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Construction of galactose assimilating, carotenoid producing yeasts by protoplast fusion Hansen, Christine S. 1988

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CONSTRUCTION  OF G A L A C T O S E  ASSIMILATING,  C A R O T E N O I D 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  FUSION  by C H R I S T I N E S. H A N S E N B.Sc., U n i v e r s i t y  of B r i t i s h Columbia,  A THESIS SUBMITTED  IN P A R T I A L F U L F I L L M E N T  T H E R E Q U I R E M E N T S FOR T H E D E G R E E M A S T E R OF  OF  SCIENCE  in T H E F A C U L T Y OF G R A D U A T E  STUDIES  (Department o f Food 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 to the required  standard  T H E U N I V E R S I T Y OF B R I T I S H June,  (P  1985  COLUMBIA  1988  C h r i s t i n e S. H a n s e n ,  1988  OF  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 w e r e p r e p a r e d f r o m two y e a s t 24202)  and  rhodozyma  JC were  containing yeast  facilitated  genera  were  fused  glycol  s e l e c t e d by g r o w t h a t  genera  similar  to  Protoplasts  8455).  by  prior  with  that of  either  treatment.  Selected single both  (ATCC  growth  S-(2-aminoethyl)-L-cysteine.  polyethylene were  fragilis  a P.  the  use  nitrogen  taxonomic  morphology  rhodozyma.  the  of  carotenoid  30°C on y e a s t  cellular  of  Protoplasts  by  Stable  fusants display  strains J \  and  rhodozyma  prepared  from  cells  a  in  from  P.  media  these  two  electrofusion  producing base p l u s  cell  carotenoid  or lines  galactose.  characteristics a  (ATCC  common  to  composition  — iii— T A B L E OF CONTENTS PAGE ABSTRACT T A B L E OF CONTENTS  ii i i i  LIST.OF TABLES  iv  L I S T OF F I G U R E S  v  DEDICATION  vii  ACKNOWLEDGEMENT  vi  INTRODUCTION  1  L I T E R A T U R E REVIEW I Genetic Manipulations II C a r o t e n o i d s I I I P h a f f i a rhodozyma IV K l u y v e r o m y c e s f r a g i 1 i s  3 7 13 16  M A T E R I A L S AND METHODS I Strains 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 .... I I I B u f f e r s and Reagents IV M e d i a 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 and R e g e n e r a t i o n 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 VII C a r o t e n o i d A n a l y s i s  18 18 18 19 20 22 23  RESULTS I C h a r a c t e r i s t i c s of the Parent Organisms 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 I I I A m e l i o r a t i o n o f the C e l l Wall o f j \ rhodozyma t o Enzyme D i g e s t i o n 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 Protoplasts V S e l e c t i o n of Fused C e l l s 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  32 33 35  DISCUSSION  55  CONCLUSIONS  63  REFERENCES  64  APPENDICES  25 27 28  - iv LIST OF TABLES PAGE Table Table  I. II.  Table III.  Table  Table  IV.  V.  Selected Biochemical test results for J \ rhodozyma, f r a q i l i s and Fusant #26  26  Effects of Various Amino Acid Analogues on Growth and Morphology of F\ rhodozyma  29  Colony Morphology on Some Presumed Fusants Grown on YNB-galactose + KC1 media  34  Pigments Isolated from Various Cultures and Their Respective TLC and Absorption Data  40  Colour Change Test of Various Carotenoids Upon the Addition of Concentrated H S0« to Acetone Solutions  53  Elution Characteristics of Pigments isolated from Fusant #26, P^. rhodozyma and R_;. toruloides on a S i l i c a Gel 60 Column  54  2  Table  VI.  -  V -  L I S T OF FIGURES PAGE Figure  Figure  Figure  1.  2A.  2B.  f\ of  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 5 mg/ml AEC i n MEA (600X m a g n i f i c a t i o n )  F u s a n t #26 grown on Y N B - g a l a c t o s e (600X m a g n f i c a t i o n ) F u s a n t #26 grown  in  plates 36  YNB-glucose broth  (1500X m a g n i f i c a t i o n ) 3.  R^ t o r u l o i d e s  Figure  4A.  JC f r a g i l i s grown on Y N B - g a l a c t o s e (600X m a g n i f i c a t i o n )  Figure  Figure  Figure  Figure  Figure  Figure  Figure  4B.  5A.  5B.  6.  7.  8.  9.  10.  grown  36  Figure  Figure  on MEA  J C f r a g i l i s grown i n (1500X m a g n f i c a t i o n )  39  j \ r h o d o z y m a grown i n (1500X m a g n f i c a t i o n )  39  YNB-glucose broth  250 ml f l a s k C u l t u r e o f F u s a n t #26 on S t a n d a r d M e d i a + g a l a c t o s e TLC o f a l l - t r a n s A s t a x a n t h i n , a n d i s o l a t e d f r o m F u s a n d #26 g r o w n on p l a t e s and l i q u i d media  grown 41 Pigments galactose 42  V i s i b l e Absorption S p e c t r a of Pigments from P . rhodozyma, 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  V i s i b l e Absorption Spectra of pigments from the 2 f r a c t i o n s grown i n l i q u i d m e d i a  8 - c a r o t e n e and o f F u s a n t #26 46  V i s i b l e Absorption  Pigments  Spectra of  (solid)  isolated  a n d R^. t o r u l o i d e s  Mass S p e c t r u m o f  Figure  12.  Mass S p e c t r u m o f P i g m e n t s r h o d o z y m a grown on MEA a t  14.  38 38  11.  Figure  plates  YNB-glucose broth  Figure  13.  37  J \ r h o d o z y m a g r o w n on MEA p l a t e s (600X m a g n i f i c a t i o n )  f r o m F u s a n t #26  Figure  31  Astaxanthin isolated 22°C  47 48  from P.  Mass S p e c t r u m o f F u s a n t #26 grown t e m p . 150°C  Pigments i s o l a t e d from on G a l a c t o s e P l a t e s - P r o b e  Mass S p e c t r u m o f F u s a n t #26 grown t e m p . 280°C  Pigments i s o l a t e d from on G a l a c t o s e P l a t e s - P r o b e  49  50  51  - vi -  ACKNOWLEDGEMENTS The a u t h o r for  his  wishes to  extend s i n c e r e g r a t i t u d e  c o n c e r n and a s s i s t a n c e i n  the  preparation  to  Dr.  P.M. Townsley  and e x e c u t i o n  of  this  thesis. She  also  wishes  D r . W.D. P o w r i e , D r . and D r . also  B. D i l l  thank  the  members  J . Richards both of  assistance  author  of  the  the  wishes  provided  to  his  research  Department  the Department of M i c r o b i o l o g y .  extended to Dr. A. F i n l a y s o n f o r The  of  of  to  of  committee,  Food S c i e n c e  Much a p p r e c i a t i o n  is  constant a s s i s t a n c e .  acknowledge  by Sherman Y e e , s e n i o r  the  valuable  technician  in  expertise the  and  Department  Food S c i e n c e . Many  Smith  thanks  and C a r o l  are Burton  extended for  to  their  Kathleen  Barber,  encouragement  and  Yvonne J o n e s , support  during  project. A special  t h a n k s t o J o y c e Tom f o r  her a s s i s t a n c e in  typing.  Donna this  - .yii  -  DEDICATION This  thesis  understanding, more t h a n  i s d e d i c a t e d t o my p a r e n t s  and  to  she knows.  my  sister,  Clo  and  for to  their  Cassie  help, who  patience  has  helped  and me  - 1 -  INTRODUCTION Previous potential  experiments  commercial  poultry  and  have  value  pen-reared  demonstrated  as  a  the m u l t i l a y e r e d ,  liberation  the  of  applications of  the  cell  walls  inapplicable et a i .  carotenoids  (Okabue  on a l a r g e  carbon  industry.  cheese  plants  producer  that  to  One  renders  at  in  of  et  al_.  1979,  of  with  regards  et  al.  animal to  rhodozyma  this  the  for  1980).  o r g a n i s m makes to  1980).  diets  has  astaxanthin  wall  or d e s t r u c t i v e  such  waste  commercial Modification  have  proven  carotenoids  to  be  (Okabue  is  whey,  oxygen  treatment  a  utilize  (BOD)  impractical  a  byproduct  demand  and i n t e r g e n e r i c  prominent.  