Microbial Production of Avermectin - ACS Symposium Series (ACS

Chapter 20

Microbial Production of Avermectin Prakash S. Masurekar

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Merck and Company, Inc., Rahway, NJ 07065

The avermectins, a group of eight closely related compounds produced by Streptomyces avermitilis, are potent, broad spectrum antiparasitic agents, effective in animals and plants. These compounds are oleandrose disaccharide derivatives of a pentacyclic 16 membered macrolide ring and act as GABA agonists. It has been shown that a hydroxyl at C-5 and the disaccharide moiety are essential for good activity. Initial fermentation medium development in shake flasks resulted in the selection of dextrose, peptonized milk and autolysed yeast as medium ingredients. Process scaleup studies were done in highly instrumented fermentors. Kinetics of the fermentation showed that the product is formed after near exhaustion of glucose and completion of cell growth. Glucose was also found to repress substantially an extracellular neutral lipase. Postulated biosynthetic scheme involves the formation of the macrolide ring from acetate, propionate and isoleucine via polyketide pathway followed by methy1ation and glycosylation. Genetic evidence in support of this scheme was obtained by characterization of mutants which have altered compositions of avermectins. Further, avermectin B2 O-methyltransferase was shown to increase coordinately with the yield of the avermectins in productivity mutants. Avermectins are very potent, broadspectrum a n t i p a r a s i t i c agents. These were detected by Merck s c i e n t i s t s i n fermentation broth of Streptomyces a v e r m i t i l i s i n a screen which involved the use of mice infected with Nematospiroides dubius. The microbial culture was o r i g i n a l l y isolated at the Kitasato Institute from a s o i l sample collected at Kawana Ito City i n Japan (1,2). Subsequent high performance l i q u i d chromatographic analysis showed that the broth contained eight closely related compounds. The four major components were designated as Ala, A2a, Bla and B2a and the four minor components were designated as Alb, A2b, Bib and B2b.

0097-6156/88/0362-0242$06.00/0 © 1988 American Chemical Society

Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Microbial Production of Avermectin

243

Ivermectin, which i s sold commercially, i s 22,23-dihydroavermectin 31. I t contains at least 80% 22,23-dihydroavermectin Bla and not more than 20% 22,23-dihydroavermectin Bib.


CHEMISTRY T^e structures of the eight components were determined by "^H and ^ C NMR and mass spectroscopy as well as X-ray crystallography (3,4). The basic structure i s shown i n Figure 1. Avermectins contain a 16-membered macrolide ring, a spiroketal couched i n two 6-membered rings, a cyclohexene d i o l fused to 5-membered c y c l i c ether and α-L-oleandrosyl-a-L-oleandrosyloxy disaccharide. The eight avermectins d i f f e r from each other i n the nature of the substituents on C-5, C-23 and C-25. In those designated as A, R5 i s methyl as compared to those designated as Β where R5 i s H. Avermectins of series 1 contain 22,23-olefin, whereas i n those of series 2 t h i s o l e f i n i s reduced. Furthermore they contain a hydroxy1 group at C-23. A sec-butyl substituent on C-25 character izes "a" group while those of "b" group contain an isopropyl substituent. Thus, a l l possible combinations of these three structural differences give r i s e to the eight components. In order to understand the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p , a number of avermectin derivatives were prepared chemically. Some of the types of reactions c a r r i e d out include acylation, (5) a l k y l a t i o n (6), hydrogénation (7) and oxidation (8). Comparisons of the a n t i p a r a s i t i c a c t i v i t i e s of the eight compounds produced i n the fermentation along with those of chemically modified avermectins provided information on the structure a c t i v i t y r e l a t i o n s h i p . The disaccharide was essential f o r good a c t i v i t y (7). The a c t i v i t y of the aglycone against g a s t r o i n t e s t i n a l helminths was reduced 30-fold as compared to that of the disaccharide. S i m i l a r l y , those which contained a 5-hydroxy group were more potent than those containing a 5-methoxy, i . e , avermectins of "B" series were more potent than those of "A" series (5,7). Reduction of 22,23 double bond had only small e f f e c t on the a c t i v i t y (1). However, differences between the a c t i v i t i e s of avermectin-"1" and "2" seemed to be dependent on the s e n s i t i v i t i e s of p a r t i c u l a r parasites and the mode of administ r a t i o n (2,9). Acylation of 4"-hydroxy group did not have a detrimental e f f e c t on the a c t i v i t y . The subtle differences between the a c t i v i t i e s of avermectins "1" and "2" indicated 22,23dihydroavermectin B l to be a more desirable product. MODE OF ACTION OF AVERMECTIN This was studied i n nematodes (10), i n lobster (11) and i n mammalian brain (12,13). For example i n the studies done with the nematode Ascaris the dorsal excitatory motoneuron was i n d i r e c t l y stimulated v i a i t s ventral nerve cord (10). Avermectin addition eliminated t h i s response (Figure 2). The GABA antagonist picrotoxin restored the response. These finding suggested that avermectins act by interference with GABA mediated neural signal transmission. Studies with lobster walking leg stretcher muscle showed that avermectins i n t e r f e r e with GABA mediated neuromuscular transmission (11). These data suggested that avermectin B l acted as a GABA agonist. This was confirmed by the studies with mammalian brain (12,13).


