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UBC Theses and Dissertations

Construction of galactose assimilating, carotenoid producing yeasts by protoplast fusion 1988

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C O N S T R U C T I O N OF G A L A C T O S E A S S I M I L A T I N G , C A R O T E N O I D PRODUCING Y E A S T S BY P R O T O P L A S T F U S I O N b y C H R I S T I N E S. HANSEN B . S c . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 8 5 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE R E Q U I R E M E N T S FOR THE D E G R E E OF MASTER OF S C I E N C E i n T H E F A C U L T Y OF GRADUATE S T U D I E S ( D e p a r t m e n t o f F o o d S c i e n c e ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A J u n e , 1 9 8 8 (P C h r i s t i n e S. H a n s e n , 1 9 8 8 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) - i i - ABSTRACT P r o t o p l a s t s were p r e p a r e d f r om two y e a s t s t r a i n s J \ rhodozyma (ATCC 24202) and JC f r a g i l i s (ATCC 8455). P r o t o p l a s t s p r e p a r e d f r o m P . rhodozyma were f a c i l i t a t e d by p r i o r g r o w t h o f t h e c e l l s i n a m e d i a c o n t a i n i n g S - ( 2 - a m i n o e t h y l ) - L - c y s t e i n e . P r o t o p l a s t s f r om t h e s e two y e a s t g e n e r a were f u s e d e i t h e r by t h e use o f e l e c t r o f u s i o n o r p o l y e t h y l e n e g l y c o l t r e a t m e n t . S t a b l e c a r o t e n o i d p r o d u c i n g c e l l l i n e s were s e l e c t e d by g r o w t h a t 30°C on y e a s t n i t r o g e n base p l u s g a l a c t o s e . S e l e c t e d s i n g l e f u s a n t s d i s p l a y t a x o n o m i c c h a r a c t e r i s t i c s common t o b o t h g e n e r a w i t h a c e l l u l a r m o r p h o l o g y and a c a r o t e n o i d c o m p o s i t i o n s i m i l a r t o t h a t o f P . r hodozyma . — i i i — TABLE OF CONTENTS PAGE ABSTRACT i i T A B L E OF CONTENTS i i i L I S T . O F TABLES i v L I S T OF FIGURES v DEDICATION v i i ACKNOWLEDGEMENT v i INTRODUCTION 1 L I TERATURE REVIEW I G e n e t i c M a n i p u l a t i o n s 3 I I C a r o t e n o i d s 7 I I I P h a f f i a r h o d o z y m a 13 IV K l u y v e r o m y c e s f r a g i 1 i s 16 MATERIALS AND METHODS I S t r a i n s 18 I I C u l t i v a t i o n o f O r g a n i s m s f o r P r o t o p l a s t P r o d u c t i o n .... 18 I I I B u f f e r s a n d R e a g e n t s 18 IV M e d i a 19 V P r o t o p l a s t F o r m a t i o n , F u s i o n a n d R e g e n e r a t i o n 20 VI S e l e c t i o n a n d G r o w t h o f F u s a n t s 22 V I I C a r o t e n o i d A n a l y s i s 23 RESULTS I C h a r a c t e r i s t i c s o f t h e P a r e n t O r g a n i s m s 25 I I P r o t o p l a s t F o r m a t i o n by P a r e n t a l S t r a i n s 27 I I I A m e l i o r a t i o n o f t h e C e l l W a l l o f j \ r h o d o z y m a t o Enzyme D i g e s t i o n 28 IV F u s i o n o f ] C f r a g i l i s a n d A E C - t r e a t e d f \ r h o d o z y m a P r o t o p l a s t s 32 V S e l e c t i o n o f F u s e d C e l l s 33 VI I d e n t i f i c a t i o n o f t h e P i g m e n t P r o d u c e d 35 DISCUSSION 55 CONCLUSIONS 63 REFERENCES 64 APPENDICES - iv - LIST OF TABLES PAGE Table I. Selected Biochemical test results for J \ rhodozyma, f raq i l i s and Fusant #26 26 Table II. Effects of Various Amino Acid Analogues on Growth and Morphology of F\ rhodozyma 29 Table III. Colony Morphology on Some Presumed Fusants Grown on YNB-galactose + KC1 media 34 Table IV. Pigments Isolated from Various Cultures and Their Respective TLC and Absorption Data 40 Table V. Colour Change Test of Various Carotenoids Upon the Addition of Concentrated H2S0« to Acetone Solutions 53 Table VI. Elution Characteristics of Pigments isolated from Fusant #26, P̂ . rhodozyma and R_;. toruloides on a S i l i c a Gel 60 Column 54 - V - L IST OF FIGURES PAGE F i g u r e 1 . f \ rhodozyma c e l l s grown i n t h e p r e s e n c e o f 5 mg/ml AEC i n MEA (600X m a g n i f i c a t i o n ) 31 F i g u r e 2 A . F u s a n t #26 grown on Y N B - g a l a c t o s e p l a t e s (600X m a g n f i c a t i o n ) 36 F i g u r e 2 B . F u s a n t #26 grown i n Y N B - g l u c o s e b r o t h (1500X m a g n i f i c a t i o n ) 36 F i g u r e 3 . R^ t o r u l o i d e s grown on MEA 37 F i g u r e 4 A . JC f r a g i l i s grown on Y N B - g a l a c t o s e p l a t e s (600X m a g n i f i c a t i o n ) 38 F i g u r e 4 B . JC f r a g i l i s grown i n Y N B - g l u c o s e b r o t h (1500X m a g n f i c a t i o n ) 38 F i g u r e 5 A . J \ rhodozyma grown on MEA p l a t e s (600X m a g n i f i c a t i o n ) 39 F i g u r e 5 B . j \ rhodozyma grown i n Y N B - g l u c o s e b r o t h (1500X m a g n f i c a t i o n ) 39 F i g u r e 6 . 250 ml f l a s k C u l t u r e o f F u s a n t #26 grown on S t a n d a r d M e d i a + g a l a c t o s e 41 F i g u r e 7 . TLC o f a l l - t r a n s A s t a x a n t h i n , and P i g m e n t s i s o l a t e d f rom Fusand #26 grown on g a l a c t o s e p l a t e s and l i q u i d med ia 42 F i g u r e 8 . V i s i b l e A b s o r p t i o n S p e c t r a o f P i g m e n t s f r o m P . r hodozyma , a l l - t r a n s a s t a x a n t h i n and F u s a n t #26 grown on S o l i d M e d i a 45 F i g u r e 9 . V i s i b l e A b s o r p t i o n S p e c t r a o f 8 - c a r o t e n e and p i g m e n t s f rom the 2 f r a c t i o n s o f F u s a n t #26 grown i n l i q u i d med ia 46 F i g u r e 1 0 . V i s i b l e A b s o r p t i o n S p e c t r a o f P i g m e n t s i s o l a t e d f r om F u s a n t #26 ( s o l i d ) and R^. t o r u l o i d e s 47 F i g u r e 1 1 . Mass S p e c t r u m o f A s t a x a n t h i n 48 F i g u r e 1 2 . Mass S p e c t r u m o f P i g m e n t s i s o l a t e d f r o m P . rhodozyma grown on MEA a t 22°C 49 F i g u r e 1 3 . Mass S p e c t r u m o f P i g m e n t s i s o l a t e d f r om F u s a n t #26 grown on G a l a c t o s e P l a t e s - P r o b e temp. 150°C 50 F i g u r e 1 4 . Mass S p e c t r u m o f P i g m e n t s i s o l a t e d f r om F u s a n t #26 grown on G a l a c t o s e P l a t e s - P r o b e temp. 280°C 51 - v i - ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x t e n d s i n c e r e g r a t i t u d e t o D r . P . M . T o w n s l e y f o r h i s c o n c e r n and a s s i s t a n c e i n t h e p r e p a r a t i o n and e x e c u t i o n o f t h i s t h e s i s . She a l s o w i s h e s t o t hank t h e members o f t h e r e s e a r c h c o m m i t t e e , D r . W.D. P o w r i e , D r . J . R i c h a r d s b o t h o f t h e Depar tmen t o f Food S c i e n c e and D r . B . D i l l o f t h e Depar tmen t o f M i c r o b i o l o g y . Much a p p r e c i a t i o n i s a l s o e x t e n d e d t o D r . A . F i n l a y s o n f o r h i s c o n s t a n t a s s i s t a n c e . The a u t h o r w i s h e s t o a c k n o w l e d g e t h e v a l u a b l e e x p e r t i s e and a s s i s t a n c e p r o v i d e d by Sherman Y e e , s e n i o r t e c h n i c i a n i n t h e Depa r tmen t o f Food S c i e n c e . Many t h a n k s a r e e x t e n d e d t o K a t h l e e n B a r b e r , Yvonne J o n e s , Donna S m i t h and C a r o l B u r t o n f o r t h e i r encou ragemen t and s u p p o r t d u r i n g t h i s p r o j e c t . A s p e c i a l t h a n k s t o J o y c e Tom f o r h e r a s s i s t a n c e i n t y p i n g . - . y i i - DEDICATION T h i s t h e s i s i s d e d i c a t e d t o my p a r e n t s f o r t h e i r h e l p , p a t i e n c e and u n d e r s t a n d i n g , and t o my s i s t e r , C l o and t o C a s s i e who has h e l p e d me more t h a n she knows . - 1 - INTRODUCTION P r e v i o u s e x p e r i m e n t s have d e m o n s t r a t e d t h a t P h a f f i a rhodozyma has p o t e n t i a l c o m m e r c i a l v a l u e as a d i e t a r y s o u r c e o f a s t a x a n t h i n f o r p o u l t r y and p e n - r e a r e d s a l m o n i d s ( J o h n s o n e t a l_ . 1 9 7 9 , 1 9 8 0 ) . U n f o r t u n a t e l y , t h e m u l t i l a y e r e d , t ough c e l l w a l l o f t h i s o r g a n i s m makes l i b e r a t i o n o f t h e c a r o t e n o i d s i m p r a c t i c a l w i t h r e g a r d s t o c o m m e r c i a l a p p l i c a t i o n s (Okabue e t a_[. 1 9 8 4 , J o h n s o n e t a l . 1 9 8 0 ) . M o d i f i c a t i o n o f t h e c e l l w a l l s p r i o r t o i n c l u s i o n i n a n i m a l d i e t s have p r o v e n t o be i n a p p l i c a b l e on a l a r g e s c a l e , o r d e s t r u c t i v e t o t h e c a r o t e n o i d s (Okabue e t a i . 1 9 8 3 ) . K l u y v e r o m y c e s i s a g e n e r a o f y e a s t t h a t i s a b l e t o u t i l i z e a v a r i e - t y o f c a r b o n s o u r c e s . One such s o u r c e i s whey , a b y p r o d u c t o f t h e c h e e s e i n d u s t r y . The h i g h b i o l o g i c a l oxygen demand (BOD) o f whey f r om c h e e s e p l a n t s r e n d e r s w a s t e t r e a t m e n t i m p r a c t i c a l ( A l - s h a b i b i e t a l_ . 1 9 8 4 ; F a r a h n a k e t a j . . 1 9 8 5 ) . A t t e m p t s a t i n t e r s p e c i e s and i n t e r g e n e r i c y e a s t p r o t o p l a s t f u s i o n s have r e c e n t l y become p r o m i n e n t . In one s t u d y t he f u s i o n o f c e r e v i s i a e and f e r m e n t a t i was p e r f o r m e d t o o b t a i n a h i g h e t h a n o l p r o d u c e r t h a t u t i l i z e d a number o f c a r b o n s o u r c e s ( P i n a e t a T . 1 9 8 6 ) . The o b j e c t i v e i n most c a s e s i s t o improve i n d u s t r i a l s t r a i n s w i t h r e s p e c t t o c a r b o n u t i l i z a t i o n , i n c r e a s e d a l c o h o l p r o d u c t i o n o r o t h e r such p a r a m e t e r s . S i n c e P± rhodozyma p r o d u c e s p o t e n t i a l l y u s e f u l c a r o t e n o i d s and JC f r a g i l i s i s a b l e t o use a v a r i e t y o f e c o n o m i c a l c a r b o n s o u r c e s such as whey , t h e ma in a i m o f t h i s s t u d y was t o o b t a i n an o r g a n i s m by - 2 - i n t e r g e n e r i c p r o t o p l a s t f u s i o n t h a t comb ines t h e s e c h a r a c t e r i s t i c s . P r o t o p l a s t f u s i o n was t he method o f c h o i c e s i n c e i t has been used e x t e n s i v e l y f o r t h i s p u r p o s e ( P i n a e t a]_. 1 9 8 6 ; F a r a h n a k e t a l_ . 1 9 8 6 ) . - 3 - LITERATURE REVIEW I. Genetic Manipulation The d r i v e to improve organisms to b e t t e r s u i t t h e i r users has been a long standing o b j e c t i v e of many researchers (Peberdy, 1980). The g e n e t i c manipulation and s t r a i n enhancement of p l a n t s and animals occurs in an ever i n c r e a s i n g a p p l i c a t i o n (Sink 1984, Yu et a/L 1984). S e l e c - t i o n and mating are c l a s s i c a l ways of a l t e r i n g the genetic content of an organism, but t h i s approach can be time consuming and not always accurate or p r a c t i c a l (Sink 1984). In order to avoid and to s i m p l i f y c o n s t r a i n t s , i n v o l v e d in formal c e l l reproduction, g e n e t i c information can be introduced to a c e l l by f u s i o n of t h e i r r e s p e c t i v e p r o t o p l a s t s (Peberdy 1980). a) P r o t o p l a s t Production The generation of v i a b l e p r o t o p l a s t s can be a very complicated and d i f f i c u l t procedure, the success of which v a r i e s g r e a t l y , depending upon the organism used (Yu et aj_. 1987, Pina et aj_. 1986). The i s o l a t i o n of p r o t o p l a s t s r e q u i r e s the d i g e s t i o n of the c e l l w a l l , r e l e a s i n g the c e l l u l a r cytoplasm encased w i t h i n the plasma membrane. To maintain the i n t e g r i t y of the plasma membrane, which i s q u i t e f r a g i l e , the e n t i r e process must be c a r r i e d out in a hypertonic environment to provide osmotic s t a b i l i t y (Peberdy, 1980). In the past, researchers were forc e d to p u r i f y t h e i r own enzymes f o r c e l l wall d i g e s t i o n which was time consuming, and often non-reproducible. Today, enzymes are commercially a v a i l a b l e not only from the t r a d i t i o n a l source, such as the s n a i l H e l i x pomatia, but a l s o - 4 - f r o m v a r i o u s m i c r o o r g a n i s m s such as t h e f u n g i A s p e r g i 1 1 us s p . and N e u r o s p o r a s p . The t e c h n i q u e i n v o l v e d i n t h e d e v e l o p m e n t o f p r o t o p l a s t s f r om f u n g i p o s e s a p a r t i c u l a r p r o b l e m when i t comes t o d i g e s t i o n o f t h e c e l l by e n z y m e s . The f u n g a l c e l l w a l l i s a p h y s i c a l and c h e m i c a l comp lex o f many d i f f e r e n t s t r u c t u r a l components such a s c h i t i n and B - g l u c a n s w h i c h r e q u i r e more t h a n one enzyme f o r t h e i r d e g r a d a t i o n . Hamlyn e t aj [ . (1981) d e m o n s t r a t e d t h a t h i g h l e v e l s o f c h i t i n a s e and B - g l u c a n a s e m i x e d i n a " s o u p " s u c c e s s f u l l y d i g e s t e d c e l l w a l l s o f v a r i o u s s p e c i e s o f A s p e r g i l l u s . T h e r e a r e a l s o o t h e r f a c t o r s a f f e c t i n g t h e p r o d u c t i o n o f p r o t o p l a s t s such as c e l l a g e , g row th c o n d i t i o n s , p r e t r e a t m e n t o f c e l l s , d u r a t i o n o f d i g e s t i o n and t h e pH o f t h e p r o t o p l a s t b u f f e r . Morgan e t a l_ . (1980) f o u n d t h a t f o r t h e y e a s t ]C f r a q i l i s , d e s i r a b l e p r o t o p l a s t s were o b t a i n e d f r o m s h a k i n g f l a s k c u l t u r e s t h a t were i n l a t e l o g , e a r l y s t a t i o n a r y g r o w t h p h a s e . P e b e r d y ( 1 9 8 0 ) , on t he o t h e r h a n d , f o u n d t h a t g r o w t h o f S t r e p t o m y c e s i n g l y c i n e , imp roved p r o t o p l a s t y i e l d s . P r e - t r e a t m e n t o f t h e i s o l a t e d c u l t u r e w i t h t h i o l compounds such as m e r c a p t o e t h a n o l a l s o enchanced t h e p e r c e n t a g e o f p r o t o p l a s t s r e c o v e r e d (Hamlyn e t a l_ . 