[ 4 C
] m e t h i o n i n e
( p r e c u r s o r s
o f
f o s f o m y c i n )
o r
H - l a b e l e d
1 ,
2 - d i h y d r o x y p r o p y l -
p h o s p h o n i c
a c i d
( d i o l
o f
f o s f o m y c i n ) w a s a d d e d
t o
f e r m e n t a t i o n
b r o t h
b e f o r e
c o - c r y s t a l l i z a t i o n , n o
r a d i o -
a c t i v i t y w a s
d e t e c t e d i n
t h e f i n a l
c r y s t a l s ,
t h e r e b y
s h o w i n g
t h e
s p e c i f i c i t y
o f
t h e
c o - c r y s t a l l i z a t i o n
p r o c e -
d u r e .
F o r
t h e
i s o l a t i o n o f
f o s f o m y c i n o f
l o w
r a d i o a c t i v i t y ,
c o l u m n
c h r o m a t o g r a p h y
w a s
u s e d t o
c o n c e n t r a t e
t h e
a n t i b i o t i c
b e f o r e t h e c o - c r y s t a l l i z a t i o n
s t e p .
T h e
pH
o f
c e l l - f r e e
f e r m e n t a t i o n
b r o t h w a s
a d j u s t e d t o
7
w i t h
8 5 %
p h o s p h o r i c a c i d .
A
s a m p l e
( 2 0
t o
4 0
m l )
w a s
a p p l i e d t o a
c o l u m n ( 0 . 9
b y 2 5
c m )
c o n t a i n i n g
D o w e x
1 - X 2
( c h l o r i d e
c y c l e )
r e s i n .
T h e c o l u m n
w a s
w a s h e d
w i t h w a t e r
a n d e l u t e d
w i t h
0 . 3 M
N a C l .
F r a c t i o n s
w e r e a s s a y e d
q u a l i t a t i v e l y
f o r
f o s f o m y c i n b y
u s i n g
0 . 2 5 - i n c h
( a b o u t
0 . 6
c m ) p a p e r d i s k s o n
a g a r medium
s e e d e d
w i t h
P r o t e u s
v u l g a r i s
( M B - 8 3 8 ) .
B i o a c t i v e
f r a c t i o n s
w e r e
c o m b i n e d ,
c o n c e n t r a t e d u n d e r
r e d u c e d
p r e s s u r e , a n d
d e s a l t e d
o n
a
B i o - G e l
P - 2
( 2 0 0
t o
4 0 0
m e s h )
c o l u m n .
T h e
c o l u m n w a s
e l u t e d
w i t h
e i t h e r
w a t e r o r 1 %
b u t a n o r .
B i o a c t i v e
f r a c t i o n s w e r e
c o m -
b i n e d a n d
c o n c e n t r a t e d .
F o s f o m y c i n
w a s
i s o l a t e d
f r o m t h e
c o n c e n t r a t e d f r a c t i o n s b y
c o - c r y s t a l l i z a t i o n
w i t h
P E A - f o s f o m y c i n a s
d e s c r i b e d
a b o v e .
C h e m i c a l
d e g r a d a t i o n
o f
r a d i o a c t i v e
f o s f o m y c i n .
T o t a l
c a r b o n
o x i d a t i o n s w e r e
c a r r i e d
o u t
b y
t h e
Van
S l y k e - F o l c h
m e t h o d
( 3 0 ) . T h e
c o m b u s t i o n
f l u i d w a s
m o d i f i e d i n
t h a t
t h e
C r O 0
c o n c e n t r a t i o n w a s
i n -
c r e a s e d
t o
1 0 0 g p e r
l i t e r .
F o r
e a c h
d e t e r m i n a t i o n ,
0 . 5
mmol
o f
s a m p l e
w a s
o x i d i z e d
a n d
t h e
r e s u l t i n g CO2
w a s
c o l l e c t e d i n
C O 2 - f r e e
2
N
NaOH.
To i s o l a t e
c a r b o n s
2
a n d 3
o f
f o s f o m y c i n ,
t h e
a n t i b i o t i c
w a s o x i d i z e d
i n i t i a l l y t o
a c e t i c
a c i d .
Two
A N T i M I C R O B .
A G .
C H E M O T H E R .
onA
pril11
,2
01
6b
yguest
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ac.a
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7/26/2019 Biosintesis de Fosfomicina
3/12
BIOSYNTHESIS
OF FOSFOMYCIN
BY S .
FRADIAE
1 2 3
m i l l i m o l e s
o f
[ 1 4 C ] P E A - f o s f o m y c i n
w e r e
i n c u b a t e d a t
r o o m t e m p e r a t u r e
f o r
1 5 h i n
5 0
m l o f
a
1 N
C r O ,
s o l u t i o n
i n
a
g r o u n d
g l a s s - s t o p p e r e d
f l a s k . T h e r e a c -
t i o n
m i x t u r e
w a s s t e a m d i s t i l l e d ,
a n d
2 5 0
m l o f
d i s t i l l a t e
w a s c o l l e c t e d a n d t i t r a t e d t o
pH
8 . 5
w i t h
C O 2 - f r e e
0 . 1
N
NaOH.
T h e
d i s t i l l a t e
w a s
c o n c e n -
t r a t e d
t o d r y n e s s u n d e r r e d u c e d
p r e s s u r e ,
s u s p e n d e d
i n
w a t e r ,
d e c o l o r i z e d
w i t h
c h a r c o a l , a n d
e v a p o r a t e d t o
d r y n e s s o v e r
a n
i n f r a r e d l a m p .
T h e r e s u l t i n g w h i t e
p o w d e r w a s d e h y d r a t e d
a t 1 2 0 C a n d c o o l e d t o r o o m
t e m p e r a t u r e
i n
a v a c u u m
d e s i c c a t o r .
T h e i s o l a t e d
m a t e r i a l
w a s i d e n t i f i e d
a s
s o d i u m a c e t a t e
b y m i c r o a -
n a l y s i s . A c e t at e o b t a i n e d
i n
t h i s
m a n n e r w a s d e -
g r a d e d t o i t s r e s p e c t i v e c a r b o n a t o ms .
C a r b o n 1 o f
a c e t a t e ( r e p r e s e n t s c a r b o n
2 o f f o s f o m y c i n ) w a s
o b -
t a i n e d
b y
t h e S c h m i d t
r e a c t i o n ( 3 4 ) a s d e s c r i b e d
b y
P h a r e s ( 2 0 ) .
O n e
m i l l i m o l e
o f s o d i u m a c e t a t e
w a s
d e g r a d e d ,
a n d t h e
r e s u l t i n g
C O ,
w a s
t r a p p e d
i n
C O , - f r e e
2
N
NaOH.
T h e m e t h y l a m i n e
w h i c h r e -
m a i n e d
i n
t h e
r e a c t i o n
m i x t u r e
( r e p r e s e n t s
f o s -
f o m y c i n c a r b o n 3 )
w a s r e c o v e r e d
b y d i s t i l l a t i o n i n t o
0 . 4 N
H 2 S O 4 . T h i s s o l u t i o n
w a s e v a p o r a t e d
t o
0 . 2 m l
b y
u s i n g
a n i n f r a r e d
l a m p ,
a n d t h e
m e t h y l a m i n e w a s
o x i d i z e d t o C O 2 b y t h e V a n S l y k e - F o l c h p r o c e d u r e
( 3 0 ) .
T h e C O 2 r e s u l t i n g f r o m
t h e v a r i o u s
o x i d a t i o n s
w a s
i s o l a t e d
f r o m t h e NaOH s a m p l e s , a n d
i t s r a d i o a c t i v -
i t y w a s
d e t e r m i n e d b y
t h e
p r o c e d u r e
o f
K o m b l a t t
e t
a l . ( 1 6 ) . R a d i o a c t i v i t y i n
f o s f o m y c i n
c a r b o n
1 w a s
d e t e r m i n e d i n d i r e c t l y
b y s u b t r ac t i n g t h e sum o f t h e
r a d i o a c t i v i t y
i n c a r b o n s
2 a n d 3 f r o m t h e t o t a l
r a d i o a c t i v i t y i n
t h e a n t i b i o t i c .
C h e m i c a l s .
R a d i o c h e m i c a l s
w e r e
p u r c h a s e d
f r o m
Ne w
E n g l a n d
N u c l e a r
C o r p . ,
B o s t o n ,
M a s s . P h o s -
p h o n a t e s
w e r e
p u r c h a s e d f r o m C a l b i o c h e m ,
L o s
A n -
g e l e s ,
C a l i f .