fermentati utilized  a  most  carbon  i s able to  of  varieof  the  whey  from  (Al-shabibi  et  al_.  1985).  become  in  that  source  biological  interspecies  and  objective  respect  was  number cases  In  utilization,  study  performed  of is  one  carbon to  yeast  to  increased  the a  (Pina  industrial  alcohol  fusions  fusion  obtain  sources  improve  protoplast  of  high et  ethanol  aT.  1986).  strains  production  or  with other  parameters. Since  fragilis whey,  Johnson  inclusion  The h i g h  recently  cerevisiae  such  1984,  i s a genera of yeast  Farahnak et aj.. Attempts  The  cell  impractical  scale,  sources.  cheese  have  to  source  (Johnson  tough  Phaffia  1983).  of  1984;  a_[.  prior  Kluyveromyces ty  et  dietary  salmonids  Unfortunately,  that  is  the  P±  rhodozyma  able main  to  produces  potentially  use a v a r i e t y  aim  of  this  of  study  useful  economical was  to  carotenoids  carbon obtain  sources an  and JC such  organism  as by  - 2 -  intergeneric  protoplast  Protoplast  fusion  extensively  for  was  this  fusion the  purpose  that  method  of  (Pina et  combines choice a]_.  1986;  these since  characteristics. it  has  Farahnak et  been  al_.  used  1986).  - 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,  Selec-  Yu et a/L 1984).  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  a c c u r a t e or p r a c t i c a l (Sink 1984). c o n s t r a i n t s , i n v o l v e d i n formal c e l l  time consuming and  not  always  In order to avoid and to s i m p l i f y 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)  Protoplast  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). protoplasts  r e q u i r e s the d i g e s t i o n of the  cell  The i s o l a t i o n of  wall, releasing  c e l l u l a r cytoplasm encased w i t h i n the plasma membrane.  To maintain  the 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  i n a hypertonic  environment to  provide  In the past, researchers were f o r c e d to p u r i f y t h e i r own  enzymes  osmotic s t a b i l i t y (Peberdy, 1980).  for  cell  wall  non-reproducible.  digestion  which  was  time  consuming,  Today, enzymes are commercially  and  a v a i l a b l e not  often 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  - 4from  various  Neurospora  microorganisms  a  The  require  in  a  the  A s p e r g i 1 1 us  fungi  than  one  demonstrated "soup"  cell  structural  more  in the development of  p r o b l e m when  fungal  many d i f f e r e n t  (1981)  involved  particular  enzymes.  as  sp.  and  sp.  The t e c h n i q u e poses  such  wall  it  comes t o  is  a  components enzyme  that  successfully  digestion  physical  and  their  l e v e l s of  digested  of  from  the  chemical  such as c h i t i n  for  high  protoplasts  fungi  cell  by  complex  of  and B - g l u c a n s  degradation.  Hamlyn  which  et  aj[.  c h i t i n a s e and B - g l u c a n a s e mixed  cell  walls  of  various  species  of  Aspergillus. There  are  also  other  protoplasts  such as c e l l  duration  digestion  al_.  of  (1980)  were  found  obtained  stationary growth  from  of  et  wall  increase of  b)  in and  of  the also  for  the  y e a s t ]C  flask  the  in  cultures  culture  enchanced the  dissolution  of  undamaged p r o t o p l a s t  of  late  log,  parameters  that  yields. such  as  recovered  involved  resulted  numbers p e r u n i t d i g e s t i o n  et  early  hand, found  compounds  has  cells,  protoplasts  protoplasts  organism  of  Morgan  protoplast  of  the  in  other  thiol  percentage  particular  were  improved  of  buffer.  desirable  on t h e  with  optimization a  that  production  pretreatment  fraqilis,  glycine,  isolated  the  protoplast  Peberdy (1980),  The  in  in an  time.  Fusion  Several  methods  organisms,  some  transformation. and  pH o f  al_. 1 9 8 1 ) .  Protoplast  cycles  the  shaking  affecting  conditions,  and  Streptomyces  mercaptoethanol  cell  age, growth  growth phase.  Pre-treatment  (Hamlyn  that  factors  have been d e v e l o p e d of  which The  restrictions  use  include:  to  alter  protoplast  of  fusion  bypasses  commonly  placed  upon  the  genetic  fusion,  the  the  hybridization  normal  pooling  material  of  cellular genetic  - 5 information.  G e n e r a l l y , the  will  compatibility.  dictate  triggering  device for  The b a s i c Protoplasts parts  to  protoplasts standing, After and  are to  Cell  involved  collected  a fusogen,  wall  pre-conjugal  concept  are  cell  wall  enable  left an  an a l l o t e d  time,  transferred  to  the  the  fusion  desired  to  of  (PEG).  the  most  association  PEG i s  f u s o g e n and t h e  an  osmotically  available  f r o m 1500 d a l t o n s well  as  fusion  final  attachment different  PEG.  1980).  Beven  inducing yeast  protoplasts. of  of  solution  Goodey and  the  to  of  the  of  and t h e  solution  cell  can  cells  be  a  to  subjected  to  with  the  are  in  equal  aggregated gently  or  membranes. are  diluted Here  completed.  polyethylene  glycol  molecular  weights  molecular weight  a  dramatic the  of  60%  induced  PEG t r e a t m e n t  on  choice  that  fusion.  and w i t h o u t  as  (w/v).  proving  successful were  effect  weight  experiment  for  fuse  is  simple.  plate.  f r o m 30% t o  cerevisiae  allowed  The  cell  wall  is  have  an  required  quite added  agar  c h o i c e of  out  of  two  different  (1983)  carried  is  fused protoplasts  ranging  These were  to  shaking  the  fusogens  variety  was  thought  and  either  concentrations  agent  receptors  associate.  G e n e r a l l y 4000 d a l t o n s  strains  Only those  a  surface  cells  stablized  used  8000 d a l t o n s  concentration (Peberdy,  used i n  to  in  of  between  the  commonly  is  strains  incubate,  p l a s m o g a m y , k a r y o g a m y and r e g e n e r a t i o n One  contact  c a u s i n g t h e membranes  then  various  activities.  in  from  and i t s  Two  to  the  an  form  presence  fused to  create  "cybrids". PEG, cells  in  itself,  together  membrane.  The  in  does  not  aggregates  fusogen  is  then  induce  plasmogamy.  creating diluted,  an  Rather,  association  allowing  it  of  plasmogamy  draws  the  the  plasma  to  occur.  -  With  this  certain  method,  cells  improved  by  fusion  (Menczel  the  medium.  The  metals  has  found  criticism  been  of  the  (Senda  (Mischel  alternating the  be  is  (DC)  is  up i n  applied  Generally,  0.01  metal  has  of  electric  Pohl  s p i n when Cells  create  reversible  apparatus  on a f l a t m i c r o s c o p e s l i d e  are  attached  most  cases,  c a p a c i t y of 1  and  is 0.01  to  to  50 mA w i t h  low-conducting, 1982). required  supply  to  1000 u F . a  to  their  of  of  two  (Pohl  between  0.25  stablized  adherations  plasmogamy to  membrane  50  to  platinum II).  1982). and  the  polarizable current a n d an  formation  At the  point current  resulting  in  wires  set  These  electrodes  The  1000  an e l e c t r i c runs plus  a r e made, a f u r t h e r  occur.  it  1983).  50 ms  buffer  One  is  direct  breakdown,  the  1981).  dipole  pulse of  to  that  electrodes  I).  be  protoplasts  other  (Appendix  can  divalent  is  coupled  mutual  with  1980).  