1 9 8 1 ) . The o p t i m i z a t i o n o f t he p a r a m e t e r s i n v o l v e d i n c e l l w a l l d i s s o l u t i o n o f a p a r t i c u l a r o r g a n i s m has r e s u l t e d i n an i n c r e a s e o f undamaged p r o t o p l a s t numbers p e r u n i t d i g e s t i o n t i m e . b) P r o t o p l a s t F u s i o n S e v e r a l methods have been d e v e l o p e d t o a l t e r t h e g e n e t i c m a t e r i a l i n o r g a n i s m s , some o f w h i c h i n c l u d e : p r o t o p l a s t f u s i o n , h y b r i d i z a t i o n and t r a n s f o r m a t i o n . The use o f f u s i o n b y p a s s e s t h e normal c e l l u l a r c y c l e s and r e s t r i c t i o n s commonly p l a c e d upon t he p o o l i n g o f g e n e t i c - 5 - i n f o r m a t i o n . G e n e r a l l y , t he c e l l w a l l and i t s v a r i o u s s u r f a c e r e c e p t o r s w i l l d i c t a t e c o m p a t i b i l i t y . C e l l w a l l c o n t a c t i s t h o u g h t t o be a t r i g g e r i n g d e v i c e f o r p r e - c o n j u g a l a c t i v i t i e s . The b a s i c c o n c e p t i n v o l v e d i n t h e f u s i o n o f c e l l s i s q u i t e s i m p l e . P r o t o p l a s t s a r e c o l l e c t e d f r om the d e s i r e d s t r a i n s and added i n e q u a l p a r t s t o a f u s o g e n , c a u s i n g t h e membranes t o a s s o c i a t e . The a g g r e g a t e d p r o t o p l a s t s a r e t h e n l e f t t o i n c u b a t e , e i t h e r s h a k i n g g e n t l y o r s t a n d i n g , t o e n a b l e an a s s o c i a t i o n be tween t h e two c e l l membranes . A f t e r an a l l o t e d t i m e , t h e f u s o g e n and t h e f u s e d p r o t o p l a s t s a r e d i l u t e d and t r a n s f e r r e d t o an o s m o t i c a l l y s t a b l i z e d a g a r p l a t e . Here p lasmogamy , karyogamy and r e g e n e r a t i o n o f t h e c e l l w a l l a r e c o m p l e t e d . One o f t h e most commonly used f u s o g e n s i s p o l y e t h y l e n e g l y c o l ( P E G ) . PEG i s a v a i l a b l e i n a v a r i e t y o f d i f f e r e n t m o l e c u l a r w e i g h t s f r o m 1500 d a l t o n s t o 8000 d a l t o n s and t h e c h o i c e o f m o l e c u l a r w e i g h t a s w e l l a s c o n c e n t r a t i o n o f t h e s o l u t i o n can have a d r a m a t i c e f f e c t on f u s i o n ( P e b e r d y , 1 9 8 0 ) . G e n e r a l l y 4000 d a l t o n s i s t h e w e i g h t o f c h o i c e used i n f i n a l s o l u t i o n c o n c e n t r a t i o n s r a n g i n g f r o m 30% t o 60% ( w / v ) . Goodey and Beven (1983) c a r r i e d o u t an e x p e r i m e n t p r o v i n g t h a t an a t t a c h m e n t i n d u c i n g a g e n t was r e q u i r e d f o r s u c c e s s f u l f u s i o n . Two d i f f e r e n t y e a s t s t r a i n s o f c e r e v i s i a e were i n d u c e d t o f o rm p r o t o p l a s t s . These were a l l o w e d t o f u s e w i t h and w i t h o u t t h e p r e s e n c e o f P E G . O n l y t h o s e c e l l s s u b j e c t e d t o t h e PEG t r e a t m e n t f u s e d t o c r e a t e " c y b r i d s " . P E G , i n i t s e l f , does no t i n d u c e p lasmogamy. R a t h e r , i t d raws t he c e l l s t o g e t h e r i n a g g r e g a t e s c r e a t i n g an a s s o c i a t i o n o f t h e p l a s m a membrane. The f u s o g e n i s t h e n d i l u t e d , a l l o w i n g p lasmogamy t o o c c u r . - 6 - W i t h t h i s m e t h o d , f u s i o n f r e q u e n c i e s o f 15-17% can be o b t a i n e d w i t h c e r t a i n c e l l s ( M e n c z e l 1 9 8 4 ) . A g g r e g a t i o n o f t h e p r o t o p l a s t s can be i m p r o v e d by t h e a d d i t i o n o f t he d i v a l e n t c a t i o n s C a 2 + and M g 2 + t o t he f u s i o n med ium. The most e f f e c t i v e c o n c e n t r a t i o n o f t h e s e d i v a l e n t m e t a l s has been f o u n d t o be a 0 .01 M s o l u t i o n ( P e b e r d y 1 9 8 0 ) . One c r i t i c i s m o f t he P E G - d i v a l e n t m e t a l i n d u c e d t y p e o f f u s i o n i s t h a t i t o c c u r s a s a v e r y random and low f r e q u e n c y e v e n t (Hamlyn e t a l_ . 1 9 8 1 ) . A n o t h e r f u s o g e n t h a t has r e c e n t l y g a i n e d i m p o r t a n c e i s t h e a p p l i c a t i o n o f a s h o r t p u l s e e l e c t r i c c u r r e n t t o c o u p l e d p r o t o p l a s t s (Senda 1 9 8 2 , B a t e s 1 9 8 3 , Poh l 1 9 8 2 ) . C e l l s and o t h e r p o l a r i z a b l e p a r t i c l e s can be seen t o s p i n when s u b j e c t e d t o an a l t e r n a t i n g c u r r e n t (AC) ( M i s c h e l e t aj_. 1 9 8 2 ) . C e l l s a r e p l a c e d be tween e l e c t r o d e s and an a l t e r n a t i n g c u r r e n t i s a p p l i e d . Due t o t h e i r mu tua l d i p o l e f o r m a t i o n t h e c e l l s a s s o c i a t e o r c r e a t e " p e a r l - c h a i n s " ( A p p e n d i x I ) . A t t h e p o i n t when c e l l s l i n e up i n c l o s e a s s o c i a t i o n , a s h o r t p u l s e o f d i r e c t c u r r e n t (DC) i s a p p l i e d c a u s i n g a r e v e r s i b l e membrane b r e a k d o w n , r e s u l t i n g i n p o i n t a d h e r a t i o n s o f p r o t o p l a s t s ( B a t e s e t a l . . 1 9 8 3 ) . G e n e r a l l y , t h e a p p a r a t u s c o n s i s t s o f two p l a t i n u m w i r e s s e t 0 . 5 - 1 mm a p a r t on a f l a t m i c r o s c o p e s l i d e ( s e e A p p e n d i x I I ) . These e l e c t r o d e s a r e t h e n a t t a c h e d t o a s u p p l y c i r c u i t ( P o h l 1 9 8 2 ) . The c a p a c i t o r , i n mos t c a s e s , i s a b l e t o d e l i v e r be tween 50 and 1000 v o l t s w i t h t h e c a p a c i t y o f 0 .01 t o 1000 u F . Once a c t i v a t e d , an e l e c t r i c c h a r g e between 1 and 50 mA w i t h a d u r a t i o n o f 0 . 2 5 t o 50 ms runs t h r o u g h a d r o p o f l o w - c o n d u c t i n g , o s m o t i c a l l y s t a b l i z e d b u f f e r p l u s p r o t o p l a s t s (Senda 1 9 8 2 ) . Once p o i n t a d h e r a t i o n s a r e made, a f u r t h e r 30 t o 60 m i n u t e s a r e r e q u i r e d f o r p lasmogamy t o o c c u r . - 7 - " E l e c t r o - f u s i o n " has a number o f a d v a n t a g e s o v e r t h e more c o n v e n t i o n a l PEG m e t h o d . I t r e q u i r e s no c h e m i c a l p r e t r e a t m e n t t o f a c i l i t a t e f u s i o n , and f u s i o n i s r a p i d and s y n c h r o n o u s ( B a t e s e t a l _ . 1 9 8 3 ) . R e p o r t s o f b e t w e e n 6 0 - 8 0 % f u s a n t s have b e e n r e g i s t e r e d ( B a t e s e t aj_. 1 9 8 3 ) . Some p r o b l e m s t h a t f a c e t h i s m e t h o d i n c l u d e o p t i m i z i n g e q u i p m e n t d e s i g n , and t h e f a c t t h a t t h e p r o t o p l a s t s t e n d t o s t i c k t o t h e m e t a l e l e c t r o d e s . The l a t t e r d i f f i c u l t y may be o v e r c o m e by t h e p l a c e m e n t o f an i n e r t membrane b e t w e e n t h e e l e c t o d e and p r o t o p l a s t s . I I . C a r o t e n o i d s C a r o t e n o i d s f o r m a g r o u p o f more t h a n 400 known n a t u r a l l y o c c u r r i n g p i g m e n t s whose c o l o u r s r a n g e f r o m y e l l o w t o r e d . T h e s e p i g m e n t s a r e r e s p o n s i b l e f o r t h e b r i l l i a n t c o l o u r s s e e n i n many f r u i t s a nd v e g e t a b l e s , C r u s t a c e a s p . , f i s h and e g g s . O n l y a l g a e , m i c r o o r g a n i s m s and p l a n t s a r e c a p a b l e o f s y n t h e s i z i n g t h e c a r o t e n o i d s ; a n i m a l s must o b t a i n them f r o m t h e i r f o o d s u p p l y . I n a d d i t i o n t o f u n c t i o n i n g a s a t t r a c t a n t s o r d e t e r r e n t s , c a r o t e n o i d s c a n a l s o p l a y a r o l e i n p h y s i o l o g i c a l f u n c t i o n s . Many a c t a s p h o t o p r o t e c t i v e a g e n t s by a b s o r b i n g p o t e n t i a l l y h a r m f u l s u n l i g h t e n e r g y o r q u e n c h i n g s i n g l e t o x y g e n m o l e c u l e s ( M u l l e r e t a]_. 1 9 8 0 ) . C a r o t e n o i d s a l s o f u n c t i o n a s a c c e s s o r y p i g m e n t s i n p h o t o s y n t h e s i s and t h u s a r e o f t e n a s s o c i a t e d w i t h t h e c h l o r o p l a s t . In a n i m a l s , c a r o t e n o i d s p l a y an i m p o r t a n t r o l e i n p h y s i o l o g i c a l f u n c t i o n . One s u c h c a r o t e n o i d , B - c a r o t e n e , i s c o n v e r t e d t o V i t a m i n A and s u b s e q u e n t l y s t o r e d i n t h e l i v e r . V i t a m i n A i s r e q u i r e d f o r t h e n o r m a l b i o s y n t h e s i s o f m u c o p o l y s a c c h a r i d e s . - 8 - a) G e n e r a l B i o s y n t h e s i s o f C a r o t e n o i d s C a r o t e n o i d s a r e s t r u c t u r e d b a s i c a l l y f r o m a h i g h l y u n s a t u r a t e d i s o p r e n e n u c l e u s . Most o f t h o s e n a t u r a l l y o c c u r r i n g a r e made up o f e i g h t i s o p r e n e r e s i d u e s . The b i o s y n t h e s i s o f c a r o t e n o i d s i s a m u l t i s t e p p r o c e s s t h a t u t i l i z e s a number o f d i f f e r e n t c e l l u l a r enzymes ( G o o d w i n 1 9 7 9 ) . I n i t i a l l y , t h r e e a c e t y l - C o A m o l e c u l e s f o r m m e v a l o n i c a c i d ( f o r m u l a 1 ) , w h i c h i s a s i x c a r b o n m o l e c u l e . T h i s i s t h e n c o n v e r t e d t o t h e f i v e c a r b o n m o l e c u l e i s o p e n t e n y l p y r o p h o s p h a t e ( I P P ) . F o u r IPP m o l e c u l e s l i n k t o f o r m t h e t w e n t y c a r b o n g e r a n y l g e r a n y l p y r o p h o s p h a t e (GGPP) ( f o r m u l a 2 ) . g e r a n y l g e r a n y l p y r o p h o s p h a t e f o r m u l a 2 Two GGPP m o l e c u l e s l i n k t a i l t o t a i l t o y i e l d t h e f i r s t f o r t y c a r b o n p r e c u r s o r c a l l e d p h y t o e n e , w h i c h i s d e s a t u r a t e d t o f o r m l y c o p e n e ( f o r m u l a 3 ) . m e v a l o n i c a c i d f o r m u l a 1 - 9 - l y c o p e n e f o r m u l a 3 From t h i s p o i n t , t h e r e a r e a number o f r o u t e s t h a t c a n be t a k e n d e p e n d i n g upon w h i c h c a r o t e n o i d i s b e i n g p r o d u c e d , s u c h a s c y c l i c c a r o t e n o i d s and x a n t h o p h y l l s ( 0 2 c o n t a i n i n g ) . O n l y t h o s e c a r o t e n o i d s c o n t a i n i n g a B - i o n e r i n g a r e c l a s s i f i e d a s p r o v i t a m i n A. T h e s e r i n g s a r e f o r m e d by s p e c i a l i z e d c y c l i z i n g enzymes t h a t work on t h e ends o f t h e l y c o p e n e m o l e c u l e . 8 - c a r o t e n e i s an e x a m p l e o f a compound w i t h s u c h e n d s ( f o r m u l a 4 ) . T h i s m o l e c u l e i s c a p a b l e o f p r o d u c i n g two m o l e c u l e s o f V i t a m i n A. 8 - c a r o t e n e f o r m u l a 4 The c a r o t e n o i d t h a t i s o f p a r t i c u l a r i n t e r e s t t o t h i s p a p e r , a s t a x a n t h i n , i s a 8 - c a r o t e n e d e r i v a t i v e ( 3 , 3 ' d i h y d r o x y - B - B - c a r o t e n e 4 , 4 ' - d i o n e ) ( f o r m u l a 5 ) . T h i s p a r t i c u l a r c a r o t e n o i d has f o u r o p t i c a l i s o m e r s t h a t c a n be p r o d u c e d n a t u r a l l y , one o f w h i c h c a n be f o u n d i n l o b s t e r a nd s h r i m p . When s h r i m p a r e i n g e s t e d by a f i s h s u c h a s s a l m o n , t h e a s t a x a n t h i n p i g m e n t s t h e f l e s h t o p r o d u c e t h e c h a r a c t e r i s t i c s r e d - o r a n g e c o l o u r ( J o h n s o n e t & ± . 1 9 8 0 ) . - 10 - o a s t a x a n t h i n f o r m u l a 5 b) C a r o t e n o i d s and M i c r o o r g a n i s m s As m e n t i o n e d p r e v i o u s l y , m i c r o o r g a n i s m s a r e c a p a b l e o f s y n t h e s i z i n g t h e i r own c a r o t e n o i d s , and b e c a u s e o f t h i s , have been t he o b j e c t o f a number o f d i f f e r e n t avenues o f r e s e a r c h . One a r e a f o c u s e d on p e r t a i n s t o t h e f u n g u s B l a k e s l e a t r i s p o r a , w h i c h p r o d u c e s B - c a r o t e n e . When t h e c e l l s were f r a c t i o n a t e d , r e s e a r c h e r s f o u n d t h a t t h e c a r o t e n o i d o c c u r r e d i n two o f t h e c e l l f r a c t i o n s , t h e s u p e r n a t a n t and t h e c e l l w a l l / m i t o c h o n d r i a f r a c t i o n (Goodwin 1 9 8 0 ) . T h i s s u g g e s t s t h a t t h e c a r o t e n o i d i s h e l d i n b o t h t h e membranes as p a r t i c u l a t e m a t t e r as w e l l as an o i l i n t h e c y t o p l a s m . In t h e f u n g u s N e u r o s p o r a c r a s s a ( M i t z k a - S c h a b e l e t a i . 1 9 8 1 ) , c e l l f r a c t i o n s c o n t a i n i n g t h e e n d o p l a s m i c r e t i c u l u m membrane, and s u p e r n a t a n t had t h e h i g h e s t c a r o t e n o i d l e v e l s . These same c e l l f r a c t i o n s a l s o h e l d t h e m a j o r i t y o f enzymes f o r c a r o t e n o g e n e s i s . A n o t h e r f r a c t i o n c o n t a i n i n g c a r o t e n o i d r e l a t e d enzymes was t h e p l a s m a membrane. T h i s s u g g e s t s t h a t t h e r e i s no t one s i n g l e s u b c e l l u l a r s i t e f o r c a r o t e n o g e n e s i s i n f u n g i , u n l i k e t h e h i g h e r p l a n t s w h e r e , f o l l o w i n g m e l v o l i c a c i d , t h e c a r o t e n o i d enzymes a r e l o c a t e d w i t h i n t he c h l o r o p l a s t s . O t h e r r e s e a r c h by R o n c e r o e t a i . (1982) l o o k e d a t the g e n e t i c s o f c a r o t e n o i d p r o d u c t i o n i n t h e f u n g u s Phycom.yces b l a k e s e c a n u s , w h i c h i s - 11 - y e l l o w due t o B - c a r o t e n e . i t was f o u n d t h r o u g h a s e r i e s o f m u t a n t m a t i n g e x p e r i m e n t s , t h a t two g e n e s ( C a r B a n d C a r RA) w e r e c l o s e l y l i n k e d a n d l o c a t e d a p p r o x i m a t e l y 10 map u n i t s f r o m t h e c e n t r o m e r e . T h e s e g e n e s p r o d u c e two e n z y m e s , p h y t o e n e d e h y d r o g e n a s e a n d l y c o p e n e c y c l a s e t h a t a r e r e s p o n s i b l e f o r t h e l a s t s i x s t e p s o f c a r o t e n o g e n e s i s . ( S i m i l a r a s s o c i a t i o n s h a v e a l s o b e e n n o t e d i n t h e y e a s t S c e r e v i s i a e i n t h e g e n e s f o r g a l a c t o s e u t i l i z a t i o n . ) One n o n - g e n e t i c f a c t o r t h a t a p p e a r s t o a f f e c t c a r o t e n o i d p r o d u c t i o n i n t h e m i c r o o r g a n i s m i s t h e c a r b o n s o u r c e . S t u d i e s by C i e g l e r e t a i . ( 1 9 7 8 ) d e m o n s t r a t e d t h a t g r o w t h on v a r i o u s g r a i n s i n c o r p o r a t e d i n t o t h e f e r m e n t a t i o n m e d i a , i n f l u e n c e d t h e c a r o t e n o i d p r o d u c t i o n o f B. t r i s p o r a w i t h c o r n g i v i n g t h e h i g h e s t y i e l d o f c a r o t e n o i d p e r gram o f d r y m y c e l i u m w e i g h t . O t h e r s t u d i e s by J o h n s o n a n d L e w i s ( 1 9 7 9 ) on P. r h o d o z y m a d e m o n s t r a t e d t h a t c a r b o n s o u r c e s s u c h a s m a l t o s e , s u c r o s e , a n d c e l l o b i o s e g a v e a h i g h e r y i e l d t h a n g l u c o s e . A s e c o n d n o n - g e n e t i c f a c t o r a f f e c t i n g c a r o t e n o g e n e s i s i s t h e pH o f t h e g r o w i n g m e d i a . M i c r o o r g a n i s m s a p p e a r t o r e s p o n d t o a d r a s t i c r e d u c t i o n o f pH p r i o r t o t h e p r o d u c t i o n o f c a r o t e n o i d s . I n o r g a n i s m s s u c h a s ^ b l a k e s l e a a n d R h o d o t o r u l a g r a c i l i s , a d r o p i n pH t o a s low a s pH 2 h a s b e e n n o t e d ( G o o d w i n 1 9 8 0 ) . I f t h e g r o w i n g medium i s b u f f e r e d a t h i g h e r pH, t h e r e i s no c a r o t e n e p r o d u c t i o n . O t h e r s t i m u l a n t s o f c a r o t e n o g e n e s i s i n c l u d e l i g h t , a n d i n t h e c a s e o f N. c r a s s a , t h e p h o t o r e c e p t o r may be 8 - c a r o t e n e i t s e l f ( M i t z k a - S c h n a b e l e t a l _ . 