F o s f o m y c i n
a n d P E A - f o s f o m y c i n w e r e
s y n t h e s i z e d
i n
t h e
P r o c e s s R e s e a r c h
D e p a r t m e n t o f
M e r c h & C o . , I n c . , R a h w a y , N . J . T h e
1 , 2 - d i h y d r o x y -
p r o p y l p h o s p h o n i c
a c i d
w a s p r e p a r e d
b y
a c i d h y d r o l y -
s i s
o f f o s f o m y c i n
a t
3 7
C ,
pH
1 , f o r
2 4
h .
RESULTS
C a r b o n
s o u r c e s
f o r
g r o w t h
a n d
f o s f o m y c i n
s y n t h e s i s .
A n u m b e r
o f
c o m p o u n d s
w e r e
t e s t e d
a s
c a r b o n
s o u r c e s
f o r
g r o w t h
a n d
f o s f o m y c i n
p r o d u c t i o n
i n
s y n t h e t i c
m e d i u m . O f
5 7
m a t e -
r i a l s
t e s t e d ,
c o r n s t a r c h , g l u c o s e ,
a n d
g l y c e r o l
w e r e t h e b e s t
f o r
s u p p o r t i n g
g r o w t h
a n d
a n t i b i -
o t i c
s y n t h e s i s .
The
o p t i m a l
c o n c e n t r a t i o n s
f o r
f o s f o m y c i n p r o d u c t i o n
w e r e
2 f o r
g l u c o s e
a n d
4 f o r
g l y c e r o l .
H i g h e r
l e v e l s
s u p p o r t e d
i n -
c r e a s e d
g r o w t h
b u t h a d l i t t l e e f f e c t
o n
f o s -
f o m y c i n
s y n t h e s i s .
G l u c o s e
was
c h o s e n
a s t h e
c a r b o n
s o u r c e
f o r
s t u d i e s i n
s y n t h e t i c
m e d i u m .
Growth
a n d
f o s f o m y c i n
p r o d u c t i o n
i n
s y n -
t h e t i c
m e d i u m .
F i g u r e
2
s h o w s t h e
t i m e c o u r s e
o f
g r o w t h
a n d
f o s f o m y c i n
p r o d u c t i o n b y
S .
f r a d i a e
i n
s y n t h e t i c
m e d i u m .
P r o d u c t i o n
f l a s k s
w e r e i n o c u l a t e d
t o
g i v e
a
c e l l
c o n c e n t r a t i o n
o f
0 . 0 9
m g
o f
d r y
c e l l s
p e r
m l
( F i g .
2 A )
o r 0 . 2
m g
o f
d r y
c e l l s
p e r
m l
( F i g .
2 B ) .
W i t h t h e s m a l l e r
i n o c u l u m , t h e r e w a s a n
i n i t i a l l a g o f 1 8
t o 2 4 h
f o l l o w e d b y
a
p h a s e o f
r a p i d
g r o w t h
a n d
s u g a r
u t i l i z a t i o n .
T h e g r o w t h
l a g
c o u l d
b e e l i m i n a t e d
b y
i n c r e a s i n g
t h e
i n o c u l u m s i z e ( F i g . 2 B ) . I n
e i t h e r
c a s e ,
f o s f o m y c i n
s y n t h e s i s
w a s i n i t i a t e d
a f t e r
a b o u t 80
o f
m a x i m a l
g r o w t h
w a s
r e a c h e d ,
a n d
m o s t
a n t i b i o t i c
s y n t h e s i s o c c u r r e d
a f t e r
t h e
r a p i d g r o w t h p h a s e . T h u s ,
i n
a n a l o g y
t o
o t h e r
s e c o n d a r y
m e t a b o l i t e s , f o s f o m y c i n
w a s
s y n t h e s i z e d d u r i n g
t h e
i d i o p h a s e
o f
g r o w t h .
T h e
d e c r e a s e
i n
c e l l d r y w e i g h t
a f t e r m a x i m a l
g r o w t h
i n d i c a t e s
t h a t t h e
c u l t u r e
u n d e r w e n t
l y s i s
i n t h e l a t e r s t a g e s
o f t h e
f e r m e n t a t i o n .
T h e
l a r g e r
i n o c u l u m
o f 0 . 2
m g
o f
d r y
c e l l s
p e r
m l
( 1
m l
o f a w a s h e d - c e l l
s u s p e n s i o n
a s d e s c r i b e d i n
M a t e r i a l s
a n d
M e t h o d s )
w a s
a d o p t e d
a s
s t a n -
d a r d
i n
a l l
e x p e r i m e n t s .
I n o r g a n i c
n u t r i t i o n a l
r e q u i r e m e n t s .
J a c k -
s o n a n d
S t a p l e y
( 1 4 ) f o u n d t h a t
i r o n
w a s
r e q u i r e d
f o r f o s f o m y c i n
s y n t h e s i s
a n d t h a t
c o -
b a l t s t i m u l a t e d a n t i b i o t i c
p r o d u c t i o n
i n
s e m i -
s y n t h e t i c
m e d i u m .
T h e e f f e c t
o f t h e s e
s u b -
s t a n c e s
o n
g r o w t h
a n d f o s f o m y c i n p r o d u c t i o n
i n
s y n t h e t i c
medium w a s e v a l u a t e d
( F i g .
3 - 5 ) .
T h e r e
w a s
a
m a r k e d
d i f f e r e n c e
i n t h e
r e s p o n s e s
t o
i n c r e a s i n g
l e v e l s
o f t h e
t w o c a t i o n s .
G r o w t h
w a s s t i m u l a t e d m a x i m a l l y b y
t h e a d d i t i o n
o f
2 0
1 6
E
1 2
E
8
e n
8
4
CULTURE
AGE
( N H R )
F I G .
2 .
G r o w t h , f o s f o m y c i n
p r o d u c t i o n ,
a n d
s u g a r
u t i l i z a t i o n
i n
s y n t h e t i c
m e d i u m .
( A )
P r o d u c t i o n
f l a s k s
w e r e
i n o c u l a t e d
w i t h
0 . 5
m l
o f
a
w a s h e d c e l l
s u s p e n s i o n
t o
g i v e
0 . 0 9
mg
o f
d r y
c e l l s
p e r
m l ,
a n d
( B )
p r o d u c t i o n f l a s k s
w e r e
i n o c u l a t e d
w i t h
1 m l
o f
t h e
c e l l
s u s p e n s i o n
t o
g i v e
0 . 2
m g
o f
d r y
c e l l s
p e r
m l .
A t
t h e
d e s i g n a t e d i n t e r v a l s ,
d u p l i c a t e
f l a s k s
were
a n a l y z e d
f o r g r o w t h
( 0 )
a n d
f o s f o m y c i n
( A ) .
I n
( A ) ,
f l a s k s
were
a n a l y z e d
a l s o
f o r
g l u c o s e
( A ) .
V O L .
5 ,
1 9 7 4
onA
pril11
,2
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yguest
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ac.a
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adedfrom
http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/ -
7/26/2019 Biosintesis de Fosfomicina
4/12
1 2 4 ROGERS
AND
BIRNBAUM
.
A
r o n
p e r m l ,
w h e r e a s
s l i g h t l y
more
i r o n
was
1 6
r e q u i r e d
f o r
m a x i m a l
f o s f o m y c i n
s y n t h e s i s
( 0 . 0 3
\
i s m o l / m l ) .
T h e s e r e s u l t s
s u g g e s t
t h a t c o b a l t
h a s
1 4
_A
a
d i r e c t r o l e
i n
a n t i b i o t i c
p r o d u c t i o n
a s i d e
f r o m
i t s
f u n c t i o n
i n
g r o w t h ,
w h e r e a s
t h e
r o l e
o f
i r o n
1 2
A
may b e i n d i r e c t a n d c o r r e l a t e d w i t h i t s
s t i m u l a -
E
/ \
t i o n o f
g r o w t h .
l o
A
o
\
T h e
e f f e c t
o f
p h o s p h a t e
on
f o s f o m y c i n s y n t h e -
8 s i s w a s s t u d i e d
b y
u s i n g n o r m a l a n d
p h o s p h a t e -
8
_
8
E ~
-
7/26/2019 Biosintesis de Fosfomicina
5/12
BIOSYNTHESIS
OF FOSFOMYCIN
BY
S .