fusion  p l a c e d between  Once a c t i v a t e d ,  duration  these  2 +  an a l t e r n a t i n g  (see Appendix  circuit  deliver  osmotically  Once p o i n t for  a  able  to  (Bates et al..  consists  of  and  close a s s o c i a t i o n , a short a  and M g  importance  to  Cells  "pearl-chains"  protoplasts  the  Due  2 +  of  gained  subjected are  protoplasts  (Hamlyn e t al_.  current  1982).  obtained  (Peberdy  type  event  recently  mm a p a r t then  induced  be  Ca  M solution  a  applied.  causing  point adherations  cations  be  1982).  current  the  concentration  1983,  aj_.  of  can  effective  pulse  seen to  et  line  the  that  c e l l s associate or  when c e l l s  divalent  to  short  Bates  can  of  random and l o w f r e q u e n c y  a  1982,  particles (AC)  of  15-17%  Aggregation  most  fusogen  application  of  1984).  PEG-divalent  occurs as a very Another  frequencies  addition  fusion  6 -  capacitor,  volts  with  charge  through  in the  between  a drop  protoplasts 30 t o  0.5-1  of  (Senda  60 m i n u t e s  are  - 7  "Electro-fusion" conventional facilitate 1983). aj_.  PEG  has  method.  f u s i o n , and  Reports  1983).  a  -  number  It  of  advantages  requires  no  over  chemical  f u s i o n i s r a p i d and  the  more  pretreatment  synchronous  (Bates  to  et al_.  of between 60-80% f u s a n t s have been r e g i s t e r e d (Bates  Some p r o b l e m s  that  face  this  method  include  et  optimizing  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 metal  electrodes.  The  latter  difficulty  may  be  overcome  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  II.  the  protoplasts.  Carotenoids Carotenoids  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  range from  yellow  to red.  responsible  brilliant  colours  seen  for  the  vegetables,  Crustacea  and  are  plants  sp.,  capable  f i s h and  In  addition  carotenoids  to  or  Carotenoids  algae,  fruits  by  as  absorbing  s i n g l e t oxygen  attractants  Vitamin  mucopolysaccharides.  (Muller  pigments  animals  is  must  or  deterrents,  to Vitamin required  A and for  Many a c t  the  et  sunlight a]_.  1980).  in photosynthesis In a n i m a l s ,  role in physiological function.  A  and  microorganisms  p o t e n t i a l l y harmful  molecules  a l s o f u n c t i o n as a c c e s s o r y  i s converted  are  supply.  functioning  agents  quenching  p l a y an i m p o r t a n t  liver.  many  carotenoids;  thus are o f t e n a s s o c i a t e d with the c h l o r o p l a s t .  B-carotene,  in  Only  the  These pigments  can 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 .  photoprotective  energy  eggs.  of s y n t h e s i z i n g  o b t a i n them f r o m t h e i r f o o d  as  by  One  such  and  carotenoids carotenoid,  subsequently  stored  in  the  normal  biosynthesis  of  - 8  a)  -  General Biosynthesis of Carotenoids Carotenoids  isoprene  are structured basically  nucleus.  Most  of those  from  a  highly  unsaturated  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 process  that utilizes  a number o f d i f f e r e n t c e l l u l a r e n z y m e s  (Goodwin  1979). I n i t i a l l y , three a c e t y l - C o A molecules form mevalonic a c i d (formula 1),  which i s a s i x carbon molecule.  carbon link  molecule t o form  T h i s i s then converted to the f i v e  isopentenyl pyrophosphate the twenty  carbon  (IPP).  geranylgeranyl  Four  IPP m o l e c u l e s  pyrophosphate  (GGPP)  (formula 2 ) .  mevalonic acid formula 1  geranylgeranyl  pyrophosphate  formula 2 Two GGPP m o l e c u l e s  link  tail  to tail  to yield  the f i r s t  carbon p r e c u r s o r c a l l e d phytoene, which i s d e s a t u r a t e d t o form (formula 3 ) .  forty  lycopene  - 9 -  lycopene formula 3 From t h i s depending  upon  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 which  carotenoid i s being  produced,  such  as  taken cyclic  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 c o n t a i n i n g ) . 2  Only those carotenoids c o n t a i n i n g a B-ione ring are c l a s s i f i e d p r o v i t a m i n A. t h a t w o r k on  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 the  ends  of  the  lycopene molecule.  e x a m p l e o f a compound w i t h s u c h e n d s  8-carotene  (formula 4).  as  enzymes i s an  This molecule 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-carotene formula 4 The  carotenoid that  astaxanthin, 4,4'-dione)  is a  i s of  8-carotene  (formula 5).  particular  derivative  interest  (3,3'  the  this  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 c a n be f o u n d i n  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  a s t a x a n t h i n pigments  paper,  dihydroxy-B-B-carotene  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 l o b s t e r and s h r i m p .  to  the  flesh  to  red-orange c o l o u r (Johnson e t &±. 1980).  produce  the  salmon,  characteristics  -  10  o  astaxanthin formula b)  C a r o t e n o i d s and As m e n t i o n e d  their  own of  to  fungus  the  cells in  previously,  different  were two  the  had t h e the  is in  the  fractions  avenues  held the  in  majority  carotenogenesis melvolic  acid,  produces  the  (Goodwin the  that  the  the  When  carotenoid and  a  the  occurred the  suggests  particulate  of  pertains  B-carotene.  This  as  object  f o c u s e d on  supernatant  1980).  membranes  been  One a r e a  fractions,  both  Neurospora crassa the  endoplasmic  carotenoid of  related  there in  is  fungi,  the  for  (Mitzka-Schabel reticulum  levels.  enzymes  carotenoid that  which  have  synthesizing  cell  that  the  as  well  1981),  cell  matter  cytoplasm.  fungus  highest  suggests  research.  found  fraction  containing  containing  of  are capable of  this,  researchers  cell  wall/mitochondria  In  and b e c a u s e o f  fractionated,  carotenoid  microorganisms  Blakeslea trispora,  of  as an o i l  Microorganisms  carotenoids,  number  5  et  ai.  membrane,  and  T h e s e same c e l l  fractions  carotenogenesis. enzymes  not unlike  one  the  enzymes  plants  are  This  site  where,  located  held  fraction  membrane.  subcellular  higher  also  Another  plasma  single  the  carotenoid  was  supernatant  for  following  within  the  chloroplasts. Other carotenoid  research  by  production  Roncero et in  the  ai.  fungus  (1982)  looked  Phycom.yces  at  the  genetics  blakesecanus,  which  of is  - 11 y e l l o w due  to B-carotene.  mating  experiments,  linked  and  These  i t was  t h a t two  genes  located approximately  genes  produce  two  found  through  (Car B and 10  map  a series  Car  RA)  u n i t s from  enzymes, phytoene  were  the  dehydrogenase  of  mutant closely  centromere. and  lycopene  c y c l a s e t h a t are r e s p o n s i b l e f o r the l a s t s i x steps of carotenogenesis. ( S i m i l a r a s s o c i a t i o n s have a l s o been n o t e d i n the y e a s t S c e r e v i s i a e i n the genes 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 in the microorganism  i s the carbon source.  S t u d i e s by C i e g l e r e t a i .  (1978) 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  fermentation media,  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.  with  corn  mycelium  giving  the  weight.  