244

T H E IMPACT O F CHEMISTRY ON BIOTECHNOLOGY

OCH3 OCH3 A

fi

H C-^0 3

>///

ÇH3

O,


H3C

ORs

Figure 1. Structure of avermectin. See text for the d e f i n i t i o n s of R5 and R26. Reproduced with permission from Réf. 1. Copyr i g h t 1984 Academic Press Inc.

AVM KftQAnl)

Plcrokwdn (Wyug/mi)

,., W

o

e

k h

Pfcrotoxin (K>/ig/mO

>

Ε

40

80

120

160

200

Time (min)

Figure 2. E f f e c t of avermectin on the response of the dorsal excitatory motoneuron of nematode Ascaris. The arrows indicate the time of additions. Reproduced with permission from Réf. 1. Copyright 1984 Academic Press Inc.


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BIOLOGICAL ACTIVITY Ivermectin which i s the marketed product, has been shown to be e f f e c t i v e against a wide range of endoparasites i n sheep, c a t t l e and horses (14,15,16). A single application either o r a l l y or parenterally was adequate for the elimination of the parasites. I t was also found to be e f f e c t i v e against a number of ectoparasites belonging to a l l the major groups l i k e f l i e s , mites, l i c e , t i c k s and fleas (1,9). In man, a c t i v i t y against Onchocerca volvulus, which causes blindness, was shown (17). In agriculture, a c t i v i t y was shown against a number of pests and insects such as two spotted spider mites on roses and beans (18,19), leaf miner on ornamentals (20), bean aphids on beans (19) and c i t r u s rust mites on oranges (19). Thus, avermectins are t r u l y broad spectrum and highly e f f e c t i v e a n t i p a r a s i t i c agents. FERMENTATION PROCESS DEVELOPMENT MEDIUM STUDIES. I n i t i a l development of the process to produce avermectin was done i n shake f l a s k s . This involved optimization of the production medium and s t r a i n improvement. For medium development studies the seed was grown i n medium containing cerelose, starch, beef extract, Ardamine PH, N-Z amine and trace elements for 1-2 days (21). The composition of i n i t i a l production medium i s described i n Table I. Table I.

Complex Medium for the Production of Dextrin D i s t i l l e r s solubles Autolyzed yeast CoCl .6H 0 D i s t i l l e d water ρ H 7.3 2

2

Avermectin

40 g 7 g 5 g 0..05 g 1 liter

Reproduced with permission from Réf. 2. Copyright 1982 Japan A n t i b i o t i c Research Association. This medium was best of a number of complex media screened. Y i e l d i n t h i s medium was 9 yg/ml and i t served as the basis f o r medium improvement studies (21). As i s customary i n these types of studies, various carbon and nitrogen sources along with vitamins and trace elements were t r i e d (21). Results of three such experiments are shown i n Table I I . In the f i r s t experiment the carbon source was varied. Dextrose was found to give the best t i t e r . In the second experiment d i f ferent nitrogen sources were t r i e d . A combination of peptonized milk and polyglycol P-2000 resulted i n increase i n production tô 53 yg/ml. Further improvement i n the y i e l d was obtained when Ardamine PH was used as a vitamin source. Optimization of dextrose, peptonized milk and Ardamine PH concentrations resulted i n medium shown i n Table I I I .


Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988. None Yeast extract Ardamine PH

Fermamine I Sheften WP100 Primatone Peptonized milk Peptonized milk Peptonized milk Peptonized milk Peptonized milk

Dextrose Dextrose Dextrose Dextrose Dextrose

Dextrose Dextrose Dextrose

2

3

Reproduced with permission from Ref. 2.

Yeast Yeast Yeast Yeast Yeast

Pharmamedia Pharmamedia Pharmamedia Pharmamedia

Dextrin Sucrose Dextrose Lactose

1

13 15 8 17 53 38 75 83

+

-+ +

5 0 30 0

Avermectin (V g/ml)

-

_

Polyglycol

Copyright 1982 Japan A n t i b i o t i c Research Association.

extract extract extract extract extract

yeast yeast yeast yeast

Primary Primary Primary Primary

Nitrogen Source

Vitamin Source

E f f e c t of Various Carbon and Nitrogen Sources on Production

Carbon Source

Experiment Number

Table I I .


n X Ζ ο ο ο

W Ο H W

Ο

H »

η χ w S

Ο

9

>

s

Η X M

fed

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Table I I I . Optimized Complex Medium for the Production of Avermectin


Cerelose Peptonized milk Autolyzed yeast Polyglycol P-2000 D i s t i l l e d water pH 7.0

45 g 24 g 2.5 g 2.5 ml 1 liter

Reproduced with permission from Ref. 2. Copyright 1982 Japan A n t i b i o t i c Research Association. Presence of P-2000 was c r i t i c a l to the success of t h i s medium. Avermectin production by the o r i g i n a l s o i l i s o l a t e i n t h i s medium was about 100 yg/ml. Thus, at t h i s point, the medium improvement resulted i n a 10-fold increase i n the y i e l d . STRAIN IMPROVEMENT. U l t r a - v i o l e t i r r a d i a t i o n was used to mutate the o r i g i n a l culture. The mutated cultures were randomly selected and tested for avermectin production. One of the mutants was found to have superior a b i l i t y to produce avermectins. I t was able to synthesize avermectin at a higher rate and for a longer time than i t s parent. This mutant designated ATCC 31271 produced 500 yg/ml as compared to 120 yg/ml by i t s parent (21). KINETICS OF FERMENTATION. Four variables, dry c e l l weight, pH, glucose u t i l i z a t i o n and avermectin production are described i n Figure 3. The growth was e s s e n t i a l l y complete i n the f i r s t 1/3 of the cycle, subsequently, the dry c e l l weight remained constant. About 85% of glucose was u t i l i z e d during the growth phase. The rest was used up i n the production phase. The pH dropped to 5.9 during the growth and increased to 6.9 as the growth slowed down. The pH then slowly declined to 6.3 during avermectin production period and f i n a l l y increased to 7.0 i n the l a s t 20% of the cycle. Thus, the pH changes were consistent with the pattern of glucose u t i l i z a t i o n . Avermectin synthesis began after growth was complete and continued l i n e a r l y u n t i l the glucose was used up. The rate of synthesis declined after the exhaustion of glucose. These r e s u l t s indicate that pH can be used to monitor the progress of fermentation. Since we noted that a limited amount of glucose was used during the production phase, we studied the p o s s i b i l i t y of other catabolic enzymes l i k e Lipases might be active at that'- time-.KINETICS OF LIPASE PRODUCTION. For t h i s purpose the culture was grown i n a production medium similar to the one described. Samples were taken p e r i o d i c a l l y and assayed for growth, pH, avermectin production, glucose u t i l i z a t i o n , neutral lipase production and phospholipase production. E x t r a c e l l u l a r neutral lipase was measured by determining release of ^^C-palmitic acid (22). The a c t i v i t y of e x t r a c e l l u l a r phospholipase was assayed by measuring the inorganic phosphate released by the enzyme (23). E s s e n t i a l l y no lipase was found as long as glucose was present (Figure 4). I t was derepressed almost 100-fold when the glucose was exhausted. I t rapidly reached the maximum and 'then declined. The reason for t h i s might be that the synthesis of lipase was not continued i n the l a s t 10% of the cycle and the enzyme synthesized up to that time was degraded. Phospholipase did not show t h i s s e n s i t i v i t y to glucose. I t s a c t i v i t y seemed to be at the maximum during the early part of the

American Chemical Society Library 1155

16th

St, N.W.