1 9 8 1 ) . An i n c r e a s e i n o x y g e n a t i o n o f c u l t u r e s h a s b e e n shown t o i n c r e a s e c a r o t e n o i d p r o d u c t i o n i n P_i r h o d o z y m a ( J o h n s o n a n d L e w i s 1 9 7 9 ) . - 12 - c ) D e t e r m i n a t i o n o f C a r o t e n o i d P i g m e n t s Due t o t h e v e r y s e n s i t i v e n a t u r e o f c a r o t e n o i d s t o w a r d s l i g h t and o x y g e n , much c a r e must be t a k e n i n t h e e x t r a c t i o n and p u r i f i c a t i o n o f t h e c a r o t e n o i d s . No one method o f e x t r a c t i o n can be c o n s i d e r e d c o m p l e t e l y s a t i s f a c t o r y as a s t a n d a r d t e c h n i q u e t o meet t he p o t e n t i a l v a r i e t y o f m a t e r i a l t o be e x t r a c t e d ( D a v i s 1 9 6 5 ) . O f t e n t h e o r g a n i c e x t r a c t i n g s o l v e n t s used must be m i s c i b l e w i t h w a t e r i n o r d e r t o e f f i c i e n t l y remove t h e c a r o t e n o i d s f r om the f r e s h t i s s u e . S e v e r a l a n a l y t i c a l p r o c e d u r e s a r e used t o e s t a b l i s h t h e i d e n t i t y o f t he c a r o t e n o i d s . G e n e r a l l y i t i s c o n c e d e d t h a t v i s i b l e and i n f a r e d s p e c t r o s c o p y , n u c l e a r m a g n e t i c r e s o n a n c e and mass s p e c t r a a f f o r d t he b e s t means o f c h e m i c a l c h a r a c t e r i z a t i o n . S e p a r a t i o n v i a t h i n l a y e r c h r o m a t o g r a p h y i s o f t e n used i n c o n j u n c t i o n w i t h some o f t h e a f o r e m e n t i o n e d t e c h n i q u e s t o p r o v i d e a r e l i a b l e p r o o f o f i d e n t i t y (Moss and Weedon, 1 9 7 6 ) . The v i s i b l e a b s o r p t i o n s p e c t r u m i s q u i t e i m p o r t a n t as s t r u c t u r a l v a r i a t i o n s w i l l e f f e c t t he shape and l o c a t i o n o f t h e s p e c t r u m ; f o r e x a m p l e , o x o - c a r o t e n o i d s do no t have t h e t y p i c a l t h r e e banded s p e c t r u m o f B - c a r o t e n e bu t r a t h e r a s i n g l e s y m m e t r i c a l band ( D a v i e s , 1 9 6 5 ) . C i s - t r a n s i s o m e r i z a t i o n may a l s o be d e t e c t e d a t s h o r t e r u l t r a - v i o l e t w a v e l e n g t h s . Mass s p e c t r o m e t r y can be c o n s i d e r e d as a v e r y p o w e r f u l t o o l f o r r e s o l v i n g c a r o t e n o i d s and t h e i r s t r u c t u r e s . A d v a n t a g e s i n c l u d e t h e r e q u i r e m e n t f o r o n l y a s m a l l amount o f m a t e r i a l . Some l i m i t a t i o n s a r e a l s o i n h e r e n t such as a r t i f a c t s c a u s e d by v o l a t i l e i m p u r i t i e s and t he r a p i d l o s s o f w a t e r m a s k i n g t h e m o l e c u l a r i o n peak (Moss and Weedon, 1 9 7 6 ) . V a r i a t i o n s can a l s o o c c u r i f d i f f e r e n c e s o f i o n i z a t i o n , t e m p e r a t u r e o r i n s e r t i o n c o n d i t i o n s e x i s t . - 13 - I I I . P h a f f i a rhodozyma P h a f f i a rhodozyma i s a r ed y e a s t genus i n t h e D e u t e r o m y c o t i n a i n t h e f o r m o r d e r B l a s t o m y c e t e s . The y e a s t was o r i g i n a l l y i s o l a t e d f rom d e c i d u o u s t r e e e x u d a t e s ( s l i m e f l u x e s ) i n J a p a n and n o r t h w e s t e r n N o r t h A m e r i c a ( M i l l e r e t a l_ . 1 9 7 6 ) . T h i s i s t h e o n l y c a r o t e n o i d p r o d u c i n g y e a s t c a p a b l e o f f e r m e n t i n g s u g a r s (Andrewes e t a l_ . 1 9 7 6 ) . One o t h e r uncommon a s p e c t o f t h i s y e a s t i s i t s a b i l i t y t o p r o d u c e a s t a x a n t h i n . A s t a x a n t h i n p r o d u c t i o n under c e r t a i n c u l t u r a l c o n d i t i o n s may r e p r e s e n t an a v e r a g e o f 85% o f t he t o t a l c a r o t e n o i d s f o u n d i n t h e c e l l (Andrewes e t a i . 1 9 7 6 ) . F\ rhodozyma i s an e l l i p s o d i a l y e a s t t h a t a p p e a r s t o l a c k a s e x u a l s t a g e w i t h i n i t s l i f e c y c l e . I t r e p r o d u c e s by b u d d i n g , w h i c h g e n e r a l l y o c c u r s a t t h e same s i t e l e a v i n g a t h i c k bud s c a r . The v e g e t a t i v e c e l l w a l l i s m u l t i l a y e r e d , c o n t a i n i n g a h i g h p r o p o r t i o n o f a 1-3 g l u c a n ( M i l l e r e t a i . 1 9 7 6 ) . The o p t i m a l t e m p e r a t u r e f o r g r o w t h and a s t a x a n t h i n p r o d u c t i o n i s 22°C ( J o h n s o n and L e w i s , 1979) bu t g r o w t h can o c c u r a t t e m p e r a t u r e s r a n g i n g f r om 0°C t o 27°C . No g r o w t h has been r e c o r d e d above 2 7 ° C . T h i s b i o t i n r e q u i r i n g y e a s t a s s i m i l a t e s c a r b o n s o u r c e s such a s D - g l u c o s e , m a l t o s e , s u c r o s e , c e l l o b i o s e and s o l u b l e s t a r c h , and w i l l f e r m e n t D - g l u c o s e , m a l t o s e and s u c r o s e . Under a u s t e r e c o n d i t i o n s , c h l a m y d o s p h o r e s a r e p r o d u c e d w h i c h g e r m i n a t e by b u d d i n g , bu t no p r o m y c e l i u m o r s p o r e f o r m a t i o n has been n o t e d , a ) C a r o t e n o i d P r o d u c t i o n o f P . rhodozyma I n v e s t i g a t i o n by A n d r e w e s , P h a f f and S t a r r (1976) c o n c e n t r a t e d p r i n c i p a l l y upon t h e i d e n t i t y o f t he p i g m e n t s p r o d u c e d by r h o d o z y m a . I t was f o u n d t h a t t h e t o t a l c a r o t e n o i d m i x t u r e made up 0 .003% o f t he t o t a l wet c e l l w e i g h t . Of t h o s e c a r o t e n o i d s , a s t a x a n t h i n c o m p r i s e d 87% - 14 - o f t h e t o t a l . O t h e r c a r o t e n o i d s p r e s e n t i n m i n o r bu t m e a s u r a b l e amounts w e r e : B - c a r o t e n e , e c h i n e n o n e , p h o e n i c o x a n t h i n and a new c a r o t e n o i d , 3 h y d r o x y - 3 ' 4 ' - d i h y d r o - 8 - \ J i - c a r o t e n - 4 - o n e . W i t h r e s p e c t t o the a s t a x a n t h i n , b o t h t h e t r a n s and t h e c h r o m a t o g r a p h i c a l l y more p o l a r c i s i s o m e r s , were i s o l a t e d f r om t h e same c e l l p r e p a r a t i o n s . In an a t t e m p t t o o p t i m i z e p i gmen t p r o d u c t i o n , J o h n s o n and L e w i s (1979) d i d e x t e n s i v e work w i t h t h e y e a s t ( see A p p e n d i x I V ) . They f o u n d t h a t when t he y e a s t was grown w i t h i n a b a t c h c u l t u r e f e r m e n t o r , a s t a x a n t h i n was p r o d u c e d m a i n l y d u r i n g t he l a t e e x p o n e n t i a l g row th p e r i o d . The opt imum pH f o r a s t a x a n t h i n f o r m a t i o n a p p e a r e d t o be 4 . 5 h o w e v e r , t h i s p i gmen t was p r o d u c e d a t a l l c u l t u r e pHs t e s t e d . V a r i o u s c a r b o n s o u r c e s e x h i b i t e d p r o n o u n c e d e f f e c t s on b o t h c e l l g r o w t h and a s t a x a n t h i n c o n t e n t . C e l l o b i o s e p r o d u c e d t h e most p i g m e n t w i t h o t h e r d i s s a c c h a r i d e s f o l l o w i n g c l o s e b e h i n d ( A p p e n d i x I V ) . Oxygen was p r o v e n t o be an i m p o r t a n t f a c t o r s i n c e c u l t u r e s p e r m i t t e d t o f e r m e n t d i s p l a y e d B - c a r o t e n e as t h e m a j o r p i g m e n t . B a s e d on c a r o t e n o i d c o n t e n t and known b i o c h e m i c a l pa thways f o r o t h e r o r g a n i s m s , Andrewes e t a l_ . (1976) p r o p o s e d a b i o s y n t h e t i c scheme f o r t h e p r o d u c t i o n o f a s t a x a n t h i n i n rhodozyma ( A p p e n d i x V ) . N e u r o s p o r e n e i s c o n v e r t e d t o p - c a r o t e n e (12) w h i c h i n t u r n i s c o n v e r t e d t o e c h i n e n o n e ( 1 4 ) . A s p e c i f i c e n z y m a t i c r e a c t i o n t h e n c o n v e r t s the e c h i n e n o n e t o h y d r o x y e c h i n e n o n e . A k e t o - i n s e r t i o n r e s u l t s i n p h o e n i c o x a n t h i n w h i c h e v e n t u a l l y w i l l f o rm t h e t e r m i n a l c a r o t e n o i d a s t a x a n t h i n ( 1 1 ) . G e n e r a l l y , t h e c i s i s o m e r i s t h e o n l y one i s o l a t e d f r o m o t h e r s o u r c e s , bu t i t i s t h o u g h t t h a t t h e p r e c u r s o r s and enzymes u t i l i z e d by t h e y e a s t a l l o w s i t t o p r o d u c e b o t h c i s and t r a n s i s o m e r s . - 15 - b) P. r h o d o z y m a a s a S o u r c e o f A s t a x a n t h i n i n S a l m o n i d D i e t s I n t r o u t , s a l m o n a n d c h a r r , a s t a x a n t h i n i s t h e m a j o r p i g m e n t r e s p o n s i b l e f o r t h e t y p i c a l r e d c o l o u r e d f l e s h ( F o s s 1 9 8 4 ) . I n t h e w i l d , t h e s e f i s h o b t a i n t h e c o l o u r t h r o u g h t h e i r d i e t b y i n g e s t i n g s h r i m p , s h r i m p l a r v a e a n d p l a n k t o n s u c h a s P± P r a s i n o p h y c e a e t h a t c o n t a i n t h e p i g m e n t . F i s h c u l t i v a t e d b y a q u a c u l t u r e do n o t h a v e t h e l u x u r y o f s u c h a d i v e r s i f i e d d i e t , t h u s t h e p i g m e n t m u s t be f e d t o them a s an i n g r e d i e n t i n t h e i r f i s h m e a l . T h i s c a n be q u i t e c o s t l y , e s p e c i a l l y i n l i e u o f i n c r e a s i n g l y s t r i c t r e g u l a t i o n s c o n c e r n i n g t h e s a f e t y o f c h e m i c a l s a s f o o d a d d i t i v e s ( J o h n s o n e t a1_. 1 9 8 0 ) . Pj_ r h o d o z y m a i s a n a t u r a l s o u r c e o f a s t a x a n t h i n a n d when f e d t o s a l m o n i d s , d e p e n d i n g upon c a r o t e n o i d l i b e r a t i n g t r e a t m e n t s , i t w i l l c o n f e r t h e p i g m e n t t o f i s h ( J o h n s o n , V i l l a , a n d L e w i s 1 9 8 0 ) . A s w i t h o t h e r y e a s t s , j \ r h o d o z y m a a l s o a c t s a s a p o t e n t i a l l y e x c e l l e n t s o u r c e o f p r o t e i n , f a t s a n d o t h e r n u t r i e n t s ( A p p e n d i x I I I ) . One d r a w b a c k t o t h e c o m m e r i c a l u s e o f t h i s y e a s t f o r f e e d , i s t h a t t h e f i s h i s u n a b l e t o d i g e s t t h e c e l l w a l l a n d t h u s r e l e a s e t h e p i g m e n t ( J o h n s o n e t a l . . 1980, Okagbue a n d L e w i s 1 9 8 3 ) . I n - v i t r o c e l l l y s i s t r e a t m e n t c o u l d be c o s t l y a n d may h a v e an a d v e r s e e f f e c t on t h e s t a b i l i t y o f t h e c a r o t e n o i d a n d n u t r i t i o n a l v a l u e . A s e c o n d d r a w b a c k r e g a r d i n g t h e u s e o f F\ r h o d o z y m a i n f i s h f e e d i s t h e r e l a t i v e l y h i g h c o s t o f f e r m e n t a t i o n . Many o f t h e s u b s t r a t e s t e s t e d by Okagbue a n d L e w i s ( 1 9 8 3 ) t h a t p r o v i d e d a d e q u a t e c e l l y i e l d a n d a s t a x a n t h i n c o n t e n t , c a n be v e r y e x p e n s i v e . T h i s s u b s t r a t e c o s t p l u s t h e a d d i t i o n a l c o s t s i n v o l v e d i n m a i n t a i n i n g t h e f e r m e n t e r a t 2 2 ° C w o u l d c o n t r i b u t e s i g n i f i c a n t l y t o t h e s e l l i n g p r i c e o f t h e f i s h f o o d . - 16 - D e p e n d i n g on g r o w t h c o n d i t i o n s , J \ rhodozyma p r o d u c e s between 30 and 800 yg a s t a x a n t h i n / g y e a s t . T h i s amount o f c a r o t e n o i d f a r e x c e e d s t h a t f o u n d i n c r u s t a c e a n s h e l l s w h i c h n o r m a l l y y i e l d 10 t o 100 yg a s t a x a n t h i n / g o f s h e l l ( J o h n s o n e t a l_ . 1 9 8 0 ) . I V . K l u v e r o m y c e s f r a g i l i s IC f r a q i 1 i s i s a l a c t o s e u t i l i z i n g y e a s t t h a t has been shown t o r e d u c e t h e c h e m i c a l oxygen demand (COD) o f c h e e s e whey by a t l e a s t 60% ( A l - s h a b i b i and Y o u n i s 1 9 8 4 ) . I n t e r e s t i n t h i s y e a s t r e v o l v e s a r o u n d i t s enzyme l a c t a s e p r o d u c i n g a b i l i t y (Mahoney e t a l_ . 1977) and i n i t s a b i l i t y t o f e r m e n t whey t o p r o d u c e e t h a n o l and s i n g l e c e l l p r o t e i n ( V i e n n e and von S t o c k a r 1 9 8 5 ) . C o l o n i e s a r e g e n e r a l l y w h i t e o r c ream and c e l l s e x h i b i t m u l t i l a t e r a l b u d d i n g as w e l l as f o r m i n g a s c o s p o r e s . IC f r a q i 1 i s i s a b l e t o grow o v e r a w i d e range o f t e m p e r a t u r e s w i t h g r o w t h o c c u r r i n g as h i g h as 4 0 ° C . a) Growth and F e r m e n t a t i o n on Whey j C f r a g i l i s i s a b l e t o p r o d u c e e t h a n o l by t h e f e r m e n t a t i o n o f c h e e s e whey . As w i t h a l l m i c r o o r g a n i s m s , c u l t u r e unde r d i f f e r e n t g rowth c o n d i t i o n s can c a u s e d i f f e r e n c e s i n end p r o d u c t s . L a c t a s e , c o n c e n t r a t i o n s i n c r e a s e s h a r p l y d u r i n g l o g phase o f g r o w t h and as t he c e l l s e n t e r s t a t i o n a r y p h a s e , t he l a c t a s e c o n c e n t r a t i o n d e c r e a s e s (Mahoney e t a_k 1 9 7 5 ) . In o r d e r t o i n c r e a s e t h e l a c t a s e p r o d u c t i o n , low a e r a t i o n i s r e q u i r e d , however low a e r a t i o n r e s u l t s i n a p o o r c e l l y i e l d . When c u l t u r e d i n whey , f o r t i f i e d w i t h s a l t s , t h i s y e a s t , no t r e c o g n i s e d a s a p r o l i f i c o i l p r o d u c e r , can be s t i m u l a t e d t o p r o d u c e up t o 22% o i l ( A l - s h a b i b i and Y o u n i s , 1 9 8 4 ) . - 17 - The opt imum pH f o r t he c o n s u m p t i o n o f t h e med ia s u b s t r a t e and e t h a n o l y i e l d l i e s be tween pH 4 and 5 , w i t h t h e most e f f i c i e n t c o n s u m p t i o n o c c u r r i n g a t a pH o f 4 . 0 ( V i e n n e and von S t o c k a r 1 9 8 5 ) . In e x p e r i m e n t s c a r r i e d ou t c o m p a r i n g t he d i f f e r e n c e between s t e r i l i z e d and n o n - s t e r i l i z e d whey p e r m e a t e ; i t was c o n c l u d e d t h a t a n o t h e r medium component o t h e r t h a n l a c t o s e was g row th l i m i t i n g and t h a t t h i s component was a f f e c t e d by h e a t s t e r i l i z a t i o n ( V i e n n e and von S t o c k e r 1 9 8 5 ) . b) P r o t o p l a s t F u s i o n i n K Iuyveromyas s p . KIuyverom.yces s p . have o f t e n been used i n s t u d i e s i n v o l v e d i n t he g e n e t i c s and b i o c h e m i s t r y o f y e a s t s as w e l l as s t u d i e s r e l a t e d t o enhanced a l c o h o l p r o d u c t i o n . These c e l l s , when s u b j e c t e d t o enzyme t r e a t m e n t a t t h e a p p r o p r i a t e s t a g e o f g r o w t h , p r o d u c e p r o t o p l a s t s w i t h r e l a t i v e e a s e ( F a r a h n a k e t a l_ . 