highest yield  Other  s t u d i e s by  of  carotenoid per  Johnson  and  Lewis  trispora  gram  of  (1979)  on  dry P.  rhodozyma 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 such as m a l t o s e , s u c r o s e , and c e l l o b i o s e gave a h i g h e r y i e l d than 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 the  growing  media.  Microorganisms  appear  to  respond  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 . such as ^  to  a  In  drastic organisms  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  1980).  I f t h e growing 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 . Other 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 , and i n the case of  N.  crassa,  the  photoreceptor  (Mitzka-Schnabel e t al_. 1981). has  been  shown  (Johnson and Lewis  to  may  be  8-carotene  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  increase carotenoid production 1979).  itself  in  P_i  rhodozyma  -  c)  Determination Due t o  oxygen, the  the  much  of  Carotenoid  very  sensitive  care  must  carotenoids.  No  completely  satisfactory  variety  material  of  solvents  efficiently  remove t h e  the  spectroscopy, best  means  mentioned Weedon,  structural  banded  for  1965).  ultra-violet Mass  also  1976).  loss  is  miscible  with  resonance  fresh  reliable  absorption effect  B-carotene  but  be  meet  the  water  order  to  tissue. the  identity  visible  mass  and  spectra  Separation with  via  some  of  spectrum  is  quite  shape  and  not  have  a  single  the  thin  layer  the  afore  (Moss  and  important  location the  of  infared  afford  identity  rather  organic  in  of  do  of  potential  the  proof  the  and  considered  Often  that  and  light  purification  establish  conjunction  a  to  1965).  conceded  and can  (Davis  oxo-carotenoids  Cis-trans  for  of  only such water  Variations  temperature  technique  towards  of  typical  the three  symmetrical  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  as  band  shorter  wavelengths.  inherent  rapid  of  extraction  from the  in  will  carotenoids  requirement  used  visible  spectrometry  resolving  it  provide  example,  spectrum  (Davies,  to  variations  spectrum;  of  characterization.  often  The  be  magnetic  techniques  extraction  p r o c e d u r e s are used to  Generally  is  1976).  must  carotenoids  the  standard  carotenoids  chemical  chromatography  in  of  extracted  used  nuclear  of  a  be  analytical  carotenoids.  nature  method  as  -  Pigments  taken  one  to  extracting  Several  be  12  or  can and  be  their  a small  as  considered  masking can  insertion  the  also  a  structures.  amount  artifacts  as  of  molecular  conditions  if  exist.  powerful  tool  for  include  the  Some l i m i t a t i o n s  are  Advantages  material.  c a u s e d by  occur  very  volatile ion  impurities  peak  differences  (Moss of  and  and  the  Weedon,  ionization,  -  III.  Phaffia Phaffia  the  form  America yeast  rhodozyma  tree  capable  ai.  occurs  at is  et  al_.  1976).  fermenting of  this  85% o f  its  the  et  occur  at  This  sugars  yeast under  the  is  is  its  certain  total  in  the  only et  ability  isolated  al_.  in  the  One  other  astaxanthin.  conditions  found  North  producing  1976).  produce  in from  northwestern  carotenoid  to  cultural  carotenoids  Deuteromycotina  originally  (Andrewes  an e l l i p s o d i a l  life  cycle.  same s i t e  production  above  sources  such  and w i l l  conditions,  22°C  This  ferment  appears  may  represent  cell  (Andrewes  by b u d d i n g ,  bud  high  scar.  to  27°C.  requiring  maltose,  D-glucose, maltose  spore formation  upon  found cell  by the  that  P.  Andrewes, identity  the  weight.  total Of  of  has been  generally  a  for  1-3  cell glucan  growth  and  but  growth  No  growth  has  assimilates  cellobiose  and s u c r o s e .  can been  carbon  and  soluble  Under  austere  by b u d d i n g ,  but  noted,  rhodozyma Phaff the  and  pigments  carotenoid  those  a sexual  1979)  yeast  sucrose,  which  of  temperature  0°C  lack  The v e g e t a t i v e  proportion  ( J o h n s o n and L e w i s , from  to  chlamydosphores are produced which germinate  Investigation  wet  a  biotin  Carotenoid Production of  was  that  optimal  ranging  D-glucose,  no p r o m y c e l i u m o r  principally  a thick  The  is  yeast  reproduces  containing  27°C. as  It  leaving  1976).  temperatures  recorded  starch,  is  ai.  astaxanthin  total  the  J a p a n and  (slime  multilayered,  (Miller  It  in  fluxes)  rhodozyma  stage w i t h i n  a)  genus  1976).  F\  wall  yeast  exudates  of  of  red  was  production  an a v e r a g e  a  The y e a s t  aspect  Astaxanthin  is  Blastomycetes.  (Miller  uncommon  et  rhodozyma  order  deciduous  13 -  Starr  produced  mixture  carotenoids,  (1976)  made  by up  astaxanthin  concentrated rhodozyma. 0.003%  of  the  c o m p r i s e d 87%  -  of  the  were:  total.  Other carotenoids  B-carotene,  echinenone,  14  -  present  in minor  phoenicoxanthin  hydroxy-3'4'-dihydro-8-\Ji-caroten-4-one. astaxanthin,  both  i s o m e r s , were In (1979)  an did  that  the  isolated  attempt  the  astaxanthin  to  The  optimum  however,  this  pigment  sources  astaxanthin  to  be an  was  pH  exhibited  important  other for  on  factor  the  to  is  echinenone  echinenone  to  from  other  utilized  the  astaxanthin  A  which  sources,  batch  cis  IV).  and  Lewis  They  found  culture  late  fermentor,  exponential appeared  culture  pHs t e s t e d .  effects  on  the  both  most  (Appendix  content et  al_.  and  growth  to  cell  be  with  O x y g e n was to  4.5  Various  growth  pigment  IV).  permitted  to  p-carotene  specific  eventually  Generally, but  it  by t h e y e a s t a l l o w s  is it  the  ferment  in  cis  a  and other  proven  displayed  (12)  which  pathways  biosynthetic  rhodozyma  A will  biochemical  proposed  enzymatic  thought to  known  (1976)  hydroxyechinenone.  (11).  polar  formation  all  astaxanthin  converted  phoenicoxanthin astaxanthin  of  (14).  more  the  pigment.  Andrewes  production  Neurosporene  during  since cultures  carotenoid  organisms,  mainly  C e l l o b i o s e produced  B - c a r o t e n e as the major Based  a  close behind  to  Johnson  (see Appendix  within  at  3  carotenoid,  respect  production,  yeast  pronounced  following  new  amounts  preparations.  pigment  produced  a  chromatographically  same c e l l  the  and With  grown  for  was  content.  dissaccharides  the  with  produced  period.  the  optimize  yeast  was  and  from  e x t e n s i v e work  when  carbon  trans  but measurable  in  reaction  then  is  the  isomer that  is the  produce both  converted the  results  in  terminal the  only  V).  converts  keto-insertion form  scheme  (Appendix  turn  for  carotenoid one  precursors c i s and t r a n s  and  isolated enzymes  isomers.  - 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 In  trout,  responsible wild,  and charr,  astaxanthin  f o r the typical red coloured  these  shrimp,  salmon  i n Salmonid  fish  shrimp  obtain larvae  the colour  contain the pigment.  i s the major  f l e s h (Foss  through  and plankton  Diets  such  1984).  