Phillips et al.; The Impact of Chemistry on Biotechnology Washington, O.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

THE IMPACT OF CHEMISTRY ON BIOTECHNOLOGY


248

Cycle Time % Max

Figure 3. Kinetics of avermectin fermentation i n shake f l a s k s . Legend: pH (O), dry c e l l weight ( • ) , avermectin ( Δ ) and glucose ( φ ) .

100 ο-

&

ο

Cycle Time %Μαχ

Figure 4. Kinetics of lipase production by Streptomyces a v e r m i t i l i s i n shake f l a s k s . Legend: glucose ( O ) , lipase and phospholipase ( Δ.) ·


(•)


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f e r m e n t a t i o n and t h e n r e d u c e d as t h e f e r m e n t a t i o n p r o c e e d e d . The maximum p h o s p h o l i p a s e a c t i v i t y o b s e r v e d was 5 0 - f o l d lower t h a n t h a t of n e u t r a l l i p a s e a c t i v i t y . These r e s u l t s s u g g e s t e d t h a t a l t e r n a t i v e c a r b o n s o u r c e s may be used d u r i n g thé p r o d u c t i o n phase. SCALE-UP STUDIES. V a r i o u s s i z e f e r m e n t o r s , 14-L t o 800-L, were used t o d e t e r m i n e t h e c r i t i c a l v a r i a b l e s f o r s c a l e - u p . These f e r m e n t o r s were i n s t r u m e n t e d t o measure pH, t e m p e r a t u r e , d i s s o l v e d oxygen, a e r a t i o n and a g i t a t i o n r a t e s , exhaust gas c o m p o s i t i o n e t c . A d i s t r i b u t e d d i g i t a l c o n t r o l network based on a Honeywell TDC-2000 was used (24). V a l u a b l e p r o c e s s i n f o r m a t i o n was o b t a i n e d from t h e s e i n s t r u m e n t e d f e r m e n t o r s , which n o t o n l y improved i t s u n d e r s t a n d i n g b u t a l s o made i t p o s s i b l e t o implement v a r i o u s c o n t r o l s t r a t e g i e s . These s t u d i e s r e s u l t e d i n a p r o c e s s w h i c h was used i n t h e p r o d u c t i o n f e r m e n t o r s t o manufacture a v e r m e c t i n . An example o f t h i s i s t a k e n from t h e work o f B u c k l a n d e t aJL (24). From t h e p r o c e s s c o n t r o l p o i n t o f view, s p e c i f i c growth r a t e i s an i m p o r t a n t v a r i a b l e . However, i t i s n o t p o s s i b l e t o measure i t on l i n e i n m y c e l i a l f e r m e n t a t i o n s by c o n v e n t i o n a l means. I f t h e r e l a t i o n s h i p between growth r a t e and oxygen uptake r a t e i s c o n s i d e r e d , i t can be seen t h a t i t i s f e a s i b l e t o e s t i m a t e t h e s p e c i f i c growth r a t e from d a t a c o l l e c t e d by exhaust gas a n a l y s i s . S i m i l a r l y , growth can be e s t i m a t e d from t h e t o t a l amount o f oxygen used. F o r example, t h e growth r a t e can be e x p r e s s e d as a c o n s t a n t t i m e s t h e oxygen uptake r a t e and t h e d r y c e l l weight t o be t h e same c o n s t a n t t i m e s i n t e g r a l o f oxygen uptake r a t e i n a p p r o p r i a t e l i m i t s . The r a t i o o f t h e s e two e q u a t i o n s g i v e s an e s t i m a t e o f t h e s p e c i f i c growth r a t e . F i g u r e 5 shows the d a t a o b t a i n e d i n an 800-L f e r m e n t e r . Total oxygen uptake and s p e c i f i c growth r a t e a r e shown as a f u n c t i o n o f time. The s p e c i f i c growth r a t e was d e t e r m i n e d from e x h a u s t gas analysis. The d r y c e l l w e i g h t i s p l o t t e d on the second Y - a x i s f o r purpose o f comparison. The growth r a t e was h i g h i n t h e f i r s t e i g h t hours i n d i c a t i n g p o s s i b l y e x p o n e n t i a l growth. A f t e r t h a t i t d e c l i n e d and r e a c h e d a v e r y low v a l u e by 24 h r s . A v e r m e c t i n p r o d u c t i o n d i d not b e g i n u n t i l a f t e r t h e s p e c i f i c growth r a t e had f a l l e n v i r t u a l l y t o z e r o , which was s i m i l a r t o t h e o b s e r v a t i o n made i n shake flasks. F o r t h e f i r s t 30 h r s , t h e r e was a good c o r r e l a t i o n between the d r y c e l l w e i g h t and t h e t o t a l oxygen u p t a k e . These r e s u l t s demonstrate t h a t t h e s p e c i f i c growth r a t e and d r y c e l l weight e s t i m a t e d from t h e exhaust gas a n a l y s i s r e p r e s e n t t r u e v a l u e s and hence can be used f o r feedback c o n t r o l o f t h e p r o c e s s . V e r y o f t e n i n p r o c e s s development t h e p r o b l e m i s as much o f s c a l e - u p as o f scale-down. P r o d u c t i o n f a c i l i t i e s cannot be m o d i f i e d w i t h o u t a l a r g e o u t l a y o f c a p i t a l , and hence i t i s e s s e n t i a l t o model p r o d u c t i o n f e r m e n t o r s i n p i l o t p l a n t f e r m e n t o r s . This i s g r e a t l y f a c i l i t a t e d by h a v i n g i n s t r u m e n t e d f e r m e n t o r s , as demons t r a t e d by t h e f o l l o w i n g example (24). A new p r o d u c t i o n medium was d e v e l o p e d i n shake f l a s k s and was found t o have a b e n e f i c i a l e f f e c t on t h e y i e l d . However, b e f o r e i t c o u l d be recommended f o r p r o d u c t i o n , i t was n e c e s s a r y t o d e t e r mine i f i t c o u l d be used i n t h e p r o d u c t i o n f e r m e n t o r s . For t h i s purpose new medium was r u n i n an 800-L f e r m e n t o r . The a g i t a t o r speed was c o n t r o l l e d t o p r e v e n t t h e d i s s o l v e d oxygen c o n c e n t r a t i o n from f a l l i n g below 20% as i t was known t h a t d i s s o l v e d oxygen c o n c e n t r a t i o n below t h a t l e v e l was d e t r i m e n t a l t o a v e r m e c t i n p r o d u c t i o n . P r e v i o u s e x p e r i e n c e i n d i c a t e d t h a t i f t h e peak oxygen demand