FRADIAE
1 2 5
I _ -
_3
01
,
2
2
3
4
5 6 7 8
9 1 0 1 1
~ ~ ~ ~ ~ ~ ~ 0
Symol a r e a s f o r
Fig3
5
5
4 l
f
tlzn
naklhshncaisa
3
3
0
0
2
3
4
5
6 7 8 9
10
I
I
K2HPO4
CONCENTRATION
( p a
MOLES
/ l n )
FIG. 6.
Effect
o f
p h o s p h a t e
on g r o w t h
and
fos-
f o m y c i n
s y n t h e s i s by:
A
phosphate-starved
c e l l s
and
B
n o n s t a r v e d c e l l s .
Cells
were
starved
fo r
p h o s p h a t e
as
described
in Materials
and Methods Growth
and
fosfomycin
weremeasured
after
72
h
o f
incubation.
Symbols
areas
fo r F ig .
3.
ble
of
u t i l i z i n g aminoalkylphosphonic
acids
as
phosphorus
sources for
growth
( 8 ,
22).
Three
aminoalkylphosphonic
acids
2-amino-3-phos-
phonopropionic a c i d ,
1 - a m i n o e t h y l p h o s p h o n i c
a c i d ,
a n d
2 - a m i n o e t h y l p h o s p h o n i c
a c i d )
w e r e
i n a c t i v e
a s
p h o s p h o r u s
sources
f o r
g r o w t h
o f
S .
f r a d i a e .
F u r t h e r m o r e ,
t h e s e
c o m p o u n d s
w e r e
w i t h o u t
e f f e c t
on
f o s f o m y c i n
s y n t h e s i s
when
a d d e d
t o m e d i u m
c o n t a i n i n g
3
, m o l
o f
K2HPO,
per m l ,
a
s u b o p t i m a l
l e v e l
o f
i n o r g an i c p h o s -
p h a t e
( s e e
F i g .
6 B ) .
I t
may
b e
t h a t
S .
f r a d i a e
i s
u n a b l e
t o
t r a n s p o r t
p h o s p h o n i c
a c i d s ,
a l t h o u g h
s e v e r a l
b a c t e r i a
p o s s e s s
a c t i v e
t r a n s p o r t s y s -
tems
f o r t h e
u p t a k e
o f
a m i n o a l k y l p h o s p h o n a t e s
( 1 0 ,
2 2 ) .
O r g a n i c
n u t r i t i o n a l
r e q u i r e m e n t s . An
e v a l -
u a t i o n
o f t h e
o r g a n i c
n u t r i t i o n a l
r e q u i r e m e n t s
f o r f o s f o m y c i n
s y n t h e s i s
w a s
made b y
d e l e t i n g
v a r i o u s c o m p o n e n t s
f r o m
t h e
s y n t h e t i c
p r o d u c -
t i o n
medium
( T a b l e
1 ) .
R e m o v a l
o f
L - a s p a r a -
g i n e p r e v e n t e d
b o t h g r o w t h a n d
f o s f o m y c i n
s y n -
t h e s i s , s u g g e s t i n g t h a t t h i s
c o m p o u n d
s e r v e s
a s
a
n i t r o g e n s o u r c e
f o r g r o w t h .
D e l e t i o n o f c i t r a t e
c a u s e d a r e d u c t i o n
i n g r o w t h
a n d
a n t i b i o t i c
p r o d u c t i o n
w h i c h may
b e
r e l a t e d
t o
pH
s i n c e ,
i n
t h e a b s e n c e o f
c i t r a t e , t h e
pH o f t h e
f e r m e n -
t a t i o n
b r o t h w a s
l o w . R e m o v a l
o f
m e t h i o n i n e
o r
g l u t a m a t e h a d
n o
e f f e c t o n
g r o w t h b u t
c a u s e d a
m a r k e d d e c r e a s e i n
f o s f o m y c i n
p r o d u c t i o n .
G l u t a m a t e
a n d
m e t h i o n i n e
r e q u i r e m e n t .
T h e e f f e c t
o f
g l u t a m a t e
a n d
m e t h i o n i n e
o n
f o s f o m y c i n
p r o d u c t i o n
w a s
e x a m i n e d
f u r t h e r b y
t i t r a t i n g
e a c h
c o m p o u n d
i n
s y n t h e t i c p r o d u c -
t i o n
medium ( F i g .
7 ) .
M a x i m a l
f o s f o m y c i n
p r o d u c t i o n
o c c u r r e d a t
a
c o n c e n t r a t i o n o f 5
a n d
0 . 7
z m o l
o f
g l u t a m a t e
a n d
m e t h i o n i n e
p e r m l ,
r e s p e c t i v e l y .
T h e
p r o n o u n c e d
s t i m u l a t i o n o f
a n t i b i o t i c
s y n t h e s i s a n d
t h e
l a c k o f g r o w t h
e f f e c t s
s u g g e s t t h a t
t h e s e
a m i n o
a c i d s
a r e
d i -
r e c t l y
i n v o l v e d i n
f o s f o m y c i n
s y n t h e s i s a s
p r e -
c u r s o r s
o r
m e t a b o l i c
r e g u l a t o r s .
T h e
i m p o r t a n c e o f
t i m e
o f
a d d i t i o n
o f L - m e t h -
i o n i n e
i s
s e e n i n
F i g . 8 .
M e t h i o n i n e w a s a d d e d
a t
t w o l e v e l s
( 0 . 3 4 a n d
0 . 6 7
g m o l / m l )
a t 0 ,
2 5 ,
3 1 ,
a n d
4 8
h . A
c o n t r o l
f l a s k
r e c e i v e d
n o
m e t h i o n i n e . A l l f l a s k s
w e r e
h a r v e s t e d
a f t e r
7 2 h ,
a n d
g r o w t h
a n d
f o s f o m y c i n
w e r e
m e a s u r e d .
A l t h o u g h n o t
s h o w n ,
g r o w t h
w a s t h e s a m e
i n a l l
f l a s k s .
T h e
a d d i t i o n
o f
m e t h i o n i n e a t z e r o
t i m e
r e s u l t e d
i n
g o o d
a n t i b i o t i c y i e l d s ( o n l y
l o w
a m o u n t s
o f f o s f o m y c i n
w e r e p r o d u c e d
i n
f l a s k s
r e c e i v i n g
n o
m e t h i o n i n e ) . When
m e t h i o n i n e
w a s a d d e d a t
2 5
h
( a p p r o x i m a t e l y t h e
t i m e o f
i n i t i a t i o n
o f
a n t i b i o t i c s y n t h e s i s ;
s e e F i g .
2 B ) ,
t h e
s y n t h e s i s
o f
f o s f o m y c i n
w a s
r e d u c e d
g r e a t l y .
When
m e t h i o n i n e
w a s
a d d e d
a t
3 1 h , n o
a n t i b i -
o t i c
w a s
made
i n
f l a s k s
r e c e i v i n g
0 . 3 4
u m o l
o f
T A B L E 1 . N u t r i t i o n a l
r e q u i r e m e n t s
f o r g r o w t h a n d
f o s f o m y c i n
s y n t h e s i s
b y
S .
f r a d i a e
i n
s y n t h e t i c
m e d i u m a
D e l e t i o n s
f r o m G r o w t h
F o s f o m y c i n
s y n t h e t i c
medium ( m g
o f
d r y pH
( p g / m l )
c e l l s / m i )
None . . . . . . . . . . . . 4 . 0 9 . 0
2 4 . 7
L - A s p a r a g i n e
. . . . .
1 . 3 8 . 8
< 2 . 0
C i t r a t e
. . . . . . . . . .
2 . 1
4 . 6
1 0 . 1
L - G l u t a m a t e
. . . . .
5 . 4
8 . 8
1 3 . 9
L - M e t h i o n i n e
. . . . 1
5 . 1
8 . 8
7 . 8
a
I n c u b a t i o n
was
f o r
7 2
h .
C i t r a t e
a n d
g l u t a m a t e
w e r e
s u p p l i e d
a s t h e t r i - an d
monosodium
s a l t s ,
r e s p e c t i v e l y .
V O L .
5 ,
1 9 7 4
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yguest
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7/26/2019 Biosintesis de Fosfomicina
6/12
1 2 6
ROGERS
ANDBIRNBAUMAG
E
l
a
2 1
1 0
0
1 8 4
1 5
1 2
9
6
3
0
0 . 4 0 . 8
1 . 2
p
MOLES/
m l
F I G .