1986 , Morgan e t a l_ . 1 9 8 0 ) . In t he m a j o r i t y o f c a s e s , t he numbers o f p r o t o p l a s t s fo rmed w i t h i n a s u s p e n s i o n a p p r o a c h e s 100% o f t h e i n i t i a l c e l l p o p u l a t i o n ( J o h a n n s e n e t a l_ . 1 9 8 4 ) . Morgan e t a l_ . (1980) used p r o t o p l a s t f u s i o n be tween h o m o t h a l l i c s t r a i n s o f ]C l a c t i s t o p r o d u c e s p o r u l a t i n g h y b r i d s . B i o c h e m i c a l l y , c y t o l o g i c a l l y and g e n e t i c a l l y , t h e s e s t u d i e s s u g g e s t t h a t t h e m a j o r i t y o f f u s i o n p r o d u c t s a r e d i p l o i d . The p r e s e n c e o f d i p l o i d y a l s o d e m o n s t r a t e s t h a t n u c l e a r f u s i o n and chromosome exchange does o c c u r i n p r o t o p l a s t f u s i o n p r o d u c t s . F u r t h e r s u p p o r t f o r t h i s p o s i t i o n was pu t f o r t h by F a r a h n a k e t a l . . (1986) when IC l a c t i s was f u s e d w i t h S_;_ c e r e v i c e a e p r o d u c i n g f u s a n t s t h a t were c a p a b l e o f b o t h a s s i m i l a t i n g l a c t o s e and p r o d u c i n g e t h a n o l i n e x c e s s o f 13% ( v o l / v o l ) . - 18 - MATERIALS AND METHODS I . S t r a i n s a) P h a f f i a r h o d o z y m a (ATCC 24202) T h i s o r g a n i s m was m a i n t a i n e d on M a l t E x t r a c t A g a r (MEA) ( D i f c o ) p l a t e s a t 2 2 ° C u n t i l t r a n s f e r t o t h e p r e - p r o t o p l a s t m e d i a (AEC-MEA) b) K l u y v e r o m y c e s f r a q i l i s (ATCC 8564) T h i s o r g a n i s m was m a i n t a i n e d on M a l t - Y e a s t - G l u c o s e - P e p t o n e (MYGP) A g a r ( D i f c o ) a t 2 2 ° C u n t i l t r a n s f e r t o t h e p r e - p r o t o p l a s t m e d i a ( Y N B - g a l a c t o s e ) ( D i f c o - B D H ) . c ) R h o d o s p o r i d i u m t o r u l o i d e s (ATCC 10788) T h i s o r g a n i s m was m a i n t a i n e d on MEA p l a t e s a t 2 2 ° C I I . C u l t i v a t i o n o f O r g a n i s m s f o r P r o t o p l a s t P r o d u c t i o n a) j \ r h o d o z y m a : One week o l d c o l o n i e s w e r e p i c k e d f r o m MEA p l a t e s and s t r e a k e d o n t o MEA + 5 mg/mL. S - ( 2 a m i n o e t h y l ) - L - c y s t e i n e (AEC) ( S i g m a ) p l a t e s a n d i n c u b a t e d a t 2 2 ° C f o r 3 weeks p r i o r t o u s e . T h i s m e t h o d i n d u c e d f i l a m e n t o u s m u t a n t s t o f o r m . b) IC f r a q i l i s : C u l t u r e s 72 h o u r s o l d w e r e s t r e a k e d o n t o y e a s t n i t r o g e n b a s e + g a l a c t o s e p l a t e s ( Y N B - g ) . T h e s e c u l t u r e s w e r e grown 48 h o u r s p r i o r t o u s e . I I I . a) P r o t o p l a s t B u f f e r ( M o r g a n e t a i . 1980) c o n s i s t e d o f : 1.0 mL A + 6.0 mL B + 6.0 mL C + 0.2 mL D w h e r e : - 19 - A = 0.05 M T r i s - H C l (pH 7.0) ( D i f c o ) B = 1.2 M KCI ( F i s h e r ) C = 0.02 M MgS0 4 .7 H 2 0 ( F i s h e r ) D = 1 M 2 - m e r c a p t o e t h a n o l ( S w a r t z / M a n n B i o t e c h ) E n zymes; B - g l u c u r o n i d a s e f r o m H e ! i x p o m a t i a ( S i g m a ) A m y l o g l u c o s i d a s e f r o m R h i z o p u s ( S i g m a ) C h i t i n a s e f r o m S t r e p t o m y c e s g r i s e u s ( S i g m a ) F u s o q e n s : w e r e p r e p a r e d a s f o l l o w s : - P o l y e t h y l e n e G l y c o l (PEG) ( S i g m a ) (mw) 4000 was a d d e d 3 0 % (w/v) t o 0.01 m C a C l 2 - 2 H 2 0 ( F i s h e r ) - F o r e l e c t r o f u s i o n 0.7 m M a n n i t o l (BDH) was u s e d a s an o s m o t i c s t a b l i z e r . M e d i a a) G r o w i n g m e d i a - M a l t E x t r a c t A g a r ( D i f c o ) was u s e d f o r m a i n t e n a n c e a n d p r o p a g a t i o n o f P i r h o d o z y m a . - M a l t - Y e a s t - G l u c o s e - P e p t o n e A g a r c o n s i s t i n g o f 3 g m a l t e x t r a c t ( D i f c o ) , 3 g y e a s t e x t r a c t ( D i f c o ) , 10 g D - g l u c o s e ( D i f c o ) , 5 g p e p t o n e ( D i f c o ) , 20 g a g a r ( D i f c o ) p e r l i t r e w i t h pH a d j u s t e d t o 5.5 was u s e d t o m a i n t a i n IC f r a g i l i s . - Y e a s t N i t r o g e n B a s e ( D i f c o ) + g a l a c t o s e (BDH) was u s e d f o r m a i n t e n a n c e a n d p r o p a g a t i o n o f IC f r a g i l i s a n d t h e f u s a n t s t r a i n s ; 1.5% a g a r was a d d e d t o t h e YNB-g ( D i f c o ) i f p l a t e s r e q u i r e d . - O s m o t i c a l l y S t a b i l i z e d R e c o v e r y M e d i a (0SRM) c o n s i s t i n g o f YNB + g a l a c t o s e + 0.6 m KCI ( F i s h e r ) + 1.5% a g a r was u s e d f o r t h e r e c o v e r y o f p r o t o p l a s t s . - 20 - - a m i n o a c i d a n a l o g u e m e d i a c o n t a i n e d 2% m a l t e x t r a c t ( D i f c o ) + 5 mg/ml o f e i t h e r s u l f a g u a n i d i n e , 5 - f l u o r o D,L t r y p t o p h a n , L t r y p t o p h o n h y d r o x a m a t e L - l y s i n e h y d r o x y a m a t e , s - h y d r o x y l y s i n e HC1 o r s - ( 2 a m i n o e t h y l ) L - c y s t e i n e + 2% B a c t o a g a r ( D i f c o ) . - S t a n d a r d Medium c o n s i s t i n g o f ( p e r l i t r e ) ( N H 4 ) 2 S 0 4 , 2 g ; K H 2 P 0 4 , 1 g; M g S 0 4 7 H 2 0 , 0.5 g ; C a C l 2 2 H 2 0 , 0.1 g ; ( F i s h e r ) y e a s t e x t r a c t ( D i f c o ) , 2 g ; 20 g o f a c a r b o n s o u r c e ; 15 g a g a r ( D i f c o ) was a d d e d t o t h e S t a n d a r d Medium i f p l a t e s r e q u i r e d . b) M e d i a f o r b i o c h e m i c a l t e s t s : - YCB ( D i f c o ) + 5 mm KN0 3 - ( B a r n e t t e t a l . 1983) - Durham f e r m e n t a t i o n t u b e s + g l u c o s e ; r a f f i n o s e ; m a l t o s e o r g a l a c t o s e o b t a i n e d f r o m M i c r o b i o l o g y M e d i a Room ( B a r n e t t e t a l _ . 1 9 8 3 ) . - U r e a B r o t h ( D i f c o ) was u s e d f o r u r e a s e t e s t . ( B a r n e t t e t a l _ . 1983) - YNB ( D i f c o ) c o n t a i n i n g n i a c i n + g a l a c t o s e (BDH) a n d YNB ( D i f c o ) w i t h o u t n i a c i n + g a l a c t o s e (BDH) we r e u s e d f o r t e s t s c o n c e r n i n g t h e r e q u i r e m e n t f o r n i a c i n . ( B a r n e t t e t a j _ . 1983) - YNB ( D i f c o ) + g l u c o s e ( D i f c o ) b r o t h was u s e d f o r f e r m e n t a t i o n t e s t s a t 2 2 ° C a n d a l i q u o t s o f t h i s m e d i a w e r e a l s o p l a c e d on a s p o t p l a t e + i o d i n e t o t e s t f o r e x t r a c e l l u r s t a r c h . ( B a r n e t t e t a j _ . 1983) V. P r o t o p l a s t R e l e a s e , F u s i o n a n d R e g e n e r a t i o n T h e p r o t o p l a s t l i b e r a t i o n p r o c e d u r e i s a s l i g h t m o d i f i c a t i o n o f t h a t d e s c r i b e d b y Morgan e t a l _ . ( 1 9 8 0 ) . T h e c e l l s w e r e grown i n t h e i r r e s p e c t i v e m e d i a a n d e a c h was i n d u c e d t o f o r m p r o t o p l a s t s s e p a r a t e l y . T he p r e t r e a t e d P h a f f i a c e l l s w e r e d i s p e r s e d i n a f i l t e r s t e r i l i z e d p r o t o p l a s t b u f f e r , c o n t a i n i n g 5 mg/mL B - g l u c u r o n i d a s e , 2 mg/mL a m y l o g l u c o s i d a s e a n d 1 mg/mL c h i t i n a s e , f o r b r e a k d o w n a n d r e m o v a l . T h i s m i x t u r e was t h e n i n c u b a t e d a t - 21 - 3 0 ° C , w i t h g e n t l e s h a k i n g f o r 4 h o u r s . T h e IC f r a g i l i s was t r e a t e d i n a s i m i l a r f a s h i o n i n a s o l u t i o n o f 5 mg/mL B - g l u c u r o n i d a s e f o r 1 1/2 h o u r s o f i n c u b a t i o n f o r c e l l w a l l r e m o v a l . A f t e r t h e i r r e s p e c t i v e i n c u b a t i o n s , t h e f r e e p r o t o p l a s t s w e r e washed t h r e e t i m e s w i t h s t e r i l e b u f f e r . A f t e r e a c h w a s h , t h e p r o t o p l a s t s were c e n t r i f u g e d f o r 5 m i n . a t 500 x g i n a F i s h e r t a b l e t o p c e n t r i f u g e a t 2 1 ° C . T h o s e p r o t o p l a s t s s l a t e d f o r e l e c t r o f u s i o n w e r e w a s h e d a f u r t h e r t h r e e t i m e s w i t h s t e r i l e 0.7 M m a n n i t o l . P r o t o p l a s t s f r o m t h e two p a r e n t a l s t r a i n s w e r e m i x e d t o g i v e 1.0 mL s u s p e n s i o n s c o n t a i n i n g b e t w e e n 1 0 6 a n d 1 0 6 p r o t o p l a s t s / m L . T h o s e s u s p e n s i o n s t o be f u s e d by PEG w e r e c e n t r i f u g e d f o r 5 m i n u t e s a t 500 x g i n t h e F i s h e r t a b l e t o p c e n t r i f u g e a t room t e m p e r a t u r e a n d t h e s u p e r n a t a n t was r e m o v e d . The m i x e d p r o t o p l a s t s w e r e r e s u s p e n d e d i n t h e 3 0 % (w/v) PEG s o l u t i o n and i n c u b a t e d f o r 30 m i n . a t 3 0 ° C . F o r r e g e n e r a t i o n , t h e PEG s u s p e n s i o n was d i l u t e d w i t h s t e r i l e p r o t o p l a s t b u f f e r , p l a t e d on OSRM a n d i n c u b a t e d a t 2 2 ° C . P r o t o p l a s t s f r o m t h e two p a r e n t a l s t r a i n s t h a t had b e e n w a s h e d i n 0.7 M m a n n i t o l w e r e m i x e d i n e q u a l p a r t s t o f o r m a s u s p e n s i o n c o n t a i n i n g b e t w e e n 1 0 s a n d 1 0 s p r o t o p l a s t s / m L . T h i s m i x t u r e was t h e n a d d e d d r o p w i s e t o a s t e r i l e c o v e r s l i p t h a t was m o u n t e d u n d e r t h e f u s i o n c e l l . T h e f u s i o n c e l l i t s e l f c o n s i s t e d o f two p a r a l l e l p l a t i n u m w i r e s 0.13 mm d i a m e t e r s e t 1 mm a p a r t m o u n t e d i n a p o l y c a r b o n a t e p l a s t i c f r a m e t h a t c o u l d be a t t a c h e d t o t h e s t a g e o f t h e m i c r o s c o p e ( w i l d , i n v e r t e d ) . T h e s e e l e c t r o d e s w e r e a t t a c h e d t o w i r e s t h a t w e r e c o n n e c t e d t o t h e f u s i o n a p p a r a t u s ( A d v a n c e d E n g i n e e r i n g C o r p . ) . The c a p a c i t o r was c h a r g e d t o t h e d e s i r e d v o l t a g e ( w h i c h r a n g e d f r o m 1500 v t o 2700 v d e p e n d i n g upon t h e e x p e r i m e n t ) . An a l t e r n a t i n g c u r r e n t o f b e t w e e n 40 a n d 1000 kHz was a p p l i e d c a u s i n g t h e p r o t o p l a s t s t o a g g r e g a t e ( B a t e s e t a l . . 1 9 8 3 ) . A t t h i s p o i n t , t h e e l e c t r i c s w i t c h was a c t i v a t e d and - 22 - a f t e r 30 ms t h e d e s i r e d v o l t a g e o f A.C. p u l s e was a p p l i e d t h r o u g h t h e s a m p l e . T h e d u r a t i o n o f t h e p u l s e was 6 u s . F o r r e g e n e r a t i o n , t h e p r o t o - p l a s t s w e r e d i l u t e d o n t o an OSRM p l a t e w i t h 0.7 M m a n n i t o l a n d i n c u b a t e d a t 2 1 ° C . V I . S e l e c t i o n o f F u s a n t s D e s i r e d f u s a n t s w e r e s e l e c t e d on t h e b a s i s o f c o l o u r p r o d u c t i o n a n d t h e i r a b i l i t y t o grow on Y N B + g a l a c t o s e m e d i a . A f t e r 7 d a y s , c o l o n i e s t h a t p r o d u c e d c o l o u r r a n g i n g f r o m y e l l o w t o o r a n g e w e r e p i c k e d f r o m t h e OSR m e d i a , a n d t r a n s f e r e d t o YNB + g a l a c t o s e m e d i a a n d i n c u b a t e d a t 3 0 ° C . C o l o n i e s t h a t g r e w a t 3 0 ° C a n d r e m a i n e d o r a n g e w e r e p i c k e d a n d m a i n t a i n e d on YNB + g a l a c t o s e p l a t e s a n d l i q u i d m e d i a . One p r e s u m e d f u s a n t , #26, was c h o s e n f o r i t s c o l o u r i n t e n s i t y , a n d q u i c k g r o w t h t o u s e f o r f u r t h e r t e s t i n g . P r e s u m e d f u s a n t s w e r e m a i n t a i n e d on Y N B + g a l a c t o s e p l a t e s , i n c u b a t e d a t 3 0 ° C . When l i q u i d c u l t u r e s w e r e u s e d , t h e m e d i a was a g a i n YNB + g a l a c t o s e . C u l t u r e s i n l i q u i d medium w i t h i n 250 mL E r l e n m e y e r f l a s k s , w e r e i n c u b a t e d a t 3 0 ° C s h a k i n g a t 200 rpm. P r e s u m e d f u s a n t s w e r e a l s o v i e w e d m i c r o s c o p i c a l l y w i t h a W i l d i n v e r t e d p h a s e c o n t r a s t m i c r o s c o p e . a) B i o c h e m i c a l t e s t s F u s a n t #26, P h a f f i a r h o d o z y m a , a n d IC f r a q i 1 i s w e r e s u b j e c t e d t o a s e l e c t e d number o f b i o c h e m i c a l t e s t s i n o r d e r t o s o r t o u t t h e r e l a t i o n s h i p o f f u s a n t #26. T h e s e t e s t s i n c l u d e d : Durham f e r m e n t a t i o n t u b e s c o n t a i n i n g : r a f f i n o s e , g a l a c t o s e , m a l t o s e a n d g l u c o s e ; u r e a b r o t h f o r u r e a s e t e s t ; YCB + N0 3 f o r u s i n g N0„ a s n i t r o g e n s o u r c e ; YNB w i t h o r w i t h o u t n i a c i n ; f e r m e n t a - t i o n t e s t s w e r e a l s o c a r r i e d o u t i n Y N B - g l u c o s e , e x t r a c e l l u l a r s t a r c h p r o d u c - t i o n was a l s o t e s t e d u s i n g Y N B - g l u c o s e a n d i o d i n e i n d i c a t o r . A l l p r o c e d u r e s f o r t h e s e t e s t s w e r e c a r r i e d o u t a s d e s c r i b e d b y B a r n e t t e t a l _ . ( 1 9 8 3 ) . - 23 - V I I . C a r o t e n o i d A n a l y s e s C e l l s grown on e i t h e r l i q u i d o r s o l i d m e d i a w e r e c o l l e c t e d a n d f r o z e n a t - 6 0 ° C i n a Forma B i o - F r e e z e r p r i o r t o c r u s h i n g . The c e l l p a s t e was f o r m e d i n t o a c y l i n d r i c a l s h a p e w i t h i n a 5 mL p l a s t i c s y r i n g e b a r r e l a n d f r o z e n f o r a t l e a s t 24 h o u r s . T h e f r o z e n c e l l s w e r e t h e n f o r c e d by means o f a p i s t o n t h r o u g h a m e t a l c y l i n d e r a t -40 t o - 6 0 ° C h a v i n g a s m a l l o r i f i c e (1 mm d i a m ) u s i n g a C a r v e r L a b o r a t o r y h y d r a u l i c p r e s s w i t h a p r e s s u r e o f up t o 10,000 l b s / i n 2 was u s e d . The c r u s h e d , f r o z e n , c e l l s w e r e i m m e d i a t e l y e x t r a c t e d w i t h c o l d a c e t o n e ( B D H ) . T h e p i g m e n t e d a c e t o n e was e v a p o r a t e d u s i n g a B u c h R o t a v a p o r R110 r o t a r y e v a p o r a t o r . T h e d r i e d p i g m e n t was t h e n r e s u s p e n d e d i n v a r i o u s s o l v e n t s d e p e n d i n g upon t h e t e s t ( i . e . a c e t o n e f o r a b s o r p t i o n s p e c t r u m a n d h e x a n e f o r c o l u m n c h r o m a t o g r a p h y ) . A l l w ork i n v o l v i n g c a r o t e n o i d s was c a r r i e d o u t i n s u b d u e d l i g h t . In a l l c a s e s , t h e r e s p e c t i v e p i g m e n t s f r o m f u s a n t #26 c u l t u r e d on b o t h l i q u i d a n d s o l i d m e d i a , r h o d o z y m a c u l t u r e d on s o l i d m e d i a , R̂ . t o r u l o i d e s c u l t u r e d on s o l i d m e d i a , B - c a r o t e n e ( S i g m a ) a n d a s t a x a n t h i n ( R o c