T H E IMPACT OF CHEMISTRY ON

250

BIOTECHNOLOGY

was below 35 mmoles/l.hr and the broth v i s c o s i t y was the same as that obtained i n the old medium then the new medium would work i n the production fermentor. Oxygen uptake rates i n the new and the old medium are plotted against time for the f i r s t 50 hrs of the fermentation i n Figure 6. The peak oxygen demand for the new medium was lower than that f o r the old medium and i t was less than 35 mmoles/l.hr. As a cross-check, power consumption was measured for the new medium i n the 800-L fermentor, which too indicated that the power per unit volume requirements for the new medium would be met in the production fermentor. These r e s u l t s showed that the new medium could be recommended f o r production.


BIOSYNTHESIS OF AVERMECTINS 13

ISOTOPE INCORPORATION STUDIES. In i n i t i a l studies, [1- C] acetate and Dt- C]propionate were used as precursors for incorporation into avermectins. At the end of the fermentation, avermectins Ala, A2a, Bla and B2a were isolated and analysed by C-NMR spectroscopy (25). The r e s u l t s showed i d e n t i c a l carbon d i s t r i b u t i o n for the macrolide ring as expected from the assumption of polyketide pathway for i t s synthesis. Seven acetate and f i v e propionate units were incorporated as shown i n Figure 7. This figure shows the p o s i t i o n where the acetate and propionate precursors are incorporated. C-25 carbon and i t s sec-butyl substituent were not labeled by ( C) acetate or propionate. These were established to be derived from L-isoleucine (1). The C-25 and i t s isopropyl substituent i n avermectin "b" series are derived from L-valine (1). The disaccharide i s derived from glucose (26). Further studies with [1- 02,1- C]propionate and [ 1 - 0 , 1 - C ] a c e t a t e incorporation and NMR analysis of four avermectins showed that oxygens attached to C-7 and C-13 of a l l avermectins and C-23 of "2" series avermectins come from propionate; and those attached to C - l , C-5, C-17 and C-19 are from acetate (25). i:i