7 . E f f e c t
o f L - g l u t a m a
on
g r o w t h
a n d
f o s f o m y c i n
s j
f o s f o m y c i n
w e r e
m e a s u r e d
a f t l
( A )
L - G l u t a m a t e
a d d e d
as
t
a n d
( B )
L - m e t h i o n i n e
a d d i t i o r
F i g .
3 .
g r o w t h p h a s e o f t h e
f e r m e n t a t i o n
t o
p r o m o t e
s
t h e s y n t h e s i s
o f
f o s f o m y c i n
d u r i n g t h e
i d i o -
7
p h a s e .
T h e
s p e c i f i c i t y
o f
t h e
m e t h i o n i n e
r e q u i r e m e n t
6
f o r
h i g h
a n t i b i o t i c
s y n t h e s i s
wa s
r e a l i z e d f r o m
5
e x p e r i m e n t s
t h a t
r e v e a l e d t h a t
t h e
f o l l o w i n g
4
c o m p o u n d s
c o u l d n o t
r e p l a c e t h e
a m i n o
a c i d :
f o l i c
a c i d ,
c y a n o c o b a l a m i n , b e t a i n e ,
c h o l i n e ,
L - s e r i n e ,
L - c y s t e i n e ,
L - t h r e o n i n e , D L - h o m o c y s -
t e i n e , a n d D - m e t h i o n i n e .
F u r t h e r , f o s f o m y c i n
_
s y n t h e s i s
w a s
i n h i b i t e d
c o m p l e t e l y
b y
e t h i o n i n e
4
~ 0
a t c o n c e n t r a t i o n s w h i c h
c a u s e d
o n l y
a
s l i g h t
O
i n h i b i t i o n
o f
g r o w t h .
E t h i o n i n e
i n h i b i t i o n
w a s
8
o v e r c o m e
b y
L - m e t h i o n i n e .
T h e s e
o b s e r v a t i o n s
L
s u p p o r t
t h e
n o t i o n
t h a t L - m e t h i o n i n e
p l a y s
a
_ 7
d i r e c t
r o l e i n
f o s f o m y c i n s y n t h e s i s
v i a i t s
f u n c -
6
t i o n
a s
a
m e t h y l
d o n o r
i n
t r a n s m e t h y l a t i o n
r e a c t i o n s ( 2 4 ) .
G l u t a m a t e c o u l d
b e
r e p l a c e d
i n t h e
medium
_
b y s e v e r a l
t r i c a r b o x y l i c
a c i d
c y c l e
i n t e r m e d i -
3
a t e s
( T a b l e
2 ) .
P y r u v a t e , a c e t a t e ,
a n d
t h e
e a r l y
2
i n t e r m e d i a t e s
o f
t h e
c y c l e ,
i s o c i t r a t e a n d c i s -
a c o n i t a t e
( c i t r a t e
b e i n g p r e s e n t
i n t h e
b a s a l
m e d i u m ) ,
w e r e
o n l y p a r t i a l l y
e f f e c t i v e
i n
r e -
1 6 2 . 0
2 4 p l a c i n g
g l u t a m a t e .
C y c l e
i n t e r m e d i a t e s ,
a - k e t o -
g l u t a r a t e
t h r o u g h
o x a l a c e t a t e ,
w e r e
a s
e f f e c t i v e
i t e a n d
L - m e t h i o n i n e
a s g l u t a m a t e
i n p r o m o t i n g
f o s f o m y c i n
s y n t h e -
y n t h e s i s .
G r o w t h
a n d
s i s .
F u m a r a t e a n d
m a l a t e c o n s i s t e n t l y
s t i m u -
e r
7 2
h
o f
i n c u b a t i o n .
l a t e d a n t i b i o t i c
p r o d u c t i o n
t o
a
g r e a t e r
e x t e n t
h e m o n o s o d i u m
s a l t ,
t h a n
d i d
g l u t a m a t e .
S e v e r a l
a m i n o
a c i d s w e r e
i .
S y m b o l s
a r e
a s
f o r
t e s t e d a n d
f a i l e d
t o
r e p l a c e
t h e
g l u t a m a t e
f u n c t i o n
i n
f o s f o m y c i n
s y n t h e s i s .
T h e s e
i n c l u d e
L - a s p a r t a t e ,
L - p r o l i n e ,
L - c i t r u l l i n e ,
L - o r n i t h i n e ,
L - a r g i n i n e ,
a n d
L - g l u t a m i n e ,
a l l o f
w h i c h
a r e
d e r i v e d
b i o s y n t h e t i c a l l y
f r o m
L - g l u t a m a t e .
T h e
a d d i t i o n o f
o r g a n i c
a c i d s
o r
a m i n o
a c i d s
d i d
n o t
a f f e c t
s i g n i f i c a n t l y
t h e
g r o w t h
o f
S .
f r a d i a e .
C i t r a t e ,
a s p a r a g i n e ,
a n d
g l y c i n e
r e -
q u i r e m e n t s .
C i t r a t e
a n d
L - a s p a r a g i n e
w e r e
r e -
T I M E
OF
A C O I T I O N OF METHIONINE
( H R )
F I G . 8 . E f f e c t
o f
t h e
t i m e o f a d d i t i o n o f
m e t h i o n i n e
on
f o s f o m y c i n s y n t h e s i s .
M e t h i o n i n e
was a d d e d t o
p r o d u c t i o n f l a s k s
t o g i v e
a f i n a l c o n c e n t r a t i o n
o f
e i t h e r
0 . 3 4
( A )
or 0 . 6 7
A m o l / m l
( @ ) .
F o s f o m y c i n was
d e t e r m i n e d
a f t e r
7 2 h
o f i n c u b a t i o n .
L - m e t h i o n i n e
p er m l , a n d o n l y 5 . 8
g g / m l
was
s y n t h e s i z e d i n
f l a s k s
r e c e i v i n g
t h e h i g h e r l e v e l
o f t h e
a m i n o
a c i d .
No
f o s f o m y c i n
was
p r o d u c e d
w h e n
m e t h i o n i n e was a d d e d
a t 4 8
h .
I t
appears
t h a t
m e t h i o n i n e must
b e
p r e s e n t
e a r l y i n t h e
T A B L E
2 .
E f f e c t
o f
o r g a n i c
a c i d s
o n f o s f o m y c i n
s y n t h e s i s
A d d i t i o n
F o s f o m y c i n
( O g / m i ) 5
N o n e
9 . 9
L - G l u t a m a t e
...
1 7 . 2
P y r u v a t e
1 2 . 0
A c e t a t e
1 2 . 0
C i s - a c o n i t a t e
1 2 . 7
I s o c i t r a t e
1 1 . 3
G l y o x y l a t e
7 . 9
a - K e t o g l u t a r a t e
. . . .
1 7 . 3
S u c c i n a t e
1 6 . 6
F u m a r a t e
1 9 . 6
M a l a t e
2 0 . 2
O x a l a c e t a t e
...
1 6 . 8
a
A t 6
m M.
' A s s a y e d
a t
7 2 h .
A v e r a g e
o f
f i v e
e x p e r i m e n t s .
A
3 2
-
2 8 _
47
2 0 L
1 6
F
1 2
F
04
2
O 0 2 3
_B
A
A N T I M I C R O B .
A G .
C H E M O T H E R .
onA
pril11
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yguest
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7/26/2019 Biosintesis de Fosfomicina
7/12
BIOSYNTHESIS
OF
FOSFOMYCIN BY S .
FRADIAE 1 2 7
q u i r e d f o r
g r o w t h a n d
f o s f o m y c i n s y n t h e s i s
( F i g .
9 ) . A n t i b i o t i c
y i e l d
c l o s e l y
p a r a l l e l e d
g r o w t h
i n
r es p o n s e
t o b o t h c o m p o n e n t s .
I n t h e
a b s e n c e o f
c i t r a t e , t h e f i n a l
pH
o f p r o d u c t i o n medium
was
l o w
( p H
6 . 9 )
a n d i n c r e a s e d
g r a d u a l l y a s
t h e
c i t r a t e
c o n c e n t r a t i o n
was
i n c r e a s e d
t o
1 0 . 2
A t m o l / m l .
A l t h o u g h
t h e
pH
r e m a i n e d
c o n s t a n t
( p H
8 . 4 )
w i t h i n
t h e c i t r a t e
c o n c e n t r a t i o n
range
o f
1 0 . 2 t o
1 7
, u m o l / m l ,
t h e r e
was a
m a r k e d e f f e c t
on
g r o w t h
a n d f o s f o m y c i n s y n t h e s i s .
