97

FACTORS

AFFECTING

CEPHALOSPORIN

C BIOSYNTHESIS

F . Marini*, L. T e a t i n i , M. F a s a n i . I s t i t u t o B i o c h i m i c o I t a l i a n o Giovanni L o r e n z i n i S. p . A . via G. L o r e n z i n i , 2 -

-

Milan (Italy)

Summary Ca p r o l a c t a m , b u t a n - 2 - o n e , b u t a n - 2 - o l , and b u t a n - 1 , 3 - d i o l w e r e found to have a s t i m u l a t i n g e f f e c t on the b i o s y n t h e s i s of c e p h a l o s p o r i n C by a m u t a n t s t r a i n of C e p h a l o s p o r i u m .

Caprolactam

w a s shown to have a s y n e r g i s t i c effect, w h e n a d d e d to the f e r m e n t a t i o n m e d i u m t o g e t h e r with e a c h of the o t h e r t h r e e c o m p o u n d s .

Introduction C e p h a l o s p o r i n C is a f l - l a c t a m a n t i b i o t i c , w h i c h is p r o d u c e d in s u b m e r g e d a e r o b i c c u l t u r e by a r e s t r i c t e d n u m b e r of m i c r o o r g a n i s m s .

It is u s e d to p r e p a r e by a

c h e m i c a l p r o c e s s 7 - a m i n o c e p h a l o s p o r a n i c acid, w h i c h is the s t a r t i n g m a t e r i a l for the p r o d u c t i o n of v a r i o u s s e m i s y n t h e t i c c e p h a l o s p o r i n s of s u p e r i o r a n t i b i o t i c a c t i v i t y and of the g r e a t e s t c l i n i c a l and i n d u s t r i a l i m p o r t a n c e .

The development

of s e m i s y n t h e t i c c e p h a l o s p o r i n s has s t i m u l a t e d m a n y s t u d i e s on the f e r m e n t a t i o n and b i o s y n t h e s i s of c e p h a l o s p o r i n C (Kanzaki and F u j i s a w a , 1976).

We h a v e

s t u d i e d s o m e f e r m e n t a t i o n c o n d i t i o n s a f f e c t i n g the b i o s y n t h e s i s of c e p h a l o s p o r i n C by a m u t a n t s t r a i n of C e p h a l o s p o r i u m ( C e p h a l o s p o r i u m sp. I . B . I . f r o m C e p h a l o s p o r i u m sp. ATCC 11550.

1131) d e r i v e d

98

Methods Our f e r m e n t a t i o n e x p e r i m e n t s w e r e c a r r i e d out in 500 m l i n d e n t e d E r l e n m e y e r f l a s k s c o n t a i n i n g 50 m l of m e d i u m . at 2 2 ° C a n d st 250 r . p . m ,

The f l a s k s w e r e i n c u b a t e d on a r o t a r y s h a k e r

for 88 h o u r s .

A complex medium was used containing: peanut meal, soy bean meal, corn meal, b e e t m o l a s s e s , D L - m e t h i o n i n e , c a l c i u m c a r b o n a t e , m e t h y l o l e a t e and l a r d oil. The q u a n t i t y of c e p h a l o s p o r i n C p r e s e n t in the final f e r m e n t a t i o n b r o t h was d e t e r m i n e d by s p e c t r o p h o t o m e t r i c a s s a y as d e s c r i b e d b y C l a r i d g e et al (1969). In o u r s c r e e n i n g p r o g r a m m e v a r i o u s s u b s t a n c e s h a v e b e e n added to the b a s a l f e r m e n t a t i o n m e d i u m in o r d e r to t e s t t h e i r e f f e c t s on the b i o s y n t h e s i s of c e p h a l o s p o r i n C. Results F o u r c o m p o u n d s , n a m e l y c a p r o l a c t a m , b u t a n - 2 - o n e , b u t a n - 2 - o l and b u t a n - 1 , 3 - d i o l a p p e a r e d to be e x t r e m e l y i n t e r e s t i n g for t h e i r e f f e c t s in e n h a n c i n g c e p h a l o s p o r i n C biosynthesis. The u s e of t h e s e four c o m p o u n d s as a d d i t i o n a l c o m p o n e n t s of f e r m e n t a t i o n m e d i a in the p r o d u c t i o n of c e p h a l o s p o r i n C h a s b e e n r e c e n t l y p a t e n t e d by I s t i t u t o B i o c h i m i c o I t a l i a n o (1978).

Table 1 s h o w s the s t i m u l a t o r y e f f e c t s on c e p h a l o s p o r [ n C

b i o s y n t h e s i s by t h e s e c o m p o u n d s and the o p t i m a l c o n c e n t r a t i o n s at w h i c h they w e r e added to the f e r m e n t a t i o n m e d i u m .

99

Table 1 - S t i m u l a t o r y e f f e c t s on c e p h a l o s p o r i n C b i o s y n t h e s i s by c a p r o l a c t a m , b u t a n - 2 - o n e , b u t a n - 2 - o l , and b u t a n - 1 , 3 - d i o l .