13

l3

13

18

13

2

BIQSYNTHETIC PATHWAY. Figure 8 shows the biosythetic scheme postulated from these data (27). Avermectin biosynthesis i s i n i t i a t e d from the precursors isoleucine, acetate, propionate and glucose and proceeds through the intermediate "X". At t h i s point, a dehydration reaction separates the biosynthesis of avermectin " l " s from the "2"s. We have not isolated any intermediates i n t h i s proposed pathway up to 6,8adeoxy-5-keto Bla algycone (28) and B2a aglycone. The reactions from either Bla aglycone and B2a aglycone to four avermectins are similar and involve glycosylation and methylation (27). One exception to t h i s s i m i l a r i t y i s that Bla cannot be converted into A l a . In addition to the labeling studies described e a r l i e r , evidence in support of t h i s pathway has come from the i s o l a t i o n of mutants which produce altered composition of avermectins and from the enzymatic studies. I have l i s t e d some of the compositional mutants in Table IV. The f i r s t class of mutants i s defective i n 5-0-methyl transferase a c t i v i t y and as a r e s u l t i t cannot convert avermectin "B"s-into "A"s and thus, accumulates avermectin Bs (29). The second class



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Figure 5. Estimation of the dry c e l l weight and the s p e c i f i c growth rate from the t o t a l oxygen consumed. Legend: t o t a l oxygen consumed and proportional dry c e l l weight (—) and s p e c i f i c growth rate (—) . Reproduced with permission from Ref. 24. Copyright 1985 Nature Publishing Co.

401

Hours

Figure 6. Comparison of oxygen uptake rates i n two production media. Legend: old medium ( Δ ) and new medium ( • ) . Reproduced with permission from Ref. 24. Copyright 1985 Nature Publishing Co.


T H E IMPACT OF CHEMISTRY ON BIOTECHNOLOGY

252


OH

1β

0= 0

CH3COOH

CH3CH2COOH

Figure 7. Incorporation of acetate and propionate units into macrolide ring of avermectin. Reproduced with permission from Ref. 1. Copyright ,1984 Academic Press Inc.

Acetate, Propionate, Isoleucine, Glucose

1 1 6,8a-Deoxy-5-Keto Β la Aglycone

I 1

Ï

(M) A2a Aglycone

Β 2a Aglycone

(G)

(G)

, (M)

B2o Monosaccharide

(G) A2a

Bla Aglycone

• Ala Aglycone

(G)

(G)

Bla Monosaccharide

6

(M)

|« ' • B2a

81a

(M)

|(G) Ala

Figure 8. Proposed scheme f o r the biosynthesis of avermectins. G = glycosylation, M = methylation. Reproduced with permission of the authors from Ref. 27.


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Table IV.


Mutant No.


253

Mutants Which Produce Different Compositions of Avermectin Defect

Product

5-O-methyltransferase Glycosyl-O-methyltransferase Glycosyltransferase Furan 'cyclase' _____

Bs A & Β desmethyls A aglycones many

of mutants lacks glycosyl-O-methyltransferase and forms avermectin A and Β desmethyls (29). The t h i r d class of mutants i s impaired i n the glycosylation reaction. They form mainly avermectin A aglycones. The fourth class of mutants i s defective i n furan "cyclase" and cannot form the 5-membered c y c l i c ether (28). These produce many d i f f e r e n t products. Thus, these classes of mutants provide genetic evidence i n support of the proposed reactions. Studies were done to i s o l a t e enzymes which catalyze the reaction i n the proposed biosynthetic pathway. One which i s catalysed by 5-O-methyltransferase i s shown i n Figure 9. Avermectin Β component i s converted into A component with S-adenosyl methionine as a methyl donor. The enzyme was detected i n the c e l l - f r e e extracts after removal of c e l l debris (30) . The assay involved conversion of avermectin B2a aglycone into A2a aglycone, which was measured by determining the incorporation C - l a b e l e d methyl from S-adenosyl methionine. The enzyme i s s p e c i f i c for C-5 hydroxy1 and does not catalyse the transfer of a methyl group to C-3' or C-3" i n the disaccharide moiety. This was independently supported by the i s o l a t i o n of mutants of classes 1 and 2 described e a r l i e r . 14

OCHs Avermectin Β Component

Avermectin A Component

Figure 9. Reaction catalysed by avermectin B2 O-methyltransferase. Reproduced with permission from Ref. 30. Copyright 1986 American Society for Microbiology.