T h u s , t h e
r o l e
o f c i t r a t e i n
a n t i b i o t i c p r o d u c t i o n
was
n o t
r e l a t e d
s o l e l y
t o
pH e f f e c t s .
M a x i m a l
g r o w t h
a n d
f o s f o m y c i n
s y n t h e s i s o c c u r r e d
a t 1 5 M m o l
o f
c i t r a t e pe r m l . H i g h e r
l e v e l s o f
c i t r a t e i n h i b i t e d
b o t h p a r a m e t e r s .
An L - a s p a r a g i n e
c o n c e n t r a t i o n
o f 3 7
u m o l / m l
r e s u l t e d
i n m a x i m a l g r o w t h
a n d a n t i b i o t i c
s y n -
t h e s i s ( F i g .
9 B ) .
The
r o l e
o f
L - a s p a r a g i n e
a p -
p e a r e d
t o b e
t h a t
o f a n i t r o g e n
s o u r c e
f o r
g r o w t h .
U r e a ,
L - g l u t a m a t e , L - g l u t a m i n e ,
L -
m e t h i o n i n e , a n d i n o r g a n i c
n i t r o g e n
c o m p o u n d s
c o u l d
n o t
r e p l a c e
L - a s p a r a g i n e
i n t h i s
c a p a c i t y .
T h o u g h L - a s p a r t i c a c i d
a n d
L - o r n i t h i n e s u p -
p o r t e d
50 o f m a x i m a l
g r o w t h i n t h e
a b s e n c e
o f
L - a s p a r a g i n e ,
t h e y c o u l d
n o t a f f e c t
f o s f o m y c i n
s y n t h e s i s .
G l y c i n e was
t h e o n l y o r g a n i c n i t r o g e n
s o ur ce t e s t e d
t h a t r e p l a c e d L - a s p a r a g i n e
f o r
0
10
20 30
p
MOLES /m l
40
F I G .
9 .
E f f e c t
o f
c i t r a t e
and
L - a s p a r a g i n e
on
g r o w t h
a n d
f o s f o m y c i n s y n t h e s i s .
( A ) E f f e c t o f
c i t r a t e
( a d d e d
a s
s o d i u m
s a l t ) ,
and
( B )
e f f e c t
o f
L - a s p a r a g i n e
( a d d e d
as
m o n o h y d r a t e ) .
S y m b o l s
a re
a s
f o r F i g .
3 .
b o t h
g r o w t h a n d
a n t i b i o t i c s y n t h e s i s .
The e f f e c t
o f g l y c i n e
i n t h e pre se nc e
a n d
a b s e n c e
o f
L -
a s p a r a g i n e
i s s h o w n
i n
F i g .
1 0 .
S i n c e f o s f o m y c i n
s y n t h e s i s
i s d e l a y e d
i n c e l l s
g r o w i n g on g l y c i n e
a s a
s o l e n i t r o g e n
so urce, g r o w t h
a n d
f o s f o m y c i n
w e r e
m e a s u r e d
a f t e r
9 6
h
o f
i n c u b a t i o n .
I n
t h e
a b s e n c e
o f
L - a s p a r a g i n e ,
no a n t i b i o t i c was s y n -
t h e s i z e d
u n t i l
t h e g l y c i n e c o n c e n t r a t i o n
ex-
c e e d e d
t h a t
r e q u i r e d
f o r m a x i m a l g r o w t h
( 3 5
, u m o l / m l ) ;
t h e g l y c i n e
c o n ce n t r a t i o n r e q u i r e d
f o r
m a x i m a l s y n t h e s i s
was a b o u t t w i c e
t h i s
l e v e l .
I n medium c o n t a i n i n g
2 0
, u m o l
o f L - a s p a r a g i n e
per m l ,
g l y c i n e c a u s e d
a
s l i g h t s t i m u l a t i o n
o f
g r o w t h
a n d d i d
p o t e n t i a t e
f o s f o m y c i n p r o d u c -
t i o n .
I n
t h i s
i n s t a n c e ,
t h e l e v e l
o f
g l y c i n e
r e -
q u i r e d
f o r
m a x i m a l
a n t i b i o t i c
s y n t h e s i s
i s
l o w e r
t h a n t h a t
r e q u i r e d
i n
L - a s p a r a g i n e - f r e e
me-
d i u m . A t
t h e
h i g h e r
l e v e l
o f
L - a s p a r a g i n e
( 4 7
, u m o l / m l ) ,
a n t i b i o t i c
s y n t h e s i s
was a t
a
m a x i -
mum, a n d f u r t h e r
a d d i t i o n
o f g l y c i n e d i d
n o t
s t i m u l a t e
a n t i b i o t i c
p r o d u c t i o n .
E i t h e r i n t h e
pre se nc e
or a b s e n c e
o f L - a s p a r a g i n e ,
g l y c i n e
i n h i b i t e d f o s f o m y c i n
s y n t h e s i s
a t c o n c e n t r a -
t i o n s w h i c h d i d
n o t i n h i b i t
g r o w t h .
T h e i n d e -
p e n d e n t
e f f e c t s
o f
g l y c i n e
o n
g r o w t h
a n d
f o s -
f o m y c i n s y n t h e s i s
s u g g e s t t h a t ,
as
w i t h L - g l u t a -
mate
a n d
L - m e t h i o n i n e ,
g l y c i n e may
h a v e a
d i r e c t
r o l e
i n a n t i b i o t i c
p r o d u c t i o n .
R a d i o i s o t o p e
i n c o r p o r a t i o n
s t u d i e s .
The
f u n c t i o n
o f c i t r a t e , L - a s p a r a g i n e ,
L - g l u t a m a t e ,
L - m e t h i o n i n e ,
g l y c i n e ,
a n d L - s e r i n e
i n
f o s -
f o m y c i n s y n t h e s i s
was e v a l u a t e d b y
u s i n g
C -
l a b e l e d c o m p o u n d s
( T a b l e
3 ) .
A f t e r
g r o w t h
on
t h e i n d i c a t e d
r a d i o a c t i v e c o m p o u n d ,
t h e
a n t i b i -
o t i c was i s o l a t e d b y
t h e
c o - c r y s t a l l i z a t i o n
proce-
d u r e ,
a n d i t s r a d i o a c t i v i t y
was
d e t e r m i n e d .
Due
t o
t h e
l o w
i n c o r p o r a t i o n
o f
r a d i o a c t i v i t y
i n t o t h e
a n t i b i o t i c
a n d
t h e
r e l a t i v e l y h i g h
l e v e l o f
t h e
m a t e r i a l s r e q u i r e d
f o r
f o s f o m y c i n s y n t h e s i s ,
i t
was more m e a n i n g f u l
t o
express
t h e
i n c o r p o r a -
t i o n
d a t a
i n t e r m s
o f
d i l u t i o n
o f
s p e c i f i c
a c t i v i t y
o f
t h e
a d d e d
s u b s t r a t e
r a t h e r t h a n
a s
p e r c e n t
i n c o r p o r a t i o n .
T h e r e
was
a
l a r g e
d i l u t i o n o f
l a b e l
i n
t h e
a n t i b i o t i c
when
L - a s p a r a g i n e - U - 4 C ,
c i t r a t e - 1 , 5 - 1
4 C ,
and
L - g l u t a m a t e - U - 1
4 C
w e r e
u s e d
as l a b e l e d
s u b s t r a t e s .
Th e l a c k
o f
i n c o r p o -
r a t i o n f r o m
L - a s p a r a g i n e
and c i t r a t e
was n o t
u n e x p e c t e d ,
b a s e d
on t h e d o s e
response
t o
t h e s e
m a t e r i a l s ;
i . e . ,
t h e s e
c o m p o u n d s p r o m o t e d
f o s -
f o m y c i n s y n t h e s i s
i n d i r e c t l y t h r o u g h
t h e i r
e f -
f e c t s
o n
g r o w t h ( s e e
F i g . 9 ) .
I t
was
u n e x p e c t e d ,
h o w e v e r ,
t h a t
L - g l u t a m a t e
d i d
n o t
c o n t r i b u t e
l a b e l
t o
t h e
a n t i b i o t i c ,
s i n c e
t h i s
a m i n o
a c i d
p r o m o t e d
f o s f o m y c i n s y n t h e s i s
w h i l e
h a v i n g
no
e f f e c t
on
g r o w t h
( s e e F i g .
7 A ) .
L - M e t h i o n i n e -
m e t h y l - 1 4 C
s e r v e d
a s t h e
b e s t r a d i o a c t i v e
p r e -
cursor.