In the  t h e i r d i e t by P±  as  pigment  ingesting  Prasinophyceae  F i s h c u l t i v a t e d by a q u a c u l t u r e  that  do n o t have 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 b e f e d t o them as  an  ingredient  especially  in their fish  in lieu  safety o f chemicals  This  of increasingly strict  depending  upon  confer the pigment t o f i s h  source  c a n be  quite  regulations  as food a d d i t i v e s (Johnson  Pj_ r h o d o z y m a i s a n a t u r a l salmonids,  meal.  costly,  concerning  the  e t a1_. 1 9 8 0 ) .  of astaxanthin  a n d when f e d t o  carotenoid  l i b e r a t i n g treatments,  i t will  (Johnson,  V i l l a , and Lewis 1980).  As with  o t h e r y e a s t s , j \ rhodozyma a l s o a c t s as a p o t e n t i a l l y e x c e l l e n t of p r o t e i n , f a t s and other n u t r i e n t s (Appendix I I I ) .  source  One d r a w b a c k t o  the commerical use o f t h i s yeast f o r feed, i s that the f i s h i s unable t o d i g e s t t h e c e l l w a l l and thus r e l e a s e t h e pigment (Johnson Okagbue and Lewis 1983). a n d may h a v e a n a d v e r s e nutritional A second  In-vitro cell  l y s i s treatment  Okagbue  astaxanthin  c o u l d be c o s t l y  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 and  value. 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  the r e l a t i v e l y high cost o f fermentation. by  e t al.. 1980,  and Lewis content,  (1983)  that  Many o f t h e s u b s t r a t e s  provided  c a n be v e r y e x p e n s i v e .  adequate  cell  This substrate  the a d d i t i o n a l costs involved i n maintaining  the fermenter  contribute  price  significantly to  the selling  of  tested  y i e l d and cost  plus  a t 22°C would  the fish  food.  - 16 D e p e n d i n g on g r o w t h yg  astaxanthin/g  found  in  is  fraqi 1 is the  chemical and  enzyme  (Vienne  and  cells  a)  whey  von S t o c k a r  exhibit is  occurring  can  concentrations cells  enter  (Mahoney e t aeration yield.  is  able  As w i t h a l l  conditions  100  budding a  are  al_.  yg  wide  1977)  single  as  range  least  and  white  to  60%  around in  cell  forming of  shown  revolves  generally  as w e l l  been  by a t  yeast  and  its  protein or  cream  ascospores.  temperatures  with  When  cultured  to  produce  sharply  In  order  however in  recognised  as a p r o l i f i c  to  (Al-shabibi  ethanol  whey, oil  the to  log  lactase  fortified  producer,  with  c a n be  1984).  fermentation  products.  phase  aeration  the  under d i f f e r e n t  end  of  of  growth Lactase,  growth  and  concentration  i n c r e a s e the  low  and Y o u n i s ,  by  culture  in  during  phase,  1975).  required,  on Whey  differences  stationary  is  22% o i l  this  ethanol  Colonies  has  c h e e s e whey  in  microorganisms,  cause  increase  a_k  to  that  as h i g h as 40°C.  fragil is  cheese whey.  of  produce  over  exceeds  10  that  et  grow  Growth and F e r m e n t a t i o n jC  yeast  (Mahoney  1985).  to  yield  ability  multilateral able  (COD)  Interest  to  far  1980).  utilizing  1984).  producing  carotenoid  normally  al_.  o x y g e n demand  ferment  fraqi 1 is  growth  lactose  Younis  lactase  to  a  of  which  (Johnson et  IC  ability  IC  shell  r h o d o z y m a p r o d u c e s b e t w e e n 30 a n d 800  amount  shells  fragilis  (Al-shabibi  and  of  This  Kluveromyces  reduce  its  yeast.  crustacean  astaxanthin/g  IV.  conditions, J\  as  the  decreases  lactase production,  results salts,  in  a  this  stimulated  to  poor  low cell  yeast, produce  not up  -  The  optimum  ethanol  yield  pH  for  lies  occurring  experiments  carried  out  non-sterilized  whey  component  than  was a f f e c t e d  b)  genetics  and  enhanced  alcohol  relative majority  approaches  by  cereviceae  stage  et  al_.  the  initial  ]C  al_.  (1980)  lactis  to  products that  fusion Farahnak  are  nuclear  between  fusion  et  producing  l a c t o s e and p r o d u c i n g  al..  ethanol  growth,  In  sterilized  and  another this  (1986) that  medium  component  1985).  as  in excess of  to  to  enzyme  1980).  (Johannsen et  fusion  between  hybrids. suggest  support  were  related  protoplasts  al_.  with  In  the  of  this  lactis  capable  the  majority  diploidy  position  was of  1984).  homothallic  exchange does  for  13%  al_.  Biochemically,  that  presence  IC  the  subjected  et  chromosome  when  studies  in  formed w i t h i n a s u s p e n s i o n  studies The  involved  produce  sporulating  and  studies  Morgan  protoplast  Further  fusants  that  when  population  these  efficient 1985).  and t h a t  well  cells,  of  diploid.  products.  as  in  protoplasts  produce  and g e n e t i c a l l y ,  used  1986,  cell  used  most  and von S t o c k a r  concluded  been  These  appropriate  the  and  sp.  yeasts  100% o f  demonstrates  forth  of  substrate  ( V i e n n e and von S t o c k e r  often  numbers o f  fusion  protoplast  have  the  cytologically of  sp.  media  with  difference  was  sterilization  cases,  of  5,  l a c t o s e was g r o w t h l i m i t i n g  (Farahnak  et  the  (Vienne  the  it  production.  the  Morgan strains  4.0  comparing  biochemistry  ease of  a pH o f  of  and  4  F u s i o n i n KIuyveromyas  KIuyverom.yces  at  pH  permeate;  by h e a t  Protoplast  treatment  at  -  consumption  between  consumption  other  the  17  fused  both  (vol/vol).  also  occur  in  was  put  with  S_;_  assimilating  - 18 MATERIALS AND I.  Strains  a)  P h a f f i a r h o d o z y m a (ATCC 2 4 2 0 2 )  b)  This organism  was  at 22°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)  on M a l t E x t r a c t A g a r (MEA)  (Difco)  plates  K l u y v e r o m y c e s f r a q i l i s (ATCC 8 5 6 4 ) This organism  was  (Difco)  22°C  at  (YNB-galactose)  c)  maintained  METHODS  maintained until  on M a l t - Y e a s t - G l u c o s e - P e p t o n e transfer  to  the  (MYGP) A g a r  pre-protoplast  media  (Difco-BDH).  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 1 0 7 8 8 ) This organism  was m a i n t a i n e d  on MEA  p l a t e s at 22°C  II.  C u l t i v a t i o n of Organisms f o r P r o t o p l a s t  a)  j \ rhodozyma:  One  s t r e a k e d o n t o MEA p l a t e s and  Production  week o l d c o l o n i e s w e r e p i c k e d  +5  incubated  mg/mL.  from  S-(2 a m i n o e t h y l ) - L - c y s t e i n e  a t 22°C f o r 3 weeks p r i o r  to use.  MEA  plates  (AEC)  and  (Sigma)  T h i s method  induced filamentous mutants to form.  b)  IC f r a q i l i s :  Cultures  base + g a l a c t o s e p r i o r to  I I I . a)  plates  72 h o u r s o l d w e r e s t r e a k e d (YNB-g).  onto yeast  nitrogen  T h e s e c u l t u r e s w e r e g r o w n 48  use.  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)  consisted of:  1.0 mL A + 6.0 mL B + 6.0 mL C + 0.2 mL D w h e r e :  hours  - 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 M g S 0  4  .7 H 0 ( F i s h e r ) 2  D = 1 M 2-mercaptoethanol  (Swartz/Mann B i o t e c h )  Enzymes; B-glucuronidase from He!ix pomatia Amyloglucosidase from Rhizopus  (Sigma)  (Sigma)  C h i t i n a s e from S t r e p t o m y c e s g r i s e u s (Sigma) Fusoqens: were p r e p a r e d as f o l l o w s : - Polyethylene Glycol  (PEG)  (Sigma)  (mw)  4 0 0 0 was  added  30%  (w/v)  t o 0.01 m C a C l - 2 H 0 ( F i s h e r ) 2  -  2  For electrofusion  0.7  m  Mannitol  (BDH)  was  used  as an  osmotic  stablizer.  Media 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 Pi rhodozyma. - Malt-Yeast-Glucose-Peptone (Difco), peptone  3 g yeast extract  Agar  consisting  (Difco),  10  g  of  3  g  D-glucose  malt  extract  (Difco),  5  ( 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  was u s e d t o m a i n t a i n IC  g 5.5  fragilis.  - 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 . - Osmotically Stabilized g a l a c t o s e + 0.6 m KCI of p r o t o p l a s t s .  Recovery  Media  (0SRM)  consisting  of  YNB  +  ( 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  - 20 - amino a c i d analogue media c o n t a i n e d mg/ml  of either  tryptophon  g; M g S 0  guanidine,  extract  5-fluoro  D,L  (Difco) + 5  tryptophan,  hydroxamate L - l y s i n e hydroxyamate, s-hydroxylysine  s-(2 aminoethyl) - Standard  sulfa  2% m a l t  L - c y s t e i n e + 2% B a c t o a g a r  L  HC1 o r  (Difco).  Medium c o n s i s t i n g o f ( p e r l i t r e ) ( N H ) S 0 , 2 g ; K H P 0 , 1 4  7 H 0 , 0.5 g ; C a C l  4  2  source;  15 g a g a r  4  extract  ( D i f c o ) was a d d e d  required.  tests:  - YCB ( D i f c o ) + 5 mm K N 0 - Durham f e r m e n t a t i o n  2  2  Medium i f p l a t e s  b) M e d i a f o r b i o c h e m i c a l  4  2 H 0 , 0.1 g ; ( F i s h e r ) y e a s t  2  ( D i f c o ) , 2 g ; 20 g o f a carbon to t h e Standard  2  - ( B a r n e t t e t a l . 1983)  3  tubes + glucose; r a f f i n o s e ; maltose  or galactose  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 . - YNB ( D i f c o ) without  containing  niacin + galactose  niacin + galactose  requirement  for niacin.  - YNB ( D i f c o ) + g l u c o s e  (Difco)  ( B a r n e t t e t aj_. 1983)  ( D i f c o ) b r o t h was used f o r f e r m e n t a t i o n  tests  a t 22°C and a l i q u o t s o f t h i s media were a l s o p l a c e d on a spot  plate  Protoplast Release, The  protoplast  l i b e r a t i o n procedure  m e d i a a n d e a c h was i n d u c e d cells  containing  were  ( B a r n e t t e t aj_. 1 9 8 3 )  Fusion and Regeneration  d e s c r i b e d by Morgan e t al_. (1980).  Phaffia  (BDH) a n d YNB  (BDH) w e 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  + iodine to test for extracellur starch.  V.  ( B a r n e t t e t al_. 1983)  o f that  T h e c e l l s were grown i n t h e i r r e s p e c t i v e  t o form  dispersed  i s a slight modification  protoplasts separately.  in a filter  5 mg/mL B - g l u c u r o n i d a s e ,  c h i t i n a s e , f o r breakdown and removal.  sterilized  protoplast  2 mg/mL a m y l o g l u c o s i d a s e This mixture  The pretreated  was t h e n  buffer,  a n d 1 mg/mL incubated a t  - 21 30°C, with  gentle  shaking  f o r 4 hours.  The  IC  f r a g i l i s was  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 incubation for cell wall After three  their  f o r 1 1/2  in a  hours of  removal.  respective  times with  treated  washed  21°C.  Those 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 were washed a f u r t h e r three  times  Protoplasts suspensions  each wash, the  were  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  w i t h s t e r i l e 0.7 M  After  free protoplasts  were  f o r 5 min.  buffer.  the  protoplasts  centrifuged  sterile  incubations,  mannitol.  from the  two  parental  c o n t a i n i n g between 10  t o be f u s e d by PEG  s t r a i n s were mixed to g i v e  and 1 0  6  were c e n t r i f u g e d  protoplasts/mL.  6  Those  f o r 5 m i n u t e s a t 500  protoplasts  incubated  were  f o r 30 m i n .  resuspended at 30°C.  in  For  the  30%  was  (w/v)  regeneration,  PEG  The  solution  and  suspension  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 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 mannitol  were mixed i n equal  10  10  s  and  s  parts  protoplasts/mL.  This  s t e r i l e c o v e r s l i p t h a t was itself consisted  o f two  a p a r t mounted i n a polycarbonate  to form a suspension m i x t u r e was  platinum  that  Corp.). 1500  The  connected  c a p a c i t o r was  to  wires  and  1000  et al.. 1983).  kHz  added  0.13  mm  the  fusion  apparatus  applied  to  fusion  diameter set  (Advanced  charged to the d e s i r e d v o l t a g e  was  dropwise The  causing  the  M  between a  cell 1  mm  to the  These e l e c t r o d e s were attached  v t o 2700 v d e p e n d i n g upon the e x p e r i m e n t ) .  b e t w e e n 40 (Bates  were  then  containing  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  stage of the microscope ( w i l d , i n v e r t e d ) . wires  at 22°C.  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  mounted under the f u s i o n c e l l .  parallel  Fisher  removed.  PEG  the  mL  suspensions  x g in the  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 and t h e s u p e r n a t a n t mixed  1.0  to  Engineering  (which ranged from  An a l t e r n a t i n g c u r r e n t protoplasts  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  to  of  aggregate  activated  and  - 22 after  30 ms  sample.  the desired  voltage  o f A.C. p u l s 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 .  was a p p l i e d  through  the  For regeneration, the proto-  p l a s t s w e r e d i l u t e d o n t o a n OSRM p l a t e w i t h 0.7 M m a n n i t o l  and incubated a t  21°C.  VI.  S e l e c t i o n o f Fusants Desired  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 g r o w on Y N B + g a l a c t o s e m e d i a . produced  A f t e r 7 days,  colonies that  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 . grew  a t 30°C  and remained  orange  galactose p l a t e s and l i q u i d media.  were  picked  Colonies that  and maintained  o n YNB  +  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 , and quick growth t o use f o r f u r t h e r t e s t i n g . Presumed f u s a n t s were m a i n t a i n e d 30°C.  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  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 m e d i u m w i t h i n 250 mL E r l e n m e y e r 30°C shaking  a t 2 0 0 rpm.  Presumed f u s a n t s were a l s o viewed  with a Wild i n v e r t e d phase c o n t r a s t  a)  Biochemical Fusant  #26.  3  microscope.  #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  These  subjected  to a  tests i n order to sort out the relationship of  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  r a f f i n o s e , galactose, maltose N0  microscopically  tests  s e l e c t e d number o f b i o c h e m i c a l fusant  f l a s k s , were incubated a t  tubes  containing:  and glucose; urea broth f o r urease  t e s t ; YCB +  f o r u s i n g N 0 „ 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 ;  fermenta-  t i o n t e s t s were a l s o c a r r i e d o u t i n YNB-glucose, e x t r a c e l l u l a r s t a r c h 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 .  All  produc-  procedures  f o r these t e s t s were c a r r i e d o u t a s d e s c r i b e d by B a r n e t t e t al_. (1983).  - 23 VII.  Carotenoid Analyses C e l l s g r o w n 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  -60°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 . into a cylindrical at  s h a p e w i t h i n a 5 mL p l a s t i c  l e a s t 24 h o u r s .  The c e l l  p a s t e was  formed  s y r i n g e b a r r e l and f r o z e n f o r  The f r o z e n c e l l s were 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  diam)  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 lbs/in  was u s e d .  2  cold acetone  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  (BDH).  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 evaporator.  