In order to further investigate i t s role i n avermectin biosynthesis, i t s a c t i v i t y was determined i n four strains of S_. a v e r m i t i l i s (Table V). These four strains are sequentially improved producers of avermec t i n obtained by UV and N-methyl-N-nitrosourethane mutagenesis (30). They were grown i n the production medium described e a r l i e r for 6 days. Each day from 48 hrs on, avermectin production and the


254

T H E IMPACT O F CHEMISTRY O N BIOTECHNOLOGY

Table V. Avermectin Β O-methyltransferase A c t i v i t y and Avermectin Production i n Strains A, Β, C, and D


Strain A Β C D

Maximum Sp. Act. of O-met. Trans. (nmol/h per mg of Protein) 0.37 0.86 1.06 1.35

Total Avermectin at 144 h (Relative Units) 0.85 2.10 2.4 3.0

Fold increase i n : Maximum Sp. Act. of Avermectin O-met. Trans. 1 1 2.5 2.3 2.8 2.9 3.5 3.6

Reproduced with permission from Ref. 30. Copyright 1986 American Society for Microbiology. a c t i v i t y of the O-methyltransferase was determined. Avermec t i n was measured by HPLC. The enzyme assay was as described. The maximum s p e c i f i c a c t i v i t y and the avermectin production i s shown here. The s p e c i f i c a c t i v i t y of the enzyme increased coordinately with the increase i n avermectin production. I t i s interesting that although the s p e c i f i c a c t i v i t y of the enzyme increased 3.5-fold, the r a t i o of Β to A components remained unchanged. These results suggest that i n any one of the strains t h i s enzyme i s not rate l i m i t i n g for the conversion of avermectin Β to A. Such information i s c r u c i a l i n the design of more r a t i o n a l approaches to improve y i e l d s . In summary, avermectin i s a potent, t r u l y broad spectrum a n t i p a r a s i t i c agent, produced by £. a v e r m i t i l i s . I t was discovered through the use of a targeted screen. The fermentation process was developed f i r s t i n shake f l a s k s , where medium optimization and s t r a i n improvements gave a 50-fold increase i n y i e l d . The process was further refined and scaled up i n highly instrumented fermentors with sophisticated process control. Biosynthetic studies with labeled precursors, i s o l a t i o n of altered compositional mutants and enzymological studies have given a f a i r l y good picture of how avermectins are synthesized.

LITERATURE CITED 1. Fisher, M.H.; Mrozik, H. In Macrolide Antibiotics Chemistry, Biology and Practice; Omura, S., Ed.; Academic: New York, 1984; Chapter 14. 2. Stapley, E.O.; Woodruff, H.B. In Trends in Antibiotic Research; Umezawa, H., Demain, A. L., Mata, T. and Huchinson, C.R., Ed.; Japan Antibiotic Research Association, 1982; p. 154. 3. Albers-Schonberg, G.; Arison, B.H.; Chabala, J.C.; Douglas, A.W.; Eskola, P.; Fisher, M.H.; Lusi, Α.; Mrozik, H., Smith, J.L.; Tolman, R.L. J. Am. Chem. Soc. 1981, 103, 4216-21. 4. Springer, J.P.; Arison, B.H.; Hirshfield, J.M.; Hoogsteen, K. J. Am. Chem. Soc. 1981, 103, 4221-24. 5. Mrozik, H.; Eskola, P.; Fisher, M.H.; Egerton, J.R.; Cifelli, S.; Ostlind, D.A. J. Med. Chem. 1982, 25, 658-63. 6. Fisher, M.H.; Lusi, Α.; Tolman, R.L. U.S. Patent 4,200,581, 1980.