h e ) w e r e u s e d a s s t a n d a r d s . a ) T h i n L a y e r C h r o m a t o g r a p h y T h e p i g m e n t s a m p l e s e x t r a c t e d f r o m f u s a n t #26, J \ r h o d o z y m a , a n d R^ t o r u l o i d e s a n d t h e p i g m e n t s B - c a r o t e n e ( S i g m a ) a n d a l l - t r a n s a s t a x a n t h i n ( R o c h e ) w e r e r e s u s p e n d e d i n a c e t o n e (BDH). A l i q u o t s w e r e t h e n s p o t t e d o n t o K e i s e l g e l 60 g l a s s p l a t e s . S e p a r a t i o n was a c h i e v e d i n a s o l v e n t s y s t e m c o n - t a i n i n g a c e t o n e r h e x a n e ( 3 0 : 7 0 ) . S a m p l e s w e r e a l s o c o - c h r o m a t o g r a p h e d w i t h a u t h e n t i c a s t a x a n t h i n ( d a t a n o t s h o w n ) . - 24 - b) C o l u m n C h r o m a t o g r a p h y T h e p i g m e n t s a m p l e s e x t r a c t e d f r o m f u s a n t #26 ( g r o w n i n l i q u i d & s o l i d m e d i a ) , F\ r h o d o z y m a , R̂ . t o r u l o i d e s , a n d a l l - t r a n s a s t a x a n t h i n w e r e d i s s o l v e d i n h e x a n e . T h e s e p i g m e n t s i n h e x a n e w e r e t h e n a p p l i e d t o a s i l i c a g e l 60 (1 cm x 24 cm) c o l u m n ( 6 0 - 2 0 0 mesh) ( B a k e r A n a l y z e d ) . The p i g m e n t s w e r e t h e n w a s h e d w i t h 30 ml o f 100% h e x a n e . I n c r e a s i n g c o n c e n t r a t i o n s o f a c e t o n e : h e x a n e c o m b i n a t i o n s w e r e a d d e d t o t h e c o l u m n s t a r t i n g a t 1:24 ( v : v ) a c e t o n e : h e x a n e a n d f i n i s h i n g w i t h 1 0 0 % a c e t o n e . E l u d e d f r a c t i o n s u n d e r vacuum c o n t a i n i n g c o l o u r e d b a n d s w e r e c o l l e c t e d a n d d r i e d i n a r o t a r y e v a p o r a t o r . c ) A b s o r p t i o n S p e c t a T h e p i g m e n t s e l u t e d f r o m t h e c o l u m n w e r e r e s u s p e n d e d i n a c e t o n e a n d t h e t o t a l s p e c t r u m was s c a n n e d on a V a r i a n C a r y 210 d o u b l e beam s p e c t r o p h o t o m e t e r . T h e maximum a b s o r p t i o n (Amax) w a s r e c o r d e d f o r e a c h s a m p l e a s w e l l a s t h e s h a p e o f t h e v i s i b l e s p e c t r u m . d) Mass S p e c t r o m e t r y P i g m e n t s a m p l e s e l u t e d f r o m t h e s i l i c a g e l 60 c o l u m n w e r e c o l l e c t e d a n d d r i e d u n d e r vacuum i n a r o t a r y e v a p o r a t o r . The s a m p l e s w e r e r e s u s p e n d e d i n a c e t o n e a n d w e r e s u b j e c t e d t o T L C t o d e t e r m i n e t h e number o f b a n d s e l u t e d . T h e s a m p l e s w e r e t h e n d r i e d w i t h n i t r o g e n g a s a n d t h e mass s p e c t r o s c o p i c a n a l y s e s w e r e c o n d u c t e d . Low r e s o l u t i o n EI was t h e s e r v i c e u s e d . e) C o l o u r r e a c t i o n s P i g m e n t s a m p l e s a n d p i g m e n t s t a n d a r d s w e r e d i s s o l v e d i n a c e t o n e . To 2 ml o f t h e m i x t u r e , 1 ml o f c o n c e n t r a t e d H 2 S 0 4 (BDH) (36 M) was a d d e d a n d t h e i m m e d i a t e c o l o u r c h a n g e was n o t e d ( K a r r e r & J u c k e r , 1 9 5 0 ) . - 25 - RESULTS I. C h a r a c t e r i s t i c s of the Parent Organisms a) P h a f f i a rhodozyma P. rhodozyma, a carotenoid producing fermentative y e a s t , produces astaxanthin as i t s major pigment under most growth c o n d i t i o n s with the best c a r o t e n o i d production o c c u r r i n g under a h i g h l y oxygenated l i q u i d medium (Johnson & Lewis 1979). As reported by M i l l e r et aj_. (1976), the optimum temperature f o r growth was 22°C with no growth o c c u r r i n g at temperatures above 27°C. The c e l l s grown at temperatures between 23°C and 27°C gave sparse, small c o l o n i e s with a l i g h t pink colour rather than the usual robust orange-red c o l o u r of the 22°C c o l o n i e s . C e l l s grown at g r e a t e r than optimum temperatures were smaller than normal, though they d i d maintain the c h a r a c t e r i s t i c " f o o t b a l l " shape, and they appeared to have much t h i c k e r c e l l walIs. Growth of P. rhodozyma was observed on a semi-synthetic media (Johnson & Lewis, 1979), with the carbon source being v a r i e d . At 22°C, growth was obtained on maltose, c e l l o b i o s e , dextrose, glucose and sucrose. There was no growth observed on the p l a t e s that contained g a l a c t o s e or l a c t o s e as the sole carbon source. When biochemical t e s t s were c a r r i e d out i n order to ensure that t h i s s t r a i n of P. rhodozyma behaved as reported in the l i t e r a t u r e (Table I ) . A l l t e s t r e s u l t s were the same as those reported in t a b l e I. During microscopic o b s e r v a t i o n , the c e l l s demonstrated p o l a r budding. In o l d e r c u l t u r e s , some lar g e c i r c u l a r chlamydospores were seen. T a b l e I . B i o c h e m i c a l t e s t r e s u l t s f o r P. rhodoxyma, IC f r a g i l i s . |L t o r u l o i d e s and F u s a n t #26. T e s t P^ rhodozyma IC f r a g i 1 i s R. t o r u l o i d e s F u s a n t #26 Y e a s t p e p t o n e + g l u c o s e Y e a s t p e p t o n e + g a l a c t o s e Y e a s t p e p t o n e + m a l t o s e Y e a s t p e p t o n e + r a f f i n o s e N0 3 YCB + N0 3 U r e a s e Growth w i t h o u t N i a c i n s t a r c h f o r m a t i o n YNB + g l u c o s e f e r m e n t a t i o n * + (no g a s ) + + + + ( g a s ) + + + + + + + + + + + + + +/- ( s l o w l y ) ro cn + ( gas ) * c a r r i e d o u t a t 2 2 ° C - 27 - b) K l u y v e r o m y c e s f r a g i l i s J C f r a g i l i s i s a y e a s t c a p a b l e o f f e r m e n t i n g whey a n d d o e s n o t p r o d u c e c a r o t e n o i d s . C u l t u r e s w e r e p l a t e d on YNB + g a l a c t o s e a n d YNB + l a c t o s e and g rown a t 3 0 ° C . T h e r e s u l t i n g c o l o n i e s r a n g e d i n c o l o u r f r o m w h i t e t o c r e a m w i t h no o b v i o u s m o r p h o l o g i c a l d i f f e r e n c e s o c c u r r i n g b e t w e e n m e d i a e x c e p t t h a t t h o s e grown on l a c t o s e c o n t a i n i n g medium w e r e l e s s s o l i d when removed w i t h a l o o p . When v i e w e d m i c r o s c o p i c a l l y , t h e c e l l s grown on l a c t o s e t e n d e d t o be h o m o g e n o u s l y e l o n g a t e w h e r e a s t h e c e l l s grown on g a l a c t o s e w e r e more c i r c u l a r . T h e c e l l w a l l s i n b o t h c a s e s w e r e t h i n a s c o m p a r e d t o t h o s e o f P h a f f i a . Some b i o c h e m i c a l t e s t s w e r e a l s o d o n e i n o r d e r t o e n s u r e t h a t t h i s s t r a i n o f K. f r a q i l i s b e h a v e d a s r e p o r t e d i n t h e l i t e r a t u r e ( T a b l e I ) . T h i s was i n d e e d t h e c a s e . I I . P r o t o p l a s t F o r m a t i o n by P a r e n t a l S t r a i n s a ) P. r h o d o z y m a When P. r h o d o z y m a c e l l w a l l s w e r e s u b j e c t e d t o d i g e s t i o n by enzymes a s o u t l i n e d by M o r g a n e t a_K ( 1 9 8 0 ) , u s i n g b o t h s i n g l e enzyme s o l u t i o n s and enzyme " s o u p s " , t h e r e w e r e no p r o t o p l a s t s f o r m e d . ( V a r i a t i o n s i n t i m e o f d i g e s t i o n d i d n o t c h a n g e t h e p r e v i o u s n e g a t i v e o u t c o m e . ) The c e l l s r e t a i n e d t h e i r " f o o t b a l l " s h a p e a n d when p l a c e d i n a h y p o t o n i c s o l u t i o n r e m a i n e d i n t a c t . When t h e s e c e l l s w e r e s u b j e c t e d t o a d i p o l e p r o d u c i n g e l e c t r i c f i e l d no s p i n n i n g was o b s e r v e d . A t t e m p t s t o p r o d u c e p r o t o p l a s t s f r o m c e l l s grown u n d e r d i f f e r e n t c o n - d i t i o n s i . e . l i q u i d m e d i a , s o l i d m e d i a , y o u n g (16 h r . o l d ) c u l t u r e s , o l d c u l t u r e s a n d a l t e r n a t i v e c a r b o n s o u r c e s , w e r e n o t s u c c e s s f u l . T h e r e s i s t a n c e o f t h e c e l l w a l l t o d i g e s t i o n i s c o n s i s t e n t w i t h p r e v i o u s l i t e r a t u r e - 28 - c o n c e r n i n g t h e d i f f i c u l t y i n e x t r a c t i o n o f c a r o t e n o i d s ( Okagbue & L e w i s 1983, O k a g b u e & L e w i s 1 9 8 4 ) . b) JC f r a g i l i s T h e p r o d u c t i o n o f p r o t o p l a s t s f r o m K l u y v e r o m y c e s s p . h a s b e e n r e l a t i v e l y w e l l d o c u m e n t e d ( M o r g a n e t a l . . 1980, F a r a h n a k e t a l _ . 1985, J o h a n n s e n e t a l _ . 1 9 8 4 ) . C e l l s f r o m g a l a c t o s e c o n t a i n i n g medium w e r e s u b j e c t e d t o d i g e s t i o n by t h e enzyme B - g l u c u r o n i d a s e f o l l o w i n g t h e g e n e r a l p r o t o c o l e s t a b l i s h e d by M o r g a n e t a l . . ( 1 9 8 0 ) . A t 3 0 ° C d i g e s t i o n o f c e l l w a l l s was a c c o m p l i s h e d i n a p p r o x i m a t e l y 1.5 h o u r s w i t h more t h a n 9 0 % o f t h e c e l l s c o n v e r t e d t o p r o t o p l a s t s . T h i s was d e m o n s t r a t e d by t h e o s m o t i c s e n s i t i v i t y o f t h e c e l l s , b y t h e i r s p h e r i c a l s h a p e i n o s m o t i c a l l y s t a b l i z e d m e d i a , a n d t h e i r a b i l i t y t o s p i n when s u b j e c t e d t o a d i p o l e p r o d u c i n g a l t e r n a t i n g c u r r e n t . U n d e r m i c r o s c o p i c o b s e r v a t i o n t h e p r o t o p l a s t s a p p e a r e d t o be i n g o o d c o n d i t i o n . T h e y w e r e t u r g i d , c i r c u l a r a n d r e g e n e r a t e d t h e i r c e l l w a l l s i n r e c o v e r y medium. I I I . A m e l i o r a t i o n o f t h e c e l l w a l l o f P. r h o d o z y m a t o enzyme d i g e s t i o n a) P r e t r e a t m e n t o f t h e c e l l s C e l l s g r o w i n g on MEA medium c o n t a i n i n g 5 mg/mL AEC s t a r t e d t o p r o d u c e " C h l a m y d o p h o r e s " ( l a r g e c i r c u l a r c e l l s ) . A f t e r t h r e e weeks t h e s e " C h l a m y d o p h o r e s " s t a r t e d t o " g e r m i n a t e " p r o d u c i n g w h at c a n be d e s c r i b e d a s " p s e u d o m y c e l i u m " a n d p l e o m o r p h i c c e l l s ( F i g . 1 ) . T h e s e c e l l s seemed t o have t h i n n e r ( a n d i n some c a s e s more i n c o m p l e t e ) c e l l w a l l s t h a n P h a f f i a . A t t h i s t i m e t h e s e c e l l s w e r e c o n s i d e r e d a s b e t t e r s u i t e d f o r p r o d u c i n g p r o t o p l a s t s t h a n t h e u n t r e a t e d p a r e n t P h a f f i a . - 29 - Table II. Effect of various amino acid analogues on the growth and cel l structure of Phaffia rhodozyma*. Analogue Growth Cell characteristics sulfa guanidine +++ ovoid and irregular ce l l s , no filaments, thin wal1 5-fluoro D,L tryptophan ++ cel l debris present no filamentous cel l s L tryptophan hydroxyamate ++ cel ls debris present a few filamentous ce l l s L-lysine hydroxyamate ++++ (white coloured) cel l s granular, thin wall, no filamentous cel l s S-hydroxylysine HC1 ++++ round and ovoid ce l l s , granula, thin wal1 S-(2 aminoethyl) L-cysteine +++ many filamentous segmented ce l l s , thin wall * 2 days growth on 2% Malt Extract (Difco) + 5 mg/ml analogue + 2% Bactoagar at 22°C - 30 - O t h e r a n a l o g u e s s u c h a s s u l f a g u a n a d i n e , 5 - f l u o r o D, L t r y p t o p h a n , s - h y d r o x y l y s i n e HC1 a n d o t h e r s w e r e a l s o e v a l u a t e d f o r t h e i r a b i l i t y t o p r o d u c e t h i n w a l l s ( T a b l e I I ) . None o f t h o s e t e s t e d w e r e a s e f f e c t i v e a s AEC. F u r t h e r work u s i n g AEC i n d i c a t e s t h a t l i q u i d medium c u l t u r e s i n a d d i t i o n t o s o l i d medium c u l t u r e s w e r e s u c c e s s f u l i n p r o d u c i n g t h i n w a l l e d c e l l s . T h e s e t h i n w a l l e d c e l l s r e q u i r e a n o s m o t i c s t a b l i z e r s u c h a s 0.7 M m a n n i t o l i n t h e medium t o m a i n t a i n t h e c e l l u l a r i n t e g r i t y d u r i n g g r o w t h (P.M. T o w n s l e y , p e r s o n a l c o m m u n i c a t i o n , 1 9 8 8 ) . b) P r o t o p l a s t f o r m a t i o n b y AEC t r e a t e d P h a f f i a I t was d e c i d e d t h a t b a s e d on m i c r o s c o p i c o b s e r v a t i o n s t h a t t h e most s u i t a b l e c e l l s f o r p r o t o p l a s t f o r m a t i o n w e r e t h o s e grown on 5 mg/mL AEC - MEA p l a t e s f o r 3 w e e k s . An enzyme m i x t u r e c o n t a i n i n g 1 mg/mL c h i t i n a s e ( S i g m a ) , 2 mg/mL a m y l o g l u c o s i d a s e ( S i g m a ) a n d 5 mg/mL B - g l u c u r o n i d a s e ( S i g m a ) was f o u n d t o be t h e m o s t e f f e c t i v e f o r d i g e s t i o n o f t h e c e l l w a l l s . A f t e r i n c u b a t i o n o f t h e P h a f f i a c e l l s a t 3 0 ° C f o r 3.5-4 h o u r s , a p p r o x i m a t e l y 5 0 % o f t h e c e l l s w e r e c o n v e r t e d t o p r o t o p l a s t s a s e v i d e n c e d b y o s m o t i c s e n s i t i v i t y a n d s p i n n i n g when s u b j e c t e d t o a l t e r n a t i n g c u r r e n t . Due t o t h e l a r g e v a r i a t i o n i n c e l l m o r p h o l o g y , i t was d i f f i c u l t t o d e t e r m i n e w h i c h c e l l t y p e p r o d u c e d t h e b e s t p r o t o p l a s t s b u t many w e r e s e e n t o o o z e f r o m t h e " p s e u d o m y c e l i u m " . When p l a t e d on OSRM, m o s t o f t h e p r o t o p l a s t s d i d r e c o v e r much more s l o w l y when c o m p a r e d t o K. f r a g i l i s . S u b s e q u e n t i n v e s t i g a t i o n s i n d i c a t e t h a t p r o t o p l a s t c a n a l s o be p r e p a r e d f r o m c e l l s o b t a i n e d f r o m l i q u i d medium c u l t u r e s . - 31 - F i g . 1. j \ r h o d o z y m a c e l l s grown i n t h e p r e s e n c e o f 5 mg/ml AEC i n MEA (600X m a g n f i c a t i o n ) - 32 - I V . F u s i o n o f K. f r a g i l i s a n d AEC T r e a t e d - P . r h o d o z y m a P r o t o p l a s t s a ) F u s i o n b y E l e c t r o f u s i o n T h o s e c e l l s t o be f u s e d by e l e c t r o f u s i o n , w e r e f i r s t s u b j e c t e d t o a r o t a t i n g a l t e r n a t i n g c u r r e n t i n s i d e t h e f u s i o n c e l l ( A d v a n c e d E n g i n e e r i n g C o r p ) . D u r i n g e x p o s u r e t o AC, t h e s p i n n i n g p r o t o p l a s t s t e n d e d t o f o r m p e a r l c h a i n s ( s i n g l e l i n e s o f a s s o c i a t e d c e l l s w h i c h o f t e n b r i d g e b e t w e e n t h e p a r a l l e l p l a t i n u m e l e c t r o d e s ) w h i c h w e r e n o t e d b y B a t e s e t §_[. 1983. Once t h e p e a r l c h a i n s had f o r m e d , f u s i o n was a c h i e v e d b y t h e a p p l i c a t i o n o f a 6 u s A . C . p u l s e r a n g i n g f r o m 1500-2700 v o l t s . A t v o l t a g e s e x c e e d i n g 2700 v many o f t h e c e l l w a l l s r u p t u r e d . S o m e t i m e s c e l l s w o u l d be p u l s e d two o r t h r e e t i m e s w i t h a p a r t i c u l a r v o l t a g e w i t h o u t s h o w i n g a d v e r s e e f f e c t s . F u s i o n o f t h e p l a s m a membranes i s p r o b a b l y i m m e d i a t e ( B a t e s e t a l _ . 