I n
c o n t r a s t ,
L - m e t h i o n i n e
l a b e l e d
i n
e i t h e r
c a r b o n
1
or
2 was
a
poor
p r e c u r s o r .
These
d a t a
s u p p o r t
f u r t h e r
t h e
r o l e
o f methionine
i n
2 1
A
0 - ;
0
1 8
15 -
1 2
6
~~~~~~~~~~~~
V O L .
5 ,
1 9 7 4
onA
pril11
,2
01
6b
yguest
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ac.a
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m.org
/
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adedfrom
http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/ -
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8/12
128
ROGERS
AND BIRNBAUM
E8
o
GLC
N E
ONAETRTIN
A - M O E S / - l
FI.1.Efeto g y i e owt
n
o f m c n
C ~ ~
2 B 4
0
2 4
6
0 20
40 60
80 100
1 2 0
GLYCINE
CONCENTRATION
~
MOLES
/ m n l )
FIG.
10.
Effect
o f g l y c i n e
on
growth
and
fosfomycin
s y n t h e s i s
in
medium
c o n t a i n i n g :
A
no
L - a s p a r a g i n e ,
( B)
20
pAmol o f L-asparaine per
m l ,
and
( C )
47
umol
o f
L - a s p a r a g i n e per
m l .
G r o w t h
a n d
f o s f o m y c i n
w ere
m e a s u r e d
a t
9 6
h .
S y m b o l s
are
as
f o r
F i g .
3 .
f o s f o m y c i n s y n t h e s i s as
a
m e t h y l
d o n o r
v i a an
S - a d e n o s y l m e t h i o n i n e
m e t h y l a t i o n mechanism
( 2 4 ) .
B e c a u s e
o f i t s f u n c t i o n i n 1 - c a r b o n
m e t a b -
o l i s m ,
t h e
i n c o r p o r a t i o n
o f
D L s e r i n e 3 1 4 C was
d e t e r m i n e d . C a r b o n 3
o f t h i s a m i n o
a c i d
was
i n c o r p o r a t e d
r a t h e r
w e l l .
G l y c i n e - U - 1 4 C
i n con-
t r a s t t o
L - a s p a r a g i n e
( b o t h
amino
a c i d s are
a l t e r n a t e
n i t r o g e n
s o u r c e s
f o r
t h e
f e r m e n t a t i o n )
d i d
c o n t r i b u t e
e f f i c i e n t l y
r a d i o a c t i v i t y t o
f o s -
f o m y c i n .
A g a i n , t h i s
i s i n
agreement
w i t h t h e
d o s e
r es p o n s e d a t a ( s e e
F i g . 1 0 ) .
R a d i o a c t i v e g l u c o s e ,
p y r u v a t e ,
and a c e t a t e
w e r e
t e s t e d
a l s o f o r i n c o r p o r a t i o n
i n t o f o s -
f o m y c i n
( T a b l e 4 ) . G o o d
i n c o r p o r a t i o n
o f g l u -
cose r a d i o a c t i v i t y
was
o b t a i n e d .
G l u c o s e - I -
4 C
a n d g l u c o s e - 6 -
1 4 C
c o n t r i b u t e d
l a b e l e q u a l l y
w e l l . G l u c o s e - 2 -
IC
w a s
o n l y
s l i g h t l y
p o o r e r .
G l u c o s e - 3 , 4 - 1 4 C
w a s t h e
p o o r e s t
o f
a l l
g l u c o s e
l a b e l s ,
y i e l d i n g
a 3 6 - f o l d
d i l u t i o n o f
s p e c i f i c
T A B L E 3 .
I n c o r p o r a t i o n
o f
r a d i o a c t i v e
a m i n o
a c i d s
a n d
c i t r a t e
i n t o f o s f o m y c i n
F o s f o -
S p
a c t
m y c i n
L a b e l e d c o m p o u n d '
( c o u n t s /
s p
a c t D i l u t i o n
o f
m i n x
( c o u n t s /
s p
a c t
1 0 3 / A m o l )
m i n x
1 0 3 / u m o l )
L - A s p a r a g i n e - U - 4 C
9 0 0
< 0 . 1
>
1 , 0 0 0
L - G l u t a m i c
a c i d - U -
3 7 0
0 . 4
9 2 5
1 4 C
L - M e t h i o n i n e - I - 4 C
1 , 3 5 0
2 . 6
5 2 0
L - M e t h i o n i n e - 2 - 4 C 1 , 3 8 0
2 . 5
5 5 2
L - M e t h i o n i n e -
1 , 0 9 0
1 3 0
8 . 4
m e t h y l - 1 4 C
G l y c i n e - U - C
8 0
6 . 1
1 3
D L - S e r i n e - 3 - 1 4 C
5 5 2
1 5 . 2
3 6 . 4
C i t r i c
a c i d - i ,
5 1 4 C
7 0
< 0 . 1
>
7 0 0
a L a b e l e d
c o m p o u n d s
w e r e
a d d e d
a t 0 h
j u s t
b e f o r e
i n o c u l a t i o n .
L - A s p a r a g i n e
w a s t h e
m a j o r
n i t r o g e n
s o u r c e
i n a l l f l a s k s
e x c e p t
t h o s e
c o n t a i n i n g
g l y c i n e - U -
1 4 C .
I n t h i s
c a s e ,
g l y c i n e
s e r v e d
a s t h e
n i t r o g e n
s o u r c e .
b
A n t i b i o t i c
w a s
i s o l a t e d
a t 7 2
h
p o s t i n o c u l a t i o n
b y
t h e c o - c r y s t a l l i z a t i o n
p r o c e d u r e
e x c e p t
f o r t h e
g l y -
c i n e - U - 4 C
f l a s k s . I n t h i s c a s e ,
i n c u b a t i o n
was
c o n t i n -
u e d
t o
9 6
h ,
a n d
i s o l a t i o n
o f t h e
a n t i b i o t i c
was
a f f e c t e d b y c o - c r y s t a l l i z a t i o n
a f t e r
p a r t i a l p u r i f i c a t i o n
b y
i o n
e x c h a n g e
a n d
g e l
f i l t r a t i o n
c h r o m a t o g r a p h y .
D e t a i l s
o f t h e
i s o l a t i o n
a r e
g i v e n
i n
M a t e r i a l s
a n d
M e t h o d s .
T A B L E
4 .
I n c o r p o r a t i o n
o f
r a d i o a c t i v e
g l u c o s e ,
p y r u v a t e ,
a n d
a c e t a t e i n t o
f o s f o m y c i n
P r e c u r s o r F o s f o m y c i n
L a b e l e d
s p
a c t
s p
a c t
D i l u t i o n
o f
c o m p o u n d
( c o u n t s / ( c o u n t s /
p
t
min
x min
x
1 0 / 4 m O l )
1 0 3 / M m o l )
G l u c o s e - 1 - 4 C
9 . 5 3 . 2 3 . 0
G l u c o s e - 2 - 1 4 C
1 1 . 0
2 . 2
5 . 0
G l u c o s e - 6 - 1 4 C
9 . 1 3 . 3 2 . 6
G l u c o s e - 3 , 4 - 4 C
1 0 . 0 0 . 2 8 3 5 . 7
P y r u v a t e - l - 4 C
1 1 1
0 . 1 3
8 5 4
P y r u v a t e - 2 -
4 C
1 5 4
0 . 2 3
6 7 0
P y r u v a t e - 3 - 4 C
1 1 8
0 . 5 1
2 3 1
A c e t a t e - i , 2 - C
3 5 7 4 . 5 7 9
A c e t a t e - 1 - 4 C
1 , 0 0 0
0 . 0 2 >
1 , 0 0 0
A c e t a t e - 2 - 1 4 C
3 2 5
8 . 9 3 6 . 5
a
L a b e l e d
c o m p o u n d s
w e r e
a d d e d
a t 0 h
j u s t b e f o r e
i n o c u l a t i o n .
b
A n t i b i o t i c
w a s
i s o l a t e d
a t 7 2 h
b y c o - c r y s t a l l i z a -
t i o n
a f t e r
p a r t i a l
p u r i f i c a t i o n b y
i o n
e x c h a n g e
a n d
g e l
f i l t r a t i o n
c h r o m a t o g r a p h y .
D e t a i l s
o f t h e i s o l a t i o n a r e
g i v e n
i n
M a t e r i a l s
a n d
M e t h o d s .
A N T I M I C R O B .
A G .