20. MASUREKAR


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7. Chabala, J.C.; Mrozik, H.; Tolman, R.L.; Eskola, P.; Lusi, A.; Peterson, L.M.; Woods, M.E.; Fisher, M.H.; Campbell, W.C.; Egerton, J.R.; Ostlind, D.A. J. Med. Chem. 1980, 23, 1134-36. 8. Chabala, J.C.; Rosegay, Α.; Walsh, M.A.R. J. Agric. Food Chem. 1981, 29, 881. 9. Campbell, W.C.; Fisher, M.H.; Stapley, E.O.; Albers-Schonberg, G.; Jacob, Τ.A. Science, 1983, 221, 823-28. 10. Kass, S.; Wang, C.C.; Walrond, J.P.; Stretton, A.O.W. Proc. Nat. Acad. Sci. 1980, 77, 6211-15. 11. Fritz, L.C.; Wang, C.C.; Gorio, A. Proc. Nat. Acad. Sci. 1979, 76, 2062-66. 12. Pong, S.S.; Wang, C.C. Neuropharmacology 1980, 19, 311-17. 13. Pong, S.S.; Wang, C.C. J. Neurochemistry 1982, 38, 375. 14. Egerton, J.R.; Birnbaum, J.; Blair, L.S.; Chabala, J.C; Conroy, J.; Fisher, M.H.; Mrozik, H.; Ostlind, D.A.; Wilkins, C.A.; Campbell, W.C. Bri. Vet. J. 1980, 136, 88-97. 15. Egerton, J.R.; Eary, C . H . ; Suhayda, D. Vet. Parasit. 1981, 8, 59-70. 16. Egerton, J.R.; Brokken, E.S.; Suhayda, D.; Eary, C.H.; Wooden, J.W.; Kilgore, R.L. Vet. Parasit. 1981, 8, 83-88. 17. Aziz, M.Α.; Diallo, S.; Diopp, I.M.; Lariviere, M.; Porta, M. Lancet 1982, 1982-II, 171-173. 18. Green, A. St.J.; Dybas, R.A. Proc. 1984 British Corp Protection Conf. 1984, 3, 11A-7. 19. Dybas, R.A. U.S. Patent 4,560,677, 1985. 20. Green, A. St.J.; Hejne, B.; Schreus, J . ; Dybas, R.A. Med. Fac. Landbouww. Rijksuniv. Gent 1985, 50/2b, 603-22. 21. Burg, R.W.; Miller, B,M.; Baker, E.E.; Birnbaum, J . ; Currie, S.A.; Hartman, R.; Kong, Y.L.; Monaghan, R.L.; Olson, G.; Putter, I.; Tunac, J.B.; Wallick, H.; Stapley, E.O.; Oiwa, R.; Omura, S. Antimicrob. Agents Chemother. 1979, 15, 361-67. 22. Khoo, J.C.; Steinberg, D. In Methods in Enzymology;-Lowenstein, J.M. Ed.; Academic: New York, 1975, Vol. 35, p.181. 23. Krug, E.L.; Kent, C. In Methods in Enzymology; Lowenstein, J.M.; Academic: New York, 1981, Vol. 72, p. 347. 24. Buckland, B.; Brix, T.; Fastert, H.; Gbewonyo, K.; Hunt, G.; Jain, D. Biotechnology, 1985, 3, 982-88. 25. Cane, D.E.; Liang, T.-C.; Kaplan, L.; Nallin, M.K.; Schulman, M.D.; Hensens, O.D.; Douglas, A.W.; Albers-Schonberg, G. J. Am. Chem. Soc. 1983, 105, 4110-12. 26. Schulman, M.D.; Valentino, D.; Hensens, O. J. Antibiotics, 1986, 39, 541-49. 27. Chen, T.S.; Inamine, E. Twenty Seventh Annual Mtg. of Am. Soc. Pharmacognosy 1986. Abst #4. 28. Goegelman, R.T.; Gullo, V.P.; Kaplan, L. U.S. Patent 4,378,353, 1983. 29. Ruby, C.L.; Schulman, M.D.; Zink, D.L.; Streicher, S.L. Proc. 6th I n t ' l . Symp. on Actinomycetes Biol. 1985, Vol. Α., p. 279. 30. Schulman, M.D.; Valentino, D., Nallin, M.; Kaplan, L. Antimocrob. Agents Chemotherap. 1986, 29, 620-24. RECEIVED July

27, 1987


Microbial Production of Avermectin - ACS Symposium Series (ACS

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