1983) and a f t e r t h e f u s i o n p u l s e , t h e f u s e d c e l l s w e r e n o t r e s u b j e c t e d t o t h e AC r o t a t - i n g c u r r e n t s o a s n o t t o d i s r u p t t h e c e l l s . A c t u a l c o a l e s c e n c e o f t h e c e l l s was n o t v i s u a l l y o b s e r v e d due t o t h e f a c t t h a t w a t e r e v a p o r a t i o n f r o m t h e m a n n i t o l s o l u t i o n t e n d s t o f o r m d i s r u p t i n g c r y s t a l s w i t h i n t h e t i m e r e q u i r - e d . However some c e l l s w e r e s e e n t h a t w e r e a d v a n c e d p a s t t h e f i r s t s t e p o f p o i n t a d h e r a t i o n s . E l e c t r o d e s w e r e t h e n g e n t l y f l u s h e d w i t h 0.7 M m a n n i t o l t o wash t h e a s s o c i a t e d f u s e d p r o t o p l a s t s o n t o t h e 0SRM. b) F u s i o n b y P o l y e t h y l e n e G l y c o l T h o s e p r o t o p l a s t s t o be f u s e d w i t h PEG w e r e i n c u b a t e d w i t h PEG i n a c e n t r i f u g e t u b e . When o b s e r v e d u n d e r a m i c r o s c o p e , t h e p r o t o p l a s t s w e r e s e e n t o a s s o c i a t e b u t no c o a l e c e n c e was o b s e r v e d on s h o r t t i m e o b s e r v a t i o n . The a s s o c i a t e d p r o t o p l a s t s w e r e d i l u t e d w i t h 0.5 ml o f p r o t o p l a s t b u f f e r a nd p l a t e d on OSRM. - 33 - V. S e l e c t i o n a n d i s o l a t i o n o f f u s e d c e l l s S i n c e P. r h o d o z y m a n e i t h e r g r o w s on g a l a c t o s e c o n t a i n i n g medium n o r 3 0 ° C , g r o w t h u n d e r t h e s e c o n d i t i o n s w e r e c o n s i d e r e d t o be t h e b e s t i n h i b i t i n g t h e g r o w t h o f P h a f f i a . S i n c e K. f r a g i l i s p r o d u c e s w h i t e t o c r e a m c o l o u r e d c o l o n i e s , t h e p r o d u c t i o n o f o r a n g e c o l o u r i n c o l o n i e s g r o w i n g on OSRM c o n t a i n i n g g a l a c t o s e s h o u l d r e p r e s e n t t h e d e s i r e d f u s o g e n . T h e c e l l s w ere a l l o w e d t o r e c o v e r u n d i s t u r b e d f o r 7 d a y s a n d t h e n m o n i t o r e d f o r c o l o u r e d c o l o n i e s . U n d e r t h e a b o v e c o n d i t i o n s , K. f r a g i l i s g r e w v e r y q u i c k l y a n d i n some c a s e s o v e r g r e w t h e p l a t e . T o a v o i d f u r t h e r o v e r g r o w t h , a t t e m p t s w e r e made t o p l a t e no more t h a n 2.5 x 1 0 2 c f u / p l a t e . A f t e r a p p r o x i m a t e l y 10 d a y s s m a l l c o l o n i e s a p p e a r e d t h a t d i d show some o r a n g e h u e . T h e s e w e r e l e f t a few d a y s l o n g e r u n t i l a d e e p e r o r a n g e c o l o u r a p p e a r e d . C o l o n i e s t h a t r a n g e d i n c o l o u r f r o m y e l l o w t o p i n k a n d o r a n g e w e r e p i c k e d a n d p l a t e d on YNB + g a l a c t o s e a n d p l a c e d i n t h e 3 0 ° C i n c u b a t o r . A p p r o x i m a t e l y 45 c o l o n i e s w e r e s e l e c t e d a n d t r e a t e d i n t h i s m a n n e r . T h e low number o f c o l o n i e s o f t h i s t y p e s u g g e s t s a low f r e q u e n c y o f s u c c e s s f u l f u s i o n s , a p p r o x i m a t e l y 1 / 1 0 6 . A s t h e f u s a n t c o l o n i e s grew, a number o f common m o r p h o l o g i e s were r e c o r d e d ( T a b l e I I I ) . T h e m o s t common c o l o n i e s a p p e a r e d a s s m a l l , c o n v e x , s h i n y w i t h s m o o t h e d g e s . The f i n a l s e l e c t e d c o l o n i e s w e r e t a k e n f r o m c o l o u r e d c o l o n i e s t h a t had u n d e r g o n e e i t h e r o f t h e f u s i o n t e c h n i q u e s . T h o s e p r e s u m e d f u s a n t s t h a t e i t h e r d i d n o t grow r a p i d l y o r f a i l e d t o p r o d u c e a p p r e c i a b l y o r a n g e p i g m e n t s , w e r e c u l l e d . T h e c e l l l i n e s t h a t w e r e e s t a b l i s h e d i n c l u d e d #3, 10, 22, 2 3 , 26, 35 a n d 4 0 . C u l t u r e #26 was s e l e c t e d a s t h e m o s t d e s i r a b l e f o r f u r t h e r s t u d y s i n c e t h e c e l l s g r e w r a p i d l y a n d p o s s e s s e d t h e m o s t p i g m e n t i n t e n s i t y . - 34 - T a b l e I I I . C o l o n y m o r p h o l o g y o f some p r e s u m e d f u s a n t s on YNB - G a l a c t o s e + KCL m e d i a . C o l o n y t y p e C o l o n y m o r p h o l o g y R e p r e s e n t a t i v e f u s a n t s 1 r e d d i s h b r o w n , l a r g e (3 mm d i a m . ) , c o n v e x , s h i n y w i t h smooth e d g e s f a s t g r o w i n g #1 2 p i n k / o r a n g e , s m a l l (1 mm d i a m . ) , c o n v e x , s m o o t h e d g e s , s l o w g r o w i n g , s h i n y #3-9, 20, 21 3 p i n k , v e r y s m a l l (.5 mm d i a m . ) , d u l l , c o n v e x , smooth e d g e s #11, 12 4 o r a n g e , l a r g e (4 mm d i a m . ) , c o n v e x , j a g g e d e d g e s #13-15 5 l i g h t o r a n g e , s m a l l (1 mm d i a m ) , s h i n y , c o n v e x , smooth e d g e s #23, 26, 30, 35 & 10 - 35 - V i s u a l o b s e r v a t i o n s w i t h a m i c r o s c o p e , i n d i c a t e d t h a t f u s a n t c e l l s w ere r o u n d o r s l i g h t l y o b l o n g d e m o n s t r a t i n g m u l t i l a t e r a l b u d d i n g ( F i g . 2 ) . T h e r e w e r e no " f o o t b a l l " s h a p e d c e l l s o r " c h a m y d o p h o r e s " o b s e r v e d i n t h e f u s a n t a s was f o u n d i n p a r e n t a l c u l t u r e s ( F i g . 3 & 4 ) . I n t h e e a r l y s t a g e s o f g r o w t h on s o l i d m e d i a , t h e r e w e r e some v a r i a t i o n s i n c o l o u r i n t e n s i t y o f f u s a n t s . D u r i n g t r a n s f e r s , t h e d a r k e s t c o l o n i e s were s e l e c t e d a n d a s r e p e a t e d t r a n s f e r s w e r e made, t h e c o l o u r v a r i a t i o n was l e s s f r e q u e n t . T h e r e w e r e no o t h e r a p p a r e n t v a r i a t i o n s w i t h r e s p e c t t o g r o w t h r a t e o p t i m a l t e m p e r a t u r e o r c a r b o n s o u r c e u t i l i z a t i o n d e m o n s t r a t e d by t h e f u s a n t . F u s a n t c e l l l i n e s i n l i q u i d g a l a c t o s e c o n t a i n i n g m e d i a g r ew r a p i d l y p r o d u c i n g p i g m e n t w i t h i n 72 h r s ( F i g . 5 ) . C e l l s v i e w e d u s i n g a m i c r o s c o p e , w e r e f o u n d t o be m o s t l y c i r c u l a r i n s h a p e . B i o c h e m i c a l t e s t s w e r e a l s o d o n e on #26 t o d e t e r m i n e w h i c h p a r e n t i t m o s t r e s e m b l e d ( T a b l e I ) . V I . I d e n t i f i c a t i o n o f P i g m e n t P r o d u c e d T h i n l a y e r c h r o m a t o g r a p h y , s p e c t r o p h o t o m e t r y , a n d mass s p e c t r u m a n a l y s i s w e r e u s e d i n an a t t e m p t t o i d e n t i f y t h e f u s a n t ' s p i g m e n t . P i g m e n t s i n #26 c e l l s grown i n l i q u i d a n d s o l i d m e d i a w e r e a n a l y z e d . P i g m e n t s f r o m P. r h o d o z y m a , R h o d o s p o r i d i u m t o r u l o i d e s , a n d a u t h e n t i c c r y s t a l l i n e a s t a x a n t h i n ( R o c h e ) w e r e a l s o e x a m i n e d f o r a c o m p a r i s o n . a) T h i n L a y e r C h r o m a t o g r a p h y T L C was u s e d a s a t e c h n i q u e f o r i d e n t i f i c a t i o n a n d i s o l a t i o n . The s o l v e n t s y s t e m u s e d was 70:30 h e x a n e : a c e t o n e a c c o r d i n g t o t h e m e t h o d s o f A n d r e w e s & S t a r r , 1976; A n d r e w e s , S t a r r , & P h a f f , 1976. - 36 - F i g . 2B F u s a n t #26 grown i n Y N B - g l u c o s e b r o t h a t 2 2 ° C (1500X m a g n i f i c a t i o n ) - 37 - - 38 - F i g . 4B IC f r a q i l i s grown on Y N B - g l u c o s e p l a t e s a t 2 2 ° C (1500X m a g n i f i c a t i o n ) - 39 - F i g . 5A P. rhodozyma grown on MEA p l a t e s a t 22°C (600X magn i f i c a t i on ) F i g . 5B F\ rhodozyma grown on YNB-glucose broth at 22°C (1500X magn i f i c a t i on ) - 40 - T a b l e I V . P i g m e n t s i s o l a t e d f r o m v a r i o u s c e l l s a n d t h e i r r e s p e c t i v e T L C a n d a b s o r p t i o n d a t a . P i g m e n t R f (Amax) V i s i b l e A b s o r p t i o n (nm) A s t a x a n t h i n ( R o c h e ) .24 470 F u s a n t #26 ( p l a t e ) .24 471 F u s a n t #26 ( l i q u i d ) A ( a c e t o n e f r a c t i o n ) .30 470 B ( h e x a n e f r a c t i o n ) .67 473 8 - c a r o t e n e ( S i g m a ) .72 447 R. t o r u l o i d e s ( p l a t e ) A ( h e x a n e : a c e t o n e ) .25 487 B ( a c e t o n e f r a c t i o n ) .64 483 P h a f f i a rhodox.yma ( p l a t e ) .24 470 - 41 - F ig . 6 250 ml flask culture of Fusant #26 grown on Standard Media + galactose at 30°C - 72 hours old. (A - blank; B - Fusant #26) - 42 F i g . 7 T L C o f a l l - t r a n s a s t a x a n t h i n ( A ) , P i g m e n t s f r o m F u s a n t # 2 6 g r o w n o n s o l i d m e d i a ( G ) a n d P i g m e n t s f r o m F u s a n t # 2 6 g r o w i n i n m e d i a ( L ) . - 43 - a ) 1. C u l t u r e s f r o m S o l i d M e d i a The c a r o t e n o i d p i g m e n t s t a k e n f r om f u s a n t s grown on s o l i d med ia were e x t r a c t e d i n t o a c e t o n e and s p o t t e d on s i l i c a g e l p l a t e s and compared t o a s t a x a n t h i n (Roche) and t o j \ rhodozyma e x t r a c t s . When run i n t h e a f o r e m e n t i o n e d s o l v e n t s , a l l t h r e e p i g m e n t s f r o m t h e t h r e e s o u r c e s had i d e n t i c a l R f v a l u e s o f 0 . 2 4 ( F i g . 6 ) . C o - c h r o m a t o g r a p h y o f t h e f u s a n t p i g m e n t s w i t h a s t a x a n t h i n r e s u l t e d i n a s i n g l e band ( d a t a no t s h o w n ) . The p i g m e n t s a l s o had a s i m i l a r o r a n g e c o l o u r . 2 . C u l t u r e s f r o m L i q u i d M e d i a The p i g m e n t s t a k e n f r o m c u l t u r e s grown on l i q u i d m e d i a were s p o t t e d on s i l i c a g e l p l a t e s and as above compared t o a s t a x a n t h i n and j \ rhodozyma ( p l a t e s ) e x t r a c t s . These p i g m e n t s s e p a r a t e d i n t o s e v e r a l b a n d s , t h e f i r s t b e i n g r e d i n c o l o u r w i t h an R f o f . 3 0 and t h e s e c o n d b e i n g o r a n g e w i t h an Rf o f 0 . 6 7 . N e i t h e r o f t h e s e p i g m e n t s w o u l d c o - c h r o m a t o g r a p h w i t h a s t a x a n t h i n , o r Pi rhodozyma p i g m e n t s . b) S i l i c a Ge l Co lumn C h r o m a t o g r a p h y When p i g m e n t s f r o m c u l t u r e s o f P h a f f i a and t h e f u s a n t #26 f r o m g a l a c t o s e c o n t a i n i n g s o l i d m e d i a were p l a c e d o n t o t h e s i l i c a g e l co lumn t h e r e was no movement o f t h e p i gmen t bands w i t h t h e hexane w a s h . As t h e i n c r e a s i n g , vo lume r a t i o s o f a c e t o n e were added t o t h e hexane s t a r t i n g w i t h 1:24 a c e t o n e : h e x a n e t h e p i gmen t s t a r t e d t o move as a s i n g l e band t h r o u g h t he c o l u m n . R e c o v e r y o f t h e band was a c h i e v e d w i t h 100% a c e t o n e ( T a b l e V I ) . When p i g m e n t s f r om f u s a n t #26 grown i n l i q u i d g a l a c t o s e c o n t a i n i n g medium were c h r o m a t o g r a p h e d , f r a c t i o n (B) was e l u t e d u s i n g 1:12 a c e t o n e : h e x a n e w h i l e t h e s e c o n d f r a c t i o n (A) c o u l d o n l y be c o m p l e t e l y removed u s i n g 100% a c e t o n e . - 44 - c ) S p e c t r o p h o t o m e t r y 1. C u l t u r e s grown on S o l i d M e d i a P i g m e n t s i s o l a t e d f r o m f u s a n t #26 grown on s o l i d m e d i a p u r i f i e d on a s i l i c a g e l c o l u m n w e r e d i s s o l v e d i n a c e t o n e a n d s c a n n e d o v e r t h e v i s i b l e r a n g e . T h e s e c e l l s p r o d u c e d a s p e c t r a t h a t c o n t a i n e d a s i n g l e p eak w i t h ^max a t 4 7 1 n m « T n i s s i n g l e b r o a d peak c o r r e s p o n d s w i t h t h a t o f t h e a s t a x a n t h i n s t a n d a r d a n d p i g m e n t s i s o l a t e d f r o m P h a f f i a s o l i d c u l t u r e t h a t had A m a x a t 470 nm ( F i g . 8 ) . T h i s d i f f e r s f r o m t h e 8 - c a r o t e n e s t a n d a r d ( R o c h e ) ( A m a x 447 nm) ( F i g . 9) t h a t h a s 3 b a n d s a n d R h o d o s p o r i d i u m t o r u l o i d e s ( F i g . 10) A m a x 487 nm f o r t h e f r a c t i o n e l u d e d w i t h a c e t o n e and 483 nm f o r t h e p i g m e n t e l u d e d w i t h h e x a n e ( T a b l e I V ) . 2. C u l t u r e s grown i n L i q u i d M e d i a T h e p i g m e n t s i s o l a t e d f r o m t h e l i q u i d c u l t u r e o f f u s a n t #26 w e r e e l u d e d f r o m t h e s i l i c a g e l c o l u m n i n 2 f r a c t i o n s , t h e f i r s t e l u d e d w i t h h e x a n e (B) a n d t h e s e c o n d w i t h a c e t o n e ( A ) . T h e s e p i g m e n t s h a v e a s p e c t r a w i t h p i g m e n t A h a v i n g A m a x a t 470 nm b u t d i s p l a y i n g a l a r g e s h o u l d e r a t 535 nm and p i g m e n t B h a v i n g A m a x a t 473 nm h a v i n g t h r e e i n f l e c t i o n s more t y p i c a l o f B - c a r o t e n e . T h e s e p i g m e n t s d i f f e r f r o m t h o s e i s o l a t e d f r o m R h o d o s p o r i d i u m t o r u l o i d e s ( F i g . 1 0 ) . d) Mass S p e c t r o m e t r y C a r o t e n o i d p i g m e n t s f r o m f u s a n t #26 c u l t u r e d i n l i q u i d & s o l i d m e d i a , P i r h o d o z y m a a n d R i t o r u l o i d e s c u l t u r e d i n s o l i d m e d i a w e r e s e p a r a t e d on a s i l i c a g e l (BDH) c o l u m n . When e a c h f r a c t i o n was t h e n c h e c k e d by t h i n l a y e r c h r o m a t o g r a p h y , i t p r o d u c e d a s i n g l e b a n d . The Mass S p e c t r a o b s e r v e d a r e shown i n F i g s . 11-14. - 45 - Fig . 8 Visible Absorption Spectra of pigments from rhodozyma, al l - trans astaxanthin and Fusant #26 grown on sol id media. o-astaxanthin ;»-P. rhodozyma ; * - f u s a n t 26 - 46 - F i g . 9 V i s i b l e A b s o r p t i o n S p e c t r a o f 8 - c a r o t e n e a n d p i g m e n t s f r o m t h e 2 f r a c t i o n s o f F u s a n t #26 grown on l i q u i d m e d i a . • -ft-carotene ; • -fraction A; A-fraction B - 47 - 0.6-f wave leng th (nm) 10 V i s i b l e A b s o r p t i o n S p e c t r a o f p i g m e n t s i s o l a t e d f r o m F u s a n t #26 ( s o l i d ) a n d R. t o r u l o i d e s . A-fusant 26 ; o- fraction A; •-fraction B 0 48 - IS I I I I I I I I I I I I I I I I I I I F i g . 