The  dried  pigment  was  then  resuspended  rotary  in various solvents  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 column  chromatography).  A l l w o r k 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 . cases, the r e s p e c t i v e pigments s o l i d media, solid  rhodozyma  media,  B-carotene  f r o m f u s a n t #26  In a l l  c u l t u r e d on b o t h l i q u i d  and  c u l t u r e d on s o l i d m e d i a , R^. t o r u l o i d e s c u l t u r e d on (Sigma)  and  astaxanthin  (Roche)  were  used  as  and  R^  standards.  a)  Thin Layer The  toruloides  pigment and  Chromatography samples  e x t r a c t e d f r o m f u s a n t #26,  the pigments  B-carotene  (Sigma)  (Roche) were r e s u s p e n d e d i n a c e t o n e (BDH). K e i s e l g e l 60 g l a s s p l a t e s . taining acetonerhexane  and  J\  rhodozyma,  all-trans  astaxanthin  A l i q u o t s were then s p o t t e d onto  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 -  (30:70).  a u t h e n t i c a s t a x a n t h i n (data not  Samples shown).  were a l s o co-chromatographed  with  - 24 b)  Column  Chromatography  The pigment  samples  m e d i a ) , F\ r h o d o z y m a , in hexane.  column  with  30  in liquid & solid  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  (60-200 mesh) (Baker A n a l y z e d ) . ml  of  100%  hexane.  acetone:hexane  combinations were added  acetone:hexane  and  vacuum  (grown  R^. 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  These pigments  cm x 24 cm) washed  e x t r a c t e d f r o m f u s a n t #26  finishing  containing coloured  with bands  The p i g m e n t s  Increasing  t o t h e column  100%  acetone.  were  collected  were  concentrations  s t a r t i n g a t 1:24 Eluded and  then  (v:v)  fractions  dried  in a  of  under rotary  evaporator.  c)  Absorption Specta The pigments  total  spectrum  e l u t e d from the column were resuspended  i n a c e t o n e and the  was  210  spectrophotometer.  scanned The  maximum  on  a  Varian  absorption  sample as w e l l as the shape o f the v i s i b l e  d)  w a s  recorded f o r  beam each  spectrum.  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 under vacuum i n a r o t a r y e v a p o r a t o r . a c e t o n e and were s u b j e c t e d t o TLC samples  The s a m p l e s w e r e r e s u s p e n d e d  in  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 .  were then d r i e d w i t h n i t r o g e n gas  a n a l y s e s were conducted.  e)  (Amax)  double  Mass S p e c t r o m e t r y Pigment  The  Cary  and  t h e mass s p e c t r o s c o p i c  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 .  Colour reactions Pigment  samples  and pigment  standards were d i s s o l v e d i n acetone.  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 S 0 2  immediate  4  (BDH)  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 ,  (36 M) was a d d e d a n d 1950).  To 2 the  - 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  yeast,  produces  a s t a x a n t h i n as i t s major pigment under most growth c o n d i t i o n s with the best carotenoid  production  o c c u r r i n g under a h i g h l y oxygenated  (Johnson & Lewis 1979). temperature  The  medium  As reported by M i l l e r et aj_. (1976), the optimum  f o r growth was  above 27°C.  liquid  22°C with no growth o c c u r r i n g a t  c e l l s grown a t temperatures  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 c o l o u r r a t h e r than the usual robust orange-red c o l o u r of the 22°C c o l o n i e s . temperatures  were  smaller  than  C e l l s grown at g r e a t e r than optimum  normal,  though  they  did  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 s e m i - s y n t h e t i c media (Johnson & Lewis,  1979), with the carbon  source being v a r i e d .  At 22°C, growth  obtained on maltose, c e l l o b i o s e , dextrose, glucose and sucrose.  was  There was no  growth observed on the p l a t e s t h a t contained g a l a c t o s e or l a c t o s e as the s o l e 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 i n 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 i n t a b l e I. o b s e r v a t i o n , the c e l l s demonstrated l a r g e c i r c u l a r chlamydospores  p o l a r budding.  were seen.  During m i c r o s c o p i c  In o l d e r c u l t u r e s , some  Table 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. r h o d o x y m a , 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  Test Yeast peptone + glucose  P^ r h o d o z y m a + (no  gas)  IC f r a g i 1 i s  R.  toruloides  #26.  Fusant  #26  +  +  +  Yeast peptone + galactose  +  +  +  Yeast peptone + maltose  +  +  +  Yeast peptone + r a f f i n o s e N0  +  3  YCB +  N0  +  3  Urease Growth w i t h o u t starch  Niacin  formation  YNB + g l u c o s e  fermentation*  * c a r r i e d out a t 22°C  +  +  +  +  + +  (gas)  +  (gas)  ro cn  +/-  (slowly)  - 27  b)  -  Kluyveromyces f r a g i l i s JC  f r a g i l i s i s a yeast  carotenoids.  capable  of fermenting  C u l t u r e s w e r e p l a t e d on YNB  grown a t 30°C.  The  whey a n d d o e s n o t  + galactose  and  YNB  produce  + lactose  and  r e s u l t i n g c o l o n i e s ranged i n c o l o u r from w h i t e to cream  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 between media except  that  t h o s e g r o w n on l a c t o s e c o n t a i n i n g m e d i u m w e r e l e s s s o l i d when r e m o v e d w i t h  a  loop. When v i e w e d m i c r o s c o p i c a l l y , homogenously circular.  elongate  The  cell  whereas walls  the  the  c e l l s g r o w n on cells  i n both cases  grown  l a c t o s e tended to  on  galactose  were  be  more  were t h i n as compared to those  of  Phaffia. Some b i o c h e m i c a l  t e s t s were a l s o  done i n o r d e r  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 was  indeed the  II. a)  enzyme " s o u p s " , t h e r e  This  Strains  (1980), using  w e r e no  "football"  shape and  when p l a c e d  t o d i g e s t i o n by e n z y m e s a s s i n g l e enzyme s o l u t i o n s  formed.  negative  When t h e s e c e l l s w e r e s u b j e c t e d  no s p i n n i n g was  both  protoplasts  d i g e s t i o n d i d not change the previous  ( V a r i a t i o n s in time  outcome.)  in a  hypotonic  The  the  retained  solution  remained  to a d i p o l e producing  electric  field  from c e l l s grown under d i f f e r e n t con-  i . e . l i q u i d media, s o l i d media, young  cell  of  cells  (16  hr.  old)  c u l t u r e s and a l t e r n a t i v e c a r b o n s o u r c e s , were not s u c c e s s f u l . of  and  observed.  Attempts to produce protoplasts ditions  i n the l i t e r a t u r e (Table I ) .  rhodozyma c e l l w a l l s were s u b j e c t e d  o u t l i n e d by M o r g a n e t a_K  intact.  this  rhodozyma  When P.  their  ensure that  case.  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 P.  to  wall  to  digestion  is  consistent  with  cultures, The  previous  old  resistance literature  - 28 concerning  the d i f f i c u l t y i n e x t r a c t i o n of carotenoids  (Okagbue & Lewis  1983,  Okagbue & Lewis 1984).  b)  JC  fragilis  The 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 . has 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, 1984). the  F a r a h n a k e t a l _ . 1985,  Johannsen et al_.  