Concentration

Cephalosporin C

I n c r e a s e d yield

(g/l)

mg/1

(%)

Added compound

None

m

1950

c apr olac tam

2.5

3015

55

butan- 2- one

8

2730

40

14

3100

59

4

3025

55

butan- 2- ol butan-1,3-diol

In t e r m s of s t i m u l a t o r y e f f i c i e n c y we have c a l c u l a t e d that c a p r o l a c t a m is the m o s t e f f e c t i v e compound, s i n c e the addition of only 9.1 m o l e s of c a p r o l a c t a m is e f f e c t i v e in giving an i n c r e a s e d p r o d u c t i o n of 1 m o l e of c e p h a l o s p o r i n C.

F o r c o m p a r i s o n we

can o b s e r v e that the s t i m u l a t o r y e f f i c i e n c y of D L - m e t h i o n i n e in our s t r a i n is 22.3 m o l e s added p e r m o l e of additional c e p h a l o s p o r i n C p r o d u c e d . We might s p e c u l a t e that c a p r o l a c t a m could be e f f e c t i v e as a p r e c u r s o r in the s y n t h e s i s of the a m i n o a d i p i c acid m o i e t y of c e p h a l o s p o r i n C.

B u t a n - 2 - o n e and

b u t a n - 2 - o l could be available for m e t a b o l i c c o n v e r s i o n into 2 - k e t o b u t y r i c acid, w h i c h is p o t e n t i a l l y involved in e n z y m a t i c r e a c t i o n s l e a d i n g to the s y n t h e s i s of valine and i s o l e u c i n e or even in the s y n t h e s i s of c y s t e i n e f r o m c y s t a t h i o n i n e .

It

has not b e e n p o s s i b l e to f o r m u l a t e any h y p o t h e s i s on the m e c h a n i s m of action of butan-1,3-diol.

100

The m o d e of a c t i o n of c a p r o l a c t a m could w e l l be d i f f e r e n t f r o m that of the t h r e e C4 c o m p o u n d s .

This c o n s i d e r a t i o n p r o m p t e d us to t e s t the e f f e c t s of the c o m b i n e d

addition of e a p r o l a e t a m with e a c h of the t h r e e C4 c o m p o u n d s , as shown in Table 2.

Table 2 - S t i m u l a t o r y e f f e c t s on cephalospor~.n C b i o s y n t h e s i s b y the c o m b i n e d addition of c a p r o l a c t a m with b u t a n - 2 - o n e , b u t a n - 2 - o l , and b u t a n - 1 , 3 - d i o l ( c o n c e n t r a t i o n s added as in Table 1).

Cephalosporin C Added c o m p o u n d s mgA

Relative increased yield (%)

Total increased y~eld (%)

None

1950

c a p r o l a c t a m alone

3015

caprolactam + butan-2-one

4200

39

115

caprolactam + butan-2-ol

4450

47

128

caprolactam + butan-1,3-diol

4655

54

139

55

The r e l a t i v e i n c r e a s e d yield r e p r e s e n t s the s i n g l e e f f e c t of e a c h of the t h r e e C4 c o m p o u n d s when added to the f e r m e n t a t i o n m e d i u m in p r e s e n c e of c a p r o l a c t a m ; the total i n c r e a s e d y i e l d r e p r e s e n t s the e f f e c t of the c o m b i n e d a d d i t i o n s . We can o b s e r v e that the s t i m u l a t o r y e f f e c t s shown in Table 2 of the c o m b i n e d a d d i t i o n of c a p r o l a c t a m with e a c h of the t h r e e C4 c o m p o u n d s a r e v e r y high, and in p a r t i c u l a r t h e y a r e h i g h e r than the s u m of the e f f e c t s of the individual s u b s t a n c e s

101

(Table 1).

This means that c a p r o l a c t a m is not only a s t i m u l a t o r y substance by

itself, but that it also has a s y n e r g i s t i c effect on the biosynthesis of cephalosporin C in p r e s e n c e of butan-2-one, butan-2-ol, or b u t a n - 1 , 3 - d i o l . We know that the tripeptide a m i n o a d i p y l - c y s t e i n y l - v a l i n e is a probable i n t e r m e d i a t e in the synthesis of cephalosporin C (Gorman and Huber, 1977).

If the mode of action

of c a p r o l a c t a m could be c o r r e l a t e d with the synthesis of the aminoadipic acid moiety of cephalosporin C and the mode of action of the th ree C4 compounds could be c o r r e l a t e d with the synthesis of valine and cy stein e, the sy n erg istic effect could have an acceptable explanation.

REFERENCES Claridge, C. A.,Vaughan, R. W . , K r e s e l , P., and Gourevitch, A. (1969). Spectrophotometric a s s a y for c e phalo sp o rin C in fermentation broths.

In:

A n t i m i c r o b i a l Agents and Chemotherapy 1969 (G. L. Hobby, e d . ) p p . 131-134 Bethesda: A m e r i c a n Society for Microbiology. Gorman, M., and Huber, F. (1977).

fl-Lactam antibiotics.

In. Annual Reports

on F e r m e n t a t i o n P r o c e s s e s (D. P e r l m a n , e d . ) V o l . 1, pp. 327-346, New York: Academic Press. Istituto B i o c h i m i c o Italiano (1978) G.B. Patent 1503851. Kanzaki, T . , and F u j i s a w a , Y. (1976).

B i o s y n t h e s i s of Cephalosporins.

In:

Advances in Applied Microbiology (D. P e r l m a n , ed.) Vol. 20 pp. 159-201, New York: Academic P r e s s .