11 Mass S p e c t r u m o f A s t a x a n t h i n . - 49 - 0 0 I II I I I ! I I I II I I II I I I I IS rr T T is is I II II I II II >(ead aseq j.o % Fig. 12 Mass Spectrum of pigments isolated from P. rhodozyma grown on MEA at 22°C. 50 i i I I i i i i i i T T T l ( T T T SI oo <s at ts s» co *o •» *s es <s Fig. 13 Mass Spectrum of pigments isolated from Fusant #26 grown on galactose plates - probe temperature 150°C - 51 - ts T T T T T T T T i I I I I I I I I >|P9d a seq j o % F i g . 14 Mass S p e c t r u m o f p i g m e n t s i s o l a t e d f rom F u s a n t #26 grown on g a l a c t o s e p l a t e s - p robe t e m p e r a t u r e 280°C - - 52 - One o b v i o u s c o n c l u s i o n f r o m mass s p e c t r o m e t r y i s t h a t none o f t h e s p e c t r a o b t a i n e d f r o m t h e c e l l e x t r a c t s c o r r e s p o n d s t o t h a t o f t h e a l l - t r a n s A s t a x a n t h i n ( R o c h e ) ( F i g . 1 1 ) . The a l l - t r a n s a s t a x a n t h i n d o e s g i v e a c h a r a c t e r i s t i c mass p e a k a t 596, h o w e v e r t h e a d d i t i o n a l b u t p e a k s c o n s i d e r e d t o be c h a r a c t e r i s t i c s u c h a s M-154, M-167 a n d M-207 a r e m i s s i n g a n d two o f t h e p e a k s t h a t a r e p r e s e n t M-219 a n d M-233 do n o t h a v e e x p e c t e d i n t e n s i t i e s . T h e p i g m e n t f r o m P h a f f i a ( F i g . 12) a n d t h e p i g m e n t f r o m f u s a n t #26 grown on g a l a c t o s e p l a t e s ( F i g . 14) a r e v e r y s i m i l a r e v e n w i t h r e s p e c t t o t h e i r h i g h m o l e c u l a r w e i g h t m a t e r i a l , a n d i n f a c t c o u l d be c o n s i d e r e d t o be t h e same ( E i g e n d o r f f , p e r s o n a l c o m m u n i c a t i o n ) . e) C o l o u r r e a c t i o n s A s t a x a n t h i n a n d p i g m e n t s f r o m b o t h l i q u i d a n d p l a t e c u l t u r e s o f f u s a n t #26 p r o d u c e d a d a r k b l u e c o l o u r ; a n d p - c a r o t e n e a l i g h t b l u e c o l o u r upon t h e a d d i t i o n o f c o n c e n t r a t e d s u l p h u r i c a c i d . P i g m e n t s f r o m R. t o r u l o i d e s t u r n e d i m m e d i a t e l y c o l o u r l e s s ( T a b l e V ) . - 53 - T a b l e V. C o l o u r c h a n g e t e s t o f v a r i o u s c a r o t e n o i d s upon t h e a d d i t i o n o f c o n c e n t r a t e d s u l p h u r i c a c i d t o a c e t o n e s o l u t i o n s . C a r o t e n o i d C o l o u r o f s o l n . b e f o r e H 2 S 0 4 C o l o u r o f s o l n . a f t e r H 2 S 0 4 a d d e d a s t a x a n t h i n d a r k o r a n g e / r e d d a r k b l u e B - c a r o t e n e y e l l o w l i g h t b l u e f u s a n t #26 p l a t e o r a n g e m e d i u m - d a r k b l u e P. r h o d o z y m a o r a n g e m e d i u m - d a r k b l u e R h o d o s p o r i d i u m s p . ( a c e t o n e : h e x a n e f r a c t i o n ) p i n k / o r a n g e c o l o u r l e s s ( a c e t o n e f r a c t i o n ) p i n k c o l o u r l e s s - 54 - T a b l e V I . E l u t i o n c h a r a c t e r i s t i c s o f p i g m e n t s i s o l a t e d f r o m f \ r h o d o z y m a F u s a n t #26, a n d R̂ . t o r u l o i d e s on a s i l i c a g e l 60 c o l u m n . C a r o t e n o i d E l u t i o n s o l v e n t a s t a x a n t h i n a c e t o n e B - c a r o t e n e h e x a n e f u s a n t #26 ( p l a t e ) a c e t o n e P. r h o d o z y m a a c e t o n e F u s a n t #26 ( l i q u i d ) f r a c t i o n B f r a c t i o n A h e x a n e : a c e t o n e a c e t o n e ( 1 2 : 1 ) R. t o r u l o i d e s f r a c t i o n A f r a c t i o n B h e x a n e : a c e t o n e h e x a n e r a c e t o n e ( 1 4 : 1 ) ( 1 : 9 ) - 55 - DISCUSSION The c h i e f a i m o f t h i s s t u d y was t o i s o l a t e a s t r a i n o f y e a s t h a v i n g c h a r a c t e r i s t i c s f a v o u r i n g t h e p r o d u c t i o n o f c a r o t e n o i d s f r o m g a l a c t o s e c o n t a i n i n g medium, w h i l e a l s o p o s s e s s i n g a t h i n w a l l t o p r o m o t e e x t r a c t i o n o f t h e p i g m e n t s . To t h i s g o a l , F\ r h o d o z y m a was c h o s e n f o r i t s a b i l i t y t o p r o d u c e a s t a x a n t h i n , a n d IC f r a g i l i s f o r i t s a b i l i t y t o grow on g a l a c t o s e c o n t a i n i n g medium. E n z y m a t i c r e m o v a l o f t h e c e l l w a l l o f j \ r h o d o z y m a grown u n d e r n o r m a l c o n d i t i o n s met w i t h v e r y l i t t l e s u c c e s s a s e x p e c t e d . T h e l i t e r a t u r e s u g g e s t s t h a t t h e r e a r e m u l t i p l e l a y e r s i n t h e c e l l w a l l ( M i l l e r e t a l _ . 1976) a n d t h i s f a c t o r , a s w e l l a s t h e a 1-3 g l u c a n s make i t r e s i s t a n t t o m o s t l y t i c e n z y m e s . When m o r p h o l o g i c a l d i f f e r e n c e s w e r e f o r c e d t o o c c u r due t o t h e e f f e c t o f t h e AEC, a r e a s o f t h e c e l l w a l l t h a t w e r e t h i n n e r m i c r o s c o p i c a l l y , w e r e t h e n a b l e t o be a t t a c k e d b y t h e l y t i c e n z y m e s . O f t e n t h e s e s u s c e p t i b l e c e l l s r e s e m b l e d c h l a m y d o p h o r e s , a n d i t a p p e a r e d t h a t t h e " g e r m i n a t i o n t u b e " o f t h e s e s t r u c t u r e s t h a t w e r e a t t a c k e d . When t h e s e c e l l s g e r m i n a t e d i n t h e p r e s e n c e o f AEC t h e r e s u l t i n g p s e u d o m y c e l i u m a n d p l e o m o r p h i c c e l l s ( F i g . 1) a l s o showed a l a c k o f r e s i s t a n c e t o e n z y m a t i c d i g e s t i o n o f t h e c e l l w a l l s u g g e s t i n g t h a t t h e c e l l s w e r e s e v e r e l y a f f e c t e d b y t h e AEC. T h i s p a r t i c u l a r c e l l t y p e r e m a i n s w h i l e t h e c e l l s grow i n AEC b u t , upon r e t u r n t o MEA t h e c h a r a c t e r i s t i c w a l l a n d s h a p e o f r h o d o z y m a r e t u r n s . T h e o t h e r a m i n o a c i d a n a l o g u e s t e s t e d ( T a b l e I I ) d i d n o t p r o d u c e s u c h d e s i r a b l e c e l l s . I t i s known t h a t many c u l t u r e s t h a t a r e r e s i s t a n t t o a n a l o g u e s s u c h a s AEC a r e a b l e t o a c c u m u l a t e L - l y s i n e due t o t h e c h a n g e i n r e g u l a t i o n o f a s p a r t o k i n a s e ( T o s k a t a e t a l . . 1 9 7 8 ) . I t i s - 56 - p o s s i b l e t h a t i n P h a f f i a c o m p o n e n t s t h a t make up t h e c e l l w a l l a r e l i k e w i s e a f f e c t e d . As c o m p a r e d t o o t h e r j u s t s p e c i e s grown on m e d i a w i t h o u t a m i n o a c i d a n a l o g u e s , t h e number o f p r o t o p l a s t s r e c o v e r e d f r o m J \ r h o d o z y m a c e l l s g rown on AEC was q u i t e l o w . A number o f i n h e r e n t f a c t o r s c o u l d be t o b l a m e f o r t h i s . T h e r e l e a s e o f t h e p r o t o p l a s t s w e r e n o t s y n c h r o n o u s a n d i n f a c t o c c u r r e d o v e r a l o n g p e r i o d o f t i m e p o s s i b l y due t o i n c o n s i s t e n c i e s i n t h e c e l l w a l l s t r u c t u r e o r c o m p o s i t i o n . B e c a u s e o f t h i s , t h e c o n c e n t r a t i o n o f t h e c e l l w a l l d i g e s t i v e enzyme m i x t u r e was h i g h , a n d t h u s p r o t o p l a s t s f o r m e d e a r l y i n t h e d i g e s t i o n p e r i o d w e r e m o s t l i k e l y d e s t r o y e d . N o t a l l o f t h e c e l l s e x p o s e d t o t h e AEC d e v e l o p e d t h i n w a l l s a n d o d d s h a p e s , t h u s a p e r c e n t a g e o f t h e p o p u l a t i o n l i k e l y may n o t be a f f e c t e d a t a l l by t h e e n z y m e s . P r o t o p l a s t s f o r m e d l a t e r i n t h e d i g e s t i o n p e r i o d w e r e c h o s e n b e c a u s e a p r o p o r t i o n a l l y g r e a t e r number w e r e r e l e a s e d a t t h e l a t e r s t a g e s . On t h e o t h e r h a n d , t h e r e c o v e r y o f p r o t o p l a s t s f r o m J C f r a g i l i s was s i m p l e . The y o u n g c e l l s w e r e c o n v e r t e d t o p r o t o p l a s t s w i t h i n 1 1/2 h o u r s d e n o t i n g one o f t h e p o s i t i v e q u a l i t i e s t o be t r a n s f e r e d t o a f u s a n t . The h i g h p r o t o p l a s t f o r m i n g e f f i c i e n c i e s o b s e r v e d i n t h i s s t u d y c o r r e l a t e d w i t h t h o s e r e p o r t e d by M organ e t a l _ . ( 1 9 8 0 ) a n d J o h a n n s e n e t a l - ( 1 9 8 4 ) . Two d i f f e r e n t a p p r o a c h e s f o r t h e f u s i o n o f t h e y e a s t p r o t o p l a s t s w e r e a t t e m p t e d . T h e c o n v e n t i o n a l PEG m e t h o d a n d t h e d i p o l e e l e c t r o p o r a t i o n m e t h o d o f R i v e r a e t a_J_. ( 1 9 8 3 ) . E l e c t r o f u s i o n h a s b e e n r e p o r t e d t o h a v e an e n h a n c e d f u s i o n e f f i c i e n c y when c o m p a r e d t o t h e c o n v e n t i o n a l PEG m e t h o d ( B a t e s e t a_l_. 1 9 8 6 ) . I n t h e e n d , t h e - 57 - e l e c t r o f u s i o n was v e r y s u c c e s s f u l p r o d u c i n g a number o f t h e s e l e c t e d p r e s u m e d f u s a n t s i n c l u d i n g f u s a n t #26. When one c o n s i d e r s t h e a c t u a l number o f p r e s u m e d f u s a n t s s e l e c t e d a s c o m p a r e d t o t h e number o f p r o t o p l a s t s , t h e number seems q u i t e low ( s e e T a b l e I I I ) . One m u s t remember t h a t t h e f u s a n t s s e l e c t e d w e r e c h o s e n f o r t h e i r s p e c i f i c p r o p e r t i e s o f g r o w t h on g a l a c t o s e a n d p i g m e n t f o r m a t i o n . O t h e r f u s i o n s t h a t may n o t h a v e p r o d u c e d t h e s t a b l e d e s i r e d c h a r a c t e r i s t i c s a r e a l s o l i k e l y t o h a v e o c c u r r e d . Low numbers o f p r e s u m e d f u s a n t s may a l s o be a t t r i b u t e d t o t h e s u s p e c t e d p o o r v i a b i l i t y o f t h e P h a f f i a p r o t o p l a s t s w h i c h may n o t h a v e b e e n a b l e t o r e g e n e r a t e t h e i r c e l l w a l l s . A r e d u c e d number o f p r e s u m e d f u s a n t s may a l s o be a t t r i b u t e d t o t h e v o l t a g e s u s e d t o i n d u c e f u s i o n . The v o l t a g e s e l e c t e d a n d a p p l i e d a t t h e e l e c t r o d e s o f t h e d i p o l e c e l l f u s e r was 2700 v o l t s . T h e d i s t a n c e b e t w e e n t h e e l e c t r o d e s d e p e n d i n g on t h e e x p e r i m e n t r a n g e d f r o m 1 t o 2 mm. T h i s h i g h v o l t a g e may h a v e a f f e c t e d t h e r e g e n e r a t i o n a b i l i t y o f t h e p r o t o p l a s t s . PEG i n c o n t r a s t t o e l e c t r o f u s i o n i s a much s l o w e r m e t h o d f o r f u s i o n . T h e PEG i t s e l f d o e s n o t c a u s e f u s i o n t o o c c u r , r a t h e r i t d r a w s t h e c e l l s i n t o c l o s e a s s o c i a t i o n a n d upon d i l u t i o n o f t h e c h e m i c a l , f u s i o n may o c c u r ( P e b e r d y , 1 9 8 0 ) . T h i s d r a w i n g t o g e t h e r o f c e l l s was a c c o m p l i s h e d more r a p i d l y a n d s u c c e s s f u l l y by AC r o t a t i n g f i e l d w i t h t h e a d d e d b e n e f i t o f i n d u c i n g f u s i o n by t h e e l e c t r i c p u l s e . Some p r e s u m e d f u s a n t s w e r e o b t a i n e d u s i n g PEG s u c h a s f u s a n t #22. T h e s e f u s a n t s d i d p o s s e s s q u a l i t i e s s u c h a s c e l l m o r p h o l o g y , g a l a c t o s e a s s i m i l a t i o n a n d p i g m e n t a t i o n , s i m i l a r t o t h o s e o b t a i n e d by e l e c t r o f u s i o n ( d a t a n o t - 58 - s h o w n ) . T h i s s u g g e s t s t h a t i n t h e s e s p e c i e s o f y e a s t s b o t h m e t h o d s c a n be u s e d w i t h some s u c c e s s . The a d v a n t a g e s a s s o c i a t e d w i t h t h e e l e c t r o f u s i o n i n c l u d e t h e s y n c h r o n y o f f u s i o n m a k i n g s e l e c t i o n e a s i e r , a n d t h e r e d u c e d h a n d l i n g o f t h e f r a g i l e p r o t o p l a s t s , a s w e l l a s a v o i d i n g t h e p o t e n t i a l l y h a r m f u l e f f e c t s o f PEG a d d i t i o n . F u s a n t s w e r e s e l e c t e d w i t h t h e p r o j e c t e d g o a l i n m i n d . Th e p a r e n t P h a f f i a was d i s c o u r a g e d f r o m g r o w t h by g r o w i n g t h e p r e s u m e d f u s a n t s a t 3 0 ° C , 3 ° C a b o v e t h e r e p o r t e d g r o w t h t e m p e r a t u r e l i m i t ( M i l l e r e t a j _ . 1 9 7 6 ) , a n d on Y N B - g a l a c t o s e s i n c e t h e p a r e n t a l s t r a i n s c a n n o t a s s i m i l a t e g a l a c t o s e . T h e p a r e n t JC f r a g i l i s on t h e o t h e r h a n d c o u l d be s e l e c t e d a g a i n s t by v i r t u e o f c o l o n y c o l o u r a s g r o w t h was e x c e l l e n t b o t h a t 3 0 ° C a n d on g a l a c t o s e . O t h e r s e l e c t i v e m e d i a w e r e a l s o u s e d s u c c e s s f u l l y t o a c h i e v e d i f f e r e n t i a t i o n o f t h e f u s a n t f r o m t h e p a r e n t c u l t u r e e . g . n i a c i n r e q u i r e m e n t o f IC f r a g i l i s . T h e p r e s u m e d f u s a n t c o l o n i e s o f t e n s t a r t e d o u t r e l a t i v e l y s m a l l w i t h v e r y l i t t l e c o l o u r t h o u g h m o r p h o l o g y v a r i e d s l i g h t l y ( T a b l e I I I ) . O f t e n IC f r a g i l i s w o u l d o v e r g r o w t h e p l a t e ( h e n c e t h e n e e d t o c o n t r o l t h e g r o w t h r a t e o f t h e IC f r a g i l i s p a r e n t . T h e a c c u m u l a t i o n o f c a r o t e n o i d p i g m e n t s i s c o n s i d e r e d t o be 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 a n d u s u a l l y o c c u r s i n l a t e s t a g e s o f g r o w t h . On r e p e a t e d s e l e c t i o n a n d t r a n s f e r o f c o l o u r e d c o l o n i e s , s t a b l e c e l l l i n e s o f t h e f u s a n t s w e r e o b t a i n e d . T h e r e i s e v i d e n c e f o r P h a f f i a b e i n g t h e r e c i p i e n t o f g e n e t i c m a t e r i a l f r o m J C f r a g i l i s a s shown i n T a b l e 1. T h e p r e s u m e d f u s a n t s h a v e a c e l l u l a r m o r p h o l o g y t h a t i s q u i t e d i f f e r e n t t h a n t h a t o f P h a f f i a ( F i g . 2 & 4 ) . T h e s e d i f f e r e n c e s i n c l u d e - 59 - t h e c i r c u l a r s h a p e o f t h e f u s a n t on Y N B - g a l a c t o s e , t h e t h i n n e r c e l l w a l l a s w e l l a s t h e a b i l i t y t o bud m u l t i l a t e r a l l y . B r i g h t s p o t s w i t h i n t h e c y t o p l a s m a r e a l s o e v i d e n t i n g a l a c t o s e m e d i a a n d may be i n t r a c e l l u l a r l i p i d s s i m i l a r t o t h o s e p r o d u c e d b y J C f r a g i l i s . T h e m o r p h o l o g y a nd b i o c h e m i c a l t e s t r e s u l t s o f t h e r e d y e a s t t o r u l o i d e s a r e u n i q u e l y d i f f e r e n t f r o m t h a t o f t h e p a r e n t P h a f f i a o r t h e r e s u l t a n t c a r o t e n o i d p r o d u c i n g f u s a n t #26 ( F i g s . 