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 m e d i u m 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  enzyme B - g l u c u r o n i d a s e  Morgan e t al.. (1980). approximately protoplasts.  1.5  protocol  established  by  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  accomplished  in  converted  to  hours  T h i s was  following  with  more  the  general  than  90%  of  d e m o n s t r a t e d by t h e o s m o t i c  the  cells  s e n s i t i v i t y of the  cells,  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 , and 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 Under m i c r o s c o p i c condition.  observation  alternating current.  the p r o t o p l a s t s appeared  They were t u r g i d , c i r c u l a r and  regenerated  t o be  in  good  their cell walls  in  r e c o v e r y medium.  III. a)  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 Pretreatment  of the  cells  C e l l s g r o w i n g on MEA "Chlamydophores"  (large  m e d i u m c o n t a i n i n g 5 mg/mL AEC circular  cells).  "Chlamydophores" s t a r t e d to "germinate" " p s e u d o m y c e l i u m " and p l e o m o r p h i c  After  producing  c e l l s ( F i g . 1).  t h i n n e r ( a n d i n some c a s e s m o r e i n c o m p l e t e ) time these c e l l s were c o n s i d e r e d than the untreated parent P h a f f i a .  s t a r t e d to produce three  weeks  these  w h a t c a n be d e s c r i b e d  as  T h e s e c e l l s seemed t o have  c e l l w a l l s than P h a f f i a .  as b e t t e r s u i t e d f o r p r o d u c i n g  At t h i s  protoplasts  - 29 -  Table  II.  Effect of various amino acid analogues on the growth and c e l l structure of Phaffia rhodozyma*.  Analogue  Growth  Cell  characteristics  sulfa guanidine  +++  ovoid and i r r e g u l a r c e l l s , no filaments, thin wal1  5-fluoro D,L tryptophan  ++  c e l l debris present no filamentous c e l l s  L tryptophan hydroxyamate  ++  c e l l s debris present a few filamentous c e l l s  L-lysine  ++++ (white coloured)  c e l l s granular, thin w a l l , no filamentous c e l l s  S-hydroxylysine HC1  ++++  round and ovoid c e l l s , granula, thin wal1  S-(2 aminoethyl)  +++  many filamentous segmented c e l l s , thin wall  hydroxyamate  L-cysteine  * 2 days growth on 2% Malt Extract Bactoagar at 22°C  (Difco)  + 5 mg/ml  analogue  + 2%  - 30 Other  analogues  s-hydroxylysine  Further  as sulfaguanadine,  HC1 a n d o t h e r s  produce t h i n walls AEC.  such  (Table  work  II).  using  were a l s o  5-fluoro  evaluated  None o f t h o s e  AEC i n d i c a t e s  These t h i n walled  mannitol  i n t h e medium  (P.M.  b)  that  liquid  medium  i n producing  cultures in thin  walled  the cellular  i n t e g r i t y during  growth  communication, 1988).  Protoplast formation I t was d e c i d e d  f o r their ability to  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 t o maintain  Townsley, personal  tryptophan,  t e s t e d were a s e f f e c t i v e a s  a d d i t i o n t o s o l i d medium c u l t u r e s were s u c c e s s f u l cells.  D, L  by AEC t r e a t e d P h a f f i a  that  based  on m i c r o s c o p i c  observations  that  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 g r o w n o n 5 mg/mL A E C - MEA p l a t e s f o r 3 weeks.  A n enzyme m i x t u r e c o n t a i n i n g  2 mg/mL a m y l o g l u c o s i d a s e  1 mg/mL c h i t i n 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  found t o be t h e most 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 After approximately  incubation  of the Phaffia  cells  50% o f t h e c e l l s were converted  a t 30°C  morphology,  ( S i g m a ) was  walls. f o r 3.5-4  hours,  t o protoplasts as evidenced  by  to alternating current.  Due  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 to t h e large v a r i a t i o n i n c e l l  (Sigma),  i t was d i f f i c u l t  t o determine  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 the  "pseudomycelium".  When p l a t e d  o n 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 m o r e 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 . Subsequent i n v e s t i g a t i o n s i n d i c a t e that p r o t o p l a s t c a n a l s o be from c e l l s obtained  from l i q u i d medium c u l t u r e s .  prepared  - 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 IV. a)  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 F u s i o n by E l e c t r o f u s i o n Those c e l l s  rotating  t o be f u s e d  alternating current  Corp).  During exposure  chains  (single lines  by e l e c t r o f u s i o n , were f i r s t inside the fusion cell  subjected  (Advanced  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 tended of associated  cells  which  often  to a  Engineering t o form pearl  bridge  between the  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 §_[. 1 9 8 3 . O n c e t h e p e a r l c h a i n s h a d 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 us A.C. p u l s e r a n g i n g  from  1500-2700 v o l t s .  2 7 0 0 v many o f t h e c e l l w a l l s r u p t u r e d . or three  times  with  At voltages  exceeding  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  a p a r t i c u l a r voltage without  showing adverse  two  effects.  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) a n d 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 ing c u r r e n t so as not t o d i s r u p t the c e l l s . was  not v i s u a l l y observed  mannitol ed.  Actual coalescence of the cells  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  s o l u t i o n tends t o form d i s r u p t i n g c r y s t a l s w i t h i n the time  from  the  requir-  H o w e v e r 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  point adherations.  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  mannitol  t o w a s h 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 by 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  c e n t r i f u g e tube.  When o b s e r v e d  PEG w e r e i n c u b a t e d  under a microscope,  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 associated  p r o t o p l a s t s were d i l u t e d w i t h  p l a t e d on OSRM.  with  PEG i n a  t h e p r o t o p l a s t s were seen  on s h o r t t i m e o b s e r v a t i o n . 0.5 ml  The  o f p r o t o p l a s t b u f f e r and  - 33 V.  S e l e c t i o n and i s o l a t i o n o f fused Since  cells  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  containing  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 the growth o f P h a f f i a . colonies,  the production  containing galactose allowed  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  to recover  o f orange  should  colour  represent  undisturbed  inhibiting  t o cream  i n colonies  coloured  growing  the desired fusogen.  on  OSRM  The c e l l s  were  f o r 7 days and then monitored  f o r coloured  colonies. 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 cases overgrew t h e p l a t e . p l a t e no m o r e t h a n  To avoid f u r t h e r overgrowth, attempts  2.5 x 1 0  cfu/plate.  2  After approximately  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 . longer u n t i l a deeper orange colour appeared.  w e r e made t o 10 d a y s  small  These were l e f t a few days  C o l o n i e s t h a t ranged  i n colour  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 placed  i n t h e 30