C H E M O T H E R .
onA
pril11
,2
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ac.a
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/
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adedfrom
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7/26/2019 Biosintesis de Fosfomicina
9/12
BIOSYNTHESIS
OF
FOSFOMYCIN BY
S .
FRADLAE
1 2 9
a c t i v i t y . T h i s
d i l u t i o n
was
7 - a n d
1 2 - f o l d
g r e a t e r
t h a n
t h a t o b t a i n e d w h e n
g l u c o s e - 2 -
4 C a n d
g l u -
c o s e - l -
C ( o r g l u c o s e - 6 -
4 C ) , r e s p e c t i v e l y ,
were
precursors. A c e t a t e
a n d
p y r u v a t e i n c o r p o r a -
t i o n was
l e s s t h a n t h a t
o b s e r v e d f o r
g l u c o s e ,
i n -
d i c a t i n g
t h a t
t h e c a r b o n
p r e c u r s or
o f
f o s f o m y -
c i n
may b e a
g l y c o l y t i c
i n t e r m e d i a t e .
Even
t h o u g h t h e
d i l u t i o n
o f
r a d i o a c t i v i t y f r o m
p y r u -
v a t e was
h i g h ,
t h e r e
was
a
p r e f e r e n t i a l
i n c o r -
p o r a t i o n
o f
c a r b o n
3 .
T h o u g h a
p o or er p r e c u r -
sor
t h a n
g l u c o s e ,
m e t h i o n i n e - m e t h y l - 4 C , and
g l y c i n e ,
a c e t a t e
was
a f a r
b e t t e r
p r o g e n i t o r
o f
f o s f o m y c i n
c a r b o n
t h a n
were
p y r u v a t e , c i t -
r a t e ,
L - g l u t a m a t e , o r
L - a s p a r a g i n e .
F u r t h e r -
m o r e ,
t h e
p a t t e r n
o f
l a b e l i n g
s h o w s
t h a t
o n l y
t h e m e t h y l c a r b o n
o f a c e t a t e was
i n c o r p o r a t e d
i n t o
f o s f o m y c i n .
The
d i s t r i b u t i o n
o f
r a d i o a c t i v i t y
was
d e t e r -
m i n e d on
f o s f o m y c i n
i s o l a t e d f r o m
b r o t h s
f e d
r a d i o a c t i v e c o m p o u n d s
( T a b l e
5 ) .
S i n c e
96
o f
t h e
l a b e l
f r o m
L - m e t h i o n i n e - m e t h y l -
4 C
was
f o u n d
i n c a r b o n
3
o f
f o s f o m y c i n ,
i t
i s
c l e a r
t h a t
t h e precursor o f t h i s c a r b o n i s
t h e S - m e t h y l o f
t h e
a m i n o
a c i d .
G l y c i n e a l s o
c o n t r i b u t e d
i t s
l a b e l
t o c a r b o n
3
o f
f o s f o m y c i n
a n d
p r o b a b l y
d i d
so
v i a
L - m e t h i o n i n e .
E i g h t y p e r c e n t o f
t h e l a b e l
f r o m
c a r b o n s 1
a n d
6
o f
g l u c o s e
was
f o u n d
i n
c a r b o n 1 o f
t h e
a n t i b i o t i c .
G l u c o s e - 2 - ' 4 C c o n -
t r i b u t e d
a b o u t 80 o f
i t s r a d i o a c t i v i t y
t o c a r b o n
2 o f
f o s f o m y c i n .
The
l a b e l f r o m
a c e t a t e - 1 , 2 - ' 4 C
a n d a c e t a t e - 2 - ' 4 C was
a b o u t
e q u a l l y
d i s t r i b u t e d
b e t w e e n
c a r b o n s
1
a n d
2 o f
t h e
a n t i b i o t i c .
I t
was
n o t
p o s s i b l e t o o b t a i n
s u f f i c i e n t
l a b e l e d
a n t i b i -
o t i c f r o m
t h e
[ I 4 C
p y r u v a t e
e x p e r i m e n t s
t o
d o
d e g r a d a t i o n s t u d i e s .
DISCUSSION
E a r l i e r n u t r i t i o n a l
s t u d i e s b y J a c k s o n
a n d
S t a p l e y ( 1 4 )
h a d s h o w n
t h a t
c e r t a i n
c o m p o u n d s
w e r e
r e q u i r e d
f o r or
s t i m u l a t e d
f o s f o m y c i n syn-
t h e s i s
i n
S .
f r a d i a e . S i n c e
f o s f o m y c i n was t h e
f i r s t
e x a m p l e
o f
t h e s y n t h e s i s
o f
a
p h o s p h o n a t e
b y
a
p r o k a r y o t i c o r g a n i s m ,
i t
was
o f
i n t e r e s t
t o
s t u d y
i t s
b i o s y n t h e s i s
i n
more d e t a i l . A s i s
t h e
c a s e w i t h
many
o t h e r
a n t i b i o t i c s ,
f o s f o m y c i n
s y n t h e s i s
was
f o u n d
t o o c c u r
m a i n l y d u r i n g
t h e
i d i o p h a s e
o f
g r o w t h ,
a n d t h u s i t
may
b e
c l a s s i -
f i e d a s a
p r o d u c t
o f
s e c o n d a r y
m e t a b o l i s m .
C a t i o n s h a v e b e e n f o u n d t o b e
i m p o r t a n t
t o
t h e
p r o d u c t i o n
o f
s e c o n d a r y
m e t a b o l i t e s . U s u -
a l l y ,
t h e
s y n t h e s i s
o f a
s e c o n d a r y
p r o d u c t
i s
a f f e c t e d
b y
a
s i n g l e k e y
m e t a l
i o n ,
a n d ,
a s
i l l u s t r a t e d
b y
W e i n b e r g
( 3 3 ) ,
p r o d u c t
y i e l d
v a r i e s
l i n e a r l y
w i t h
t h e
l o g
o f t h e m e t a l c o n c e n -
t r a t i o n
a t
m e t a l
l e v e l s
w h i c h
d o
n o t
a f f e c t
g r o w t h .
T h i s
r e l a t i o n s h i p
was
o b s e r v e d
f o r
t h e
c o b a l t s t i m u l a t i o n
o f
f o s f o m y c i n
p r o d u c t i o n
( s e e F i g .
4 ) . T h u s ,
c o b a l t
c a n
b e
c o n s i d e r e d t h e
T A B L E 5 . D i s t r i b u t i o n
o f r a d i o a c t i v i t y
i n
f o s f o m y c i n a
P e r c e n t
d i s t r i b u t i o n
o f
r a d i o a c t i v i t y i n
f o s f o m y c i n
L a b e l e d
c o m p o u n d
C 2
C 1
C 2
C 3
+
C 3
L - M e t h i o n i n e - m e t h y l - 4 C
4 0
9 6
G l y c i n e - U - ' 4 C
8
5
8 7
G l u c o s e - 2 - ' 4 C 1 4 7 8
8
G l u c o s e - l 1 - 4 C
8 0 c
2 0 c
G l u c o s e - 6 -
1 4 C
8 0 c
2 0 c
A c e t a t e -
1 , 2 -' 4 C
5 3
4 2
5
A c e t a t e - 2 -
' 4 C
4 9 4 6 4
a
S t r u c t u r e a n d
c a r b o n
n u m b e r i n g :
1 2 3
H 2 0 3 P - C H - C H - C H 3 .
\ 0
D i s t r i b u t i o n
o f l a b e l w a s d e t e r m i n e d a s
d e s c r i b e d
i n M a t e r i a l s a n d M e t h o d s .
c L a b e l e d
f o s f o m y c i n
f o r m e d
i n t h e
p r e s e n c e
o f
g l u c o s e - 1 - 4 C
a n d
g l u c o s e - 6 - 4 C
w a s
d e g r a d e d o n l y
t o
t h e
a c e t a t e
s t a g e . T h e
r a d i o a c t i v i t y
o f t h e
a c e t a t e
w a s
d e t e r m i n e d a n d
r e p r e s e n t s
t h e
t o t a l
l a b e l i n
f o s -
f o m y c i n c a r b o n s
2 a n d
3 . T h e
r a d i o a c t i v i t y
o f
f o s -
f o m y c i n
c a r b o n 1
w a s
c a l c u l a t e d
a s d e s c r i b e d
i n
M a t e r i a l s a n d
M e t h o d s .
k e y
m e t a l
i n
f o s f o m y c i n
s y n t h e s i s . I n
t h e
l i s t o f
k e y
m e t a l s
a f f e c t i n g
s e c o n d a r y
m e t a b o l i s m
c o m p i l e d
b y
W e i n b e r g
( m a n g a n e s e , i r o n , a n d
z i n c ) ,
c o b a l t
w a s n o t
i n c l u d e d
( 3 3 ) . H o w e v e r , i n
a d d i t i o n
t o
a f f e c t i n g f o s f o m y c i n p r o d u c t i o n ,
l o w
l e v e l s
o f
c o b a l t h a v e
b e e n s h o w n t o c a u s e
a
m a r k e d
s t i m u l a t i o n o f
g e n t a m i c i n p r o d u c t i o n
a t
c o n c e n t r a t i o n s
h a v i n g n o a f f e c t
o n g r o w t h
( W .