2, 3 & 4 ) . T h e b i o c h e m i c a l t e s t s t h a t w e r e done w e r e c h o s e n f o r t h e i r a b i l i t y t o d i f f e r e n t i a t e b e t w e e n t h e p a r e n t a l s t r a i n s a s w e l l a s o f f e r i n g some d i f f e r e n c e s f r o m p o s s i b l e c a r o t e n o i d - c o n t a i n i n g y e a s t c o n t a m i n a n t s s u c h a s R^. t o r u l o i d e s . T h e p r e s u m e d f u s a n t s a p p e a r t o d e m o n s t r a t e c h a r a c t e r i s t i c s t h a t c a n be c o n s i d e r e d t y p i c a l t o e a c h p a r e n t a l t y p e ( T a b l e I ) . T h i s i s n o t u n e x p e c t e d s i n c e f u s i o n was a random p r o c e d u r e o f f e r i n g no c o n t r o l e x e r t e d on t h e r e c o m b i n a t i o n o f c h r o m o s o m e s . The d a t a o b t a i n e d f r o m t h e b i o c h e m i c a l t e s t s , T a b l e I s u g g e s t s t h a t i s i s d i f f i c u l t t o d e t e r m i n e w h i c h p a r e n t a l s t r a i n i s t h e d o n o r a n d w h i c h i s t h e r e c i p i e n t . V i e w i n g e v i d e n c e s u c h a s c a r o t e n o i d p r o d u c t i o n , u r e a s e a c t i v i t y a n d c e l l g r o w t h w i t h o u t n i a c i n , one may v e n t u r e t o s u g g e s t t h a t P h a f f i a i s t h e r e c i p i e n t . C h a r a c t e r i s t i c s s u c h a s g a l a c t o s e a s s i m i l a t i o n a n d h e a t t o l e r a n c e w e r e t r a n s f e r r e d p o s s i b l y f r o m J C f r a g i l i s . One c h a r a c t e r i s t i c common t o b o t h J \ r h o d o z y m a a n d JC f r a g i 1 i s i s t h e i r i n a b i l i t y t o u s e n i t r a t e a s a s o u r c e o f n i t r o g e n . F u s a n t #26 i s a l s o u n a b l e t o u t i l i z e n i t r a t e . T h i s d i f f e r s f r o m R^ t o r u l o i d e s a common r e d y e a s t w h i c h i s a b l e t o u s e n i t r a t e . C e l l s grown i n v a r i o u s m e d i a w i l l a l s o c h a n g e t h e i r m o r p h o l o g y ; f o r e x a m p l e , when t h e p r e s u m e d - 60 - f u s a n t s a r e grown i n g a l a c t o s e t h e y a r e c i r c u l a r u n l i k e e i t h e r p a r e n t o r R i t o r u l o i d e s . T h e s e v e r y same c e l l s when grown i n g l u c o s e b r o t h , d e v e l o p a s h a p e t h a t a p p e a r s t o be t h e same a s r h o d o z y m a grown u n d e r s i m i l a r c o n d i t i o n s o n l y s m a l l e r ( s e e F i g s . 2B & 5 B ) . In g l u c o s e b r o t h J C f r a g i l i s i s p e a r s h a p e d a n d R i t o r u l o i d e s i s e l o n g a t e ( s e e F i g s . 3 & 4 B ) . T h i s a g a i n s u p p o r t s t h e i d e a o f P h a f f i a b e i n g t h e r e c i p i e n t . T h e l i t e r a t u r e s u g g e s t s t h a t 8 5 % o f t h e c a r o t e n o i d s f o r m e d by P i r h o d o y z m a u n d e r m o s t a m b i e n t g r o w t h c o n d i t i o n s a r e a s t a x a n t h i n ( A n d r e w e s e t a l _ . 1976, J o h n s o n & L e w i s 1 9 7 9 ) . B e c a u s e o f t h i s , a u t h e n t i c a l l - t r a n s a s t a x a n t h i n was c h o s e n t o be t h e c a r o t e n o i d t o u s e f o r c o m p a r i s o n s . C a r o t e n o i d a n a l y s e s f r o m T L C , c o l u m n c h r o m a t o g r a p h y , c h e m i c a l r e a c t i o n s , a b s o r p t i o n s p e c t r a a n d mass s p e c t r o p h o t o m e t r y i n d i c a t e t h a t t h e p i g m e n t s o b t a i n e d f r o m j \ r h o d o z y m a a n d f u s a n t #26 grown on s o l i d m e d i a a r e t h e same ( F i g s . 7, 8, 12, 14 a n d T a b l e s I V , V ) . T h e s e p i g m e n t s i n t u r n , t e n d t o c o r r e l a t e c l o s e l y w i t h t h e a l l - t r a n s a s t a x a n t h i n ( R o c h e ) w i t h t h e e x c e p t i o n o f t h e mass s p e c t r o m e t r y d a t a . T h e r e s u l t s a l s o i n d i c a t e t h a t t h e p i g m e n t s f r o m f u s a n t #26 ( s o l i d m e d i a ) a n d j \ r h o d o z y m a a r e n o t t h e same a s t h o s e i s o l a t e d f r o m t h e common r e d y e a s t R i t o r u l o i d e s ( F i g . 10 a n d T a b l e s I V , V ) . O x o - c a r o t e n o i d s h a v e a c h a r a c t e r i s t i c a b s o r b t i o n s p e c t r a , a s do t h e B - c a r o t e n e s . Due t o t h e h i g h l e v e l s o f u n s a t u r a t i o n w i t h i n t h e m o l e c u l e t h e s e p e a k s a l s o t e n d t o be b r o a d ( D a v i e s 1 9 6 5 ) . T h e p i g m e n t s i s o l a t e d f r o m f_i r h o d o z y m a a n d f u s a n t #26 grown on s o l i d m e d i a h a v e t h i s b r o a d c h a r a c t e r i s t i c s i n g l e p e a k o f t h e x a n t h o p h y l l s ( F i g . 8 ) . T h e s h a p e o f t h e a b s o r p t i o n c u r v e p l u s t h e i r A m a x c o r r e l a t e s c l o s e l y w i t h a u t h e n t i c - 61 - a s t a x a n t h i n ; i n f a c t , t h e s p e c t r a f r o m f u s a n t #26 c a n a l m o s t be s u p e r i m p o s e d on t h a t f r o m a s t a x a n t h i n ( F i g . 8 ) . O t h e r x a n t h o p h y l 1 i c - 1 i k e s t r u c t u r e s c a n a l s o be o b s e r v e d i n t h e f r a c t i o n (A) f r o m t h e f u s a n t #26's l i q u i d c u l t u r e a s w e l l a s f r a c t i o n (B) f r o m t o r u l o i d e s . T h e l i q u i d c u l t u r e s o f f u s a n t #26 w e r e u n a b l e t o p r o d u c e p i g m e n t s t h a t r e s e m b l e s t h o s e o f F\ r h o d o z y m a grown on s o l i d m e d i a . H owever, on l i q u i d medium u n d e r l i m i t e d o x y g e n s u p p l y P h a f f i a a l t e r s i t s c a r o t e n o i d s y n t h e s i s , p r o d u c i n g B - c a r o t e n e i n e x c e s s . T h u s t h e c a r o t e n o i d s p r o d u c e d by t h e s e c u l t u r e s a p p e a r s t o be a f u n c t i o n o f t h e c u l t u r e c o n d i t i o n s . J o h n s o n & L e w i s ( 1 9 79) s u g g e s t t h a t u n d e r f e r m e n t a t i v e o r a u s t e r e c o n d i t i o n s an a c c u m u l a t i o n o f a s t a x a n t h i n p r e c u r s o r s may o c c u r i n a d d i t i o n t o a d e c r e a s e i n a s t a x a n t h i n l e v e l s . I t i s p o s s i b l e t h a t a s i m i l a r e v e n t i s o c c u r r i n g i n t h e s e l i q u i d c u l t u r e s o f f u s a n t #26 due t o i n s u f f i c i e n t a e r a t i o n o r o t h e r s u c h u n d i s c o v e r e d l i m i t a t i o n s . T h e two p i g m e n t s o b t a i n e d may be a s t a x a n t h i n p r e c u r s o r s ; f u r t h e r work a l o n g t h i s l i n e i s t o be d o n e i n t h e f u t u r e . T h e a n o m a l y o f t h e mass s p e c t r o m e t r y d a t a c a n be c o n s i d e r e d t o be n o t l e s s t h a n c o n f u s i n g . One h i g h l y i r r e g u l a r r e s u l t was t h e l a c k o f t h e mass p e a k a t 596 i n t h e p i g m e n t i s o l a t e d f r o m j \ r h o d o z y m a w h i c h a c c o r d i n g t o t h e l i t e r a t u r e ( A n d r e w e s e t a l _ . 1976; J o h n s o n & L e w i s 1 9 7 9 ) , s h o u l d be p r e s e n t due t o h i g h p e r c e n t a g e s o f a s t a x a n t h i n r e p o r t e d i n t h e c e l l s ( F i g . 1 2 ) . ( T h e s e c e l l s w e r e grown a n d t h e p i g m e n t i s o l a t e d u n d e r a p p a r e n t l y i d e n t i c a l c o n d i t i o n s a s r e p o r t e d by A n d r e w e s e t a j . . ( 1 9 7 6 ) . ) E v e n t h e a u t h e n t i c a s t a x a n t h i n u s e d g a v e l e s s t h a n e n c o u r a g i n g r e s u l t s s i n c e t h r e e o f t h e c h a r a c t e r i s t i c p e a k s w e r e m i s s i n g ( s e e F i g . 1 1 ) . - 62 - The r e s u l t s f r o m t h e mass s p e c t r a l d a t a show t h a t when t h e samp les were run a t two d i f f e r e n t t e m p e r a t u r e s , t h e r e a r e q u i t e l a r g e d i f f e r e n c e s be tween t h e s e r u n s . T h i s d e n o t e s one o f t h e i n h e r e n t p r o b l e m s o f mass s p e c t r o m e t r y as s u g g e s t e d by Moss & Weedon ( 1 9 7 6 ) . I r r e g u l a r i t i e s may have o c c u r r e d due t o samp le i m p u r i t i e s , p robe t e m p e r a t u r e s , i n s e r t i o n c o n d i t i o n s o r i o n i z a t i o n (Moss & Weedon 1 9 7 6 ) . A g a i n , t hough one c a n n o t c o n c l u d e t h a t t h e c a r o t e n o i d i s a s t a x a n t h i n , f r om t h e mass s p e c t r a d a t a , t he o t h e r t e s t s do s u g g e s t t h a t p i g m e n t s f r om J \ rhodozyma and f u s a n t #26 ( s o l i d m e d i a ) a r e s i m i l a r . C a r o t e n o i d s can a l s o be d i s t i n g u i s h e d by c o l o u r r e a c t i o n s o f p i g m e n t s when s u b j e c t e d t o s t r o n g a c i d s and b a s e s ( K a r r e r & J u c k e r 1 9 5 0 ) . In t h e p r e s e n c e o f a i r a s t a x a n t h i n i s c o n v e r t e d t o i t s o x i d i z e d d e r i v a t i v e , a s t a c e n e ( f o r m u l a 6 ) . A s t a c e n e upon t h e a d d i t i o n o f CH, CH, CH, CH, C CH, CH, CH, CH, C C ^ , X C - C H = CH-C=CHCH-CH-C=CHCH=CHCH=C-CH=.CHCH-C-CH-CH-(f / CH, OC C-CH, H.C-I CO- CO CO a s t a c e n e F o r m u l a 6 c o n c e n t r a t e d s u l p h u r i c a c i d w i l l t u r n a deep b l u e ( K a r r e r & J u c k e r 1 9 5 0 ) . R e s u l t s o b t a i n e d f r o m T a b l e V i n d i c a t e t h i s o c c u r r a n c e . C o l o u r d i f f e r e n c e s be tween d a r k b l u e and medium b l u e may be a t t r i b u t e d t o c o n c e n t r a t i o n . A g a i n a r e a c t i o n d i f f e r e n c e can be n o t e d when f u s a n t #26 i s compared t o R^ t o r u l o i d e s . - 63 - CONCLUSIONS D u r i n g t h e c o u r s e o f t h i s s t u d y , an i n t e r g e n e r i c c r o s s o f IC f r a g i l i s a n d J \ r h o d o z y m a was c a r r i e d o u t . The r e s u l t i n g p r e s u m e d f u s a n t s e x h i b i t c h a r a c t e r i s t i c s common t o b o t h p a r e n t a l t y p e s s u c h a s g r o w t h on g a l a c t o s e , h e a t t o l e r a n c e a n d c a r o t e n o i d p i g m e n t p r o d u c t i o n . M o r p h o l o g i c a l l y t h e p r e s u m e d f u s a n t s a r e m o s t l i k e j \ r h o d o z y m a s u g g e s t i n g t h a t F\ r h o d o z y m a i s t h e r e c i p i e n t s t r a i n . T h o u g h t h e c a r o t e n o i d p i g m e n t s i s o l a t e d f r o m f u s a n t #26 a n d J \ r h o d o z y m a c a n n o t be c a l l e d a s t a x a n t h i n due t o t h e a n o m a l l y o f t h e mass s p e c t r o m e t r y d a t a , t h e y a r e s i m i l a r t o e a c h o t h e r a c c o r d i n g t o a l l o t h e r t e s t s . T L C , v i s i b l e s p e c t r a , c o l u m n c h r o m a t o g r a p h y a n d c o l o u r r e a c t i o n s i n d i c a t e t h a t t h e p i g m e n t c o u l d p o s s i b l y be a s t a x a n t h i n . T h e p r e s u m e d f u s a n t c e l l l i n e #26 h a s a m i c r o s c o p i c a l l y t h i n c e l l w a l l t h a t i s e a s y t o r u p t u r e t h u s l i b e r a t i n g t h e c e l l u l a r c a r o t e n o i d s p r o m p t l y . - 64 - REFERENCES A l - S h a b i b i , M.M.A. a n d N . A . J . Y o u n i s . 1984. F e r m e n t a t i o n o f Whey F i l t r a t e b y K l u v e r o m y c e s f r a g i l i s ; t h e e f f e c t on c e l l l i p i d s . C a n . I n s t . F o o d S c i . T e c h n o l . J . V o l . 17, p . 117. A n d r e w e s , A.G., J . J . P h a f f a n d M.P. S t a r r . 1976. C a r o t e n o i d s o f P h a f f i a R h a l o z y m a , a r e d - p i g m e n t e d f e r m e n t i n g y e a s t . P h y t o c h e m . , V o l . 15, p . 1003. 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Mass S p e c t r o m e t r i c S t u d i e s o f C a r o t e n o i d s . 2. A S u r v e y o f F r a g m e n t a t i o n R e a c t i o n s . A c t a Chem. S c a n d . 2 3 , p.2 F a r a h n a k , F . , T. S e k i , D.D.Y. Ryu a n d D. O g r y d z i a k . 1986. C o n s t r u c t i o n o f L a c t o s e A s s i m i l a t i n g a n d H i g h - E t h a n o l - P r o d u c i n g Y e a s t s by P r o t o p l a s t F u s i o n . A p p l i e d a n d E n v i r o n m e n t a l M i c r o b i o l o g y , V o l . 51 , p . 3 6 2 . de F i g u e r o a , L . I . , M.F. de R i c h a r d a n d M.R. de v a n B r o o k . 1984. I n t e r s p e c i f i c P r o t o p l a s t F u s i o n o f t h e B a k e r ' s Y e a s t S a c c h a r o m y a s c e r e v i s i a e a n d S a c c h a r o m y c e s d i a s t a t i c u s . B i o t e c h n o l o g y L e t t e r s , V o l . 6, p . 2 6 9 . F o s s , P., R.R.L. G u i l l a r d a n d S. L i a a e n - J e n s e n . 1986. 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K o s i k o w s k i . 1972. L a c t a s e P o t e n t i a l o f V a r i o u s M i c r o o r g a n i s m s Grown i n Whey. J . D a i r y S c i e n c e , V o l . 56, p . 2 6 . - 68 - APPENDIX I - 69 - 1 2 i i i i i i i n | 1 i i i i 111| I—i i l 11 • 11 1—i i i i 1—i l l I MI FREQUENCY/kHl 17 S p i n ranges o f i n d i v i d u a l unbudded y e a s t c e l l s . f r om Poh l e t a l . , 1982 - 70 - F i g . 1 8 . A model f o r t h e d i p o l e moments i n a y e a s t c e l l s . f r o m Poh l e t a l . , 1982 - 71 - APPENDIX I I F i g . 1 9 . F u s i o n a p p a r a t u s - 73 - APPENDIX I I I - 74 - The growth medium was 50 ml 0 1 M-phthalate-bufTered yeast nitrogen base medium (see Methods), containing 0-6 ° 0 (w/v) Bacto-peptone and 200 mg carbon (supplied as the various sugars). The values represent the mean of two determinations. Carbon sources were sterilized separately from the basal medium. N o growth occurred in the basal medium without the addition of a carbon source. Growth Yeast Astaxanthin Astaxanthin rate, fi yield Yeast yield yield yield Carbon source O r ' ) (mg m l - 1 ) [mg (mg carbon) " 1 ] * (Mg m l " 1 ) l/ig (g yeast)"1] D-Maltose 0 1 4 3-63 0 91 1 86 512 D-Cellobiose 0 10 3-48 0 87 2-27 652 Sucrose 0 19 3-72 093 1 89 508 Succinate 0 0 9 2-66 0-67 1 33 500 D-Marui itol 0 16 3-68 0 9 2 1 80 489 D-Xylose 0 0 4 1-21 0 30 0 58 479 L-Arabinose 0 0 5 3 30 083 1 25 379 Glucono-<S-lactone 0 10 1 48 0-37 0 80 541 D-Glucose 0 2C 3 85 0 9 6 1 62 421 D -G lucose + 021 6 4 6 0 81 111 171 • Assuming all carbon utilized. t 800 mg carbon [4°„ (w/v) glucose]. T a b l e V I I . E f f e c t o f c a r b o n s o u r c e on g r o w t h and p i g m e n t a t i o n o f P. r h o d o z y m a i n S h a k e F l a s k C u l t u r e . f r o m J o h n s o n & L e w i s , 1979. - 75 - APPENDIX IV - 76 - Fig. 21. Possible biochemical pathways from the B-end group (12) to the astaxanthin end group (II). from Andrewes et al_., 1976. - 77 - APPENDIX V - 78 - Constituents Content (%) Phaffia Brewer's rhodozy ma yeast Ash 5.6 6.5 Total carbohydrate 40.3 33.4 Total nitrogen 4.82 8.71 Protein (N x 6.25) 30.1 54.4 Protein (Folin reagent) 25.0 — R N A 8.2 9.2 Total lipid 17.0 4.03 Astaxanthin .06 0 T a b l e V I I I P r o x i m a t e c o m p o s i t i o n o f P h a f f i a rhodozyma and b r e w e r s y e a s t ( dwb ) . f r om J o h n s o n et a ] , . , 1980.

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