C h a r n e y ,
U . S .
P a t e n t
3 , 1 3 6 , 7 0 4 , 1 9 6 4 ) ,
a n d
f u r t h e r m o r e ,
c o b a l t h a s
b e e n s h o w n t o
c a u s e a
d r a m a t i c
s h i f t
i n
p r o d u c t
y i e l d
i n t h e
c o u m e r -
m y c i n
A
f e r m e n t a t i o n
( 4 ) .
I n
b o t h t h e
g e n t a m i -
c i n
a n d
c o u m e r m y c i n s t u d i e s ,
i t
w a s
c o n c l u d e d
t h a t
c o b a l t f u n c t i o n s
b y
s t i m u l a t i n g
o r
a c t i v a t -
i n g
a
s p e c i f i c
e n z y m e
n e c e s s a r y
f o r
a n t i b i o t i c
s y n t h e s i s . T h e
l e v e l s
o f
c o b a l t
r e q u i r e d
f o r
m a x i m a l
f o s f o m y c i n s y n t h e s i s a r e 3 . 6 - a n d
2 , 0 0 0 - f o l d
h i g h e r
t h a n
t h o s e
r e q u i r e d
f o r
s y n t h e -
s i s
o f g e n t a m i c i n a n d
c o u m e r m y c i n
A l , r e s p e c -
t i v e l y .
I t
i s o f
i n t e r e s t t h a t t h e l e v e l s
o f c o b a l t
w h i c h
s t i m u l a t e d
f o s f o m y c i n
s y n t h e s i s
m a x i -
m a l l y
a r e a b o u t
t h r e e f o l d
g r e a t e r
t h a n t h o s e
r e q u i r e d
f o r v i t a m i n B 1 2
o v e r p r o d u c t i o n
b y
P s e u d o m o n a s
d e n i t r i f i c a n s
e v e n
t h o u g h
c o b a l t
m a k e s
u p
a n
i n t e g r a l
p a r t
o f t h e
v i t a m i n
m o l e c u l e
( L . K a p l a n ,
p e r s o n a l
c o m m u n i c a -
t i o n ) .
T h e
f u n c t i o n
o f
t h e
r e l a t i v e l y
h i g h
l e v e l s
o f
c o b a l t
i n
f o s f o m y c i n s y n t h e s i s
i s u n k n o w n .
I n
b a c t e r i a
( 2 8 )
a n d
s t r e p t o m y c e t e s ( 1 9 ) ,
a
c o b a l a -
m i n - d e p e n d e n t
t r a n s m e t h y l a s e
c a t a l y z i n g
t h e
V O L .
5 ,
1 9 7 4
onA
pril11
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http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/http://aac.asm.org/ -
7/26/2019 Biosintesis de Fosfomicina
10/12
1 3 0
ROGERS
AND BIRNBAUM
f i n a l s t e p
i n
m e t h i o n i n e
b i o s y n t h e s i s h a s
b e e n
d e m o n s t r a t e d .
A l t h o u g h
m e t h i o n i n e
i s
a
p r e -
c u r s o r
o f f o s f o m y c i n
( s e e
b e l o w ) , i t i s
u n l i k e l y
t h a t
c o b a l t s t i m u l a t e s
a n t i b i o t i c
s y n t h e s i s
a t
t h e
l e v e l
o f t h e
c o b a l a m i n - d e p e n d e n t
m e t h i o -
n i n e
s y n t h e s i z i n g
s y s t e m ,
s i n c e
h i g h
l e v e l s
o f
m e t h i o n i n e d i d n o t
s p a r e t h e
c o b a l t
r e q u i r e -
m e n t f o r
a n t i b i o t i c
s y n t h e s i s .
S e v e r a l
e n z y m e s
h a v e
b e e n s h o w n
t o
r e q u i r e
c o b a l t o r
o t h e r
d i v a l e n t
c a t i o n s f o r a c t i v a t i o n
( 5 , 3 1 ) .
I t
may
b e
t h a t a n
e n z y m e ( s )
s p e c i f i c t o
f o s f o m y c i n
s y n -
t h e s i s
r e q u i r e s c o b a l t
f o r i t s
a c t i v i t y
o r
s y n t h e -
S i S .
M e t h i o n i n e
c a u s e d
a
d r a m a t i c s t i m u l a t i o n
o f
f o s f o m y c i n
s y n t h e s i s
w h i l e
h a v i n g
l i t t l e
e f f e c t
o n
g r o w t h ,
i n d i c a t i n g
a d i r e c t
r o l e
o f
t h i s
a m i n o
a c i d
i n
a n t i b i o t i c s y n t h e s i s .
I n c o r p o r a t i o n
s t u d -
i e s
u s i n g
m e t h i o n i n e
l a b e l e d i n
v a r i o u s
p o s i -
t i o n s
s h o w e d
t h a t
t h e m e t h y l c a r b o n i s
i n c o r p o -
r a t e d
e x c l u s i v e l y
i n t o t h e
m e t h y l c a r b o n
o f
f o s f o m y c i n .
T h e m e t h y l
d o n o r
f u n c t i o n
o f
t h e
a m i n o
a c i d i n
f o s f o m y c i n
b i o s y n t h e s i s
i s
s u p -
p o r t e d
f u r t h e r
b y t h e d a t a
s h o w i n g
t h a t
e t h i o -
n i n e
i n h i b i t s
a n t i b i o t i c s y n t h e s i s
w i t h o u t
a f -
f e c t i n g g r o w t h . T h e
b i o l o g i c a l
r o l e
o f m e t h i o -
n i n e
a s a
m e t h y l d o n o r
( v i a
S - a d e n o s y l m e t h i o -
n i n e )
i n
t h e
b i o s y n t h e s i s
o f s e v e r a l
s e c o n d a r y
m e t a b o l i t e s
( 1 , 1 3 )
a s
w e l l a s
i n
t h e
p r o d u c t i o n
o f
p r i m a r y m e t a b o l i t e s
( 2 4 )
i s
w e l l
e s t a b l i s h e d .
I n a d d i t i o n
t o i t s
p r e c u r s o r
r o l e
i n
f o s f o m y c i n
p r o d u c t i o n ,
m e t h i o n i n e
a p p e a r s t o
f u n c t i o n a l s o
i n
t h e
r e g u l a t i o n
o f
a n t i b i o t i c
s y n t h e s i s . T h i s
w a s
c o n c l u d e d f r o m
e x p e r i m e n t s
w h i c h
s h o w e d
t h a t
m e t h i o n i n e m u s t b e
p r e s e n t
d u r i n g
t h e
g r o w t h p h a s e
( t r o p h o p h a s e )
i n
o r d e r
t o
p r o m o t e
f o s f o m y c i n
s y n t h e s i s
d u r i n g t h e
i d i o p h a s e .
T h i s
s i t u a t i o n
i s
a n a l o g o u s
t o
t h e
s t i m u l a t o r y
e f f e c t
o f
t r y p t o p h a n
i n
e r g o l i n e
a l k a l o i d
s y n t h e s i s
i n
C l a v i c e p s ( 7 ) .
T r y p t o p h a n ,
a
p r e c u r s o r o f
a l k a -
l o i d
s y n t h e s i s ,
i s
t h o u g h t t o
b e
r e q u i r e d
d u r i n g
t h e
t r o p h o p h a s e
t o
i n d u c e a n
e n z y m e ( s )
n e c e s -
s a r y
f o r
p r o d u c t
s y n t h e s i s
d u r i n g
i d i o p h a s e .
I t
may b e t h a t
m e t h i o n i n e
p l a y s
a
s i m i l a r i n d u c -
t i v e r o l e
i n
t h e
b i o s y n t h e s i s
o f
f o s f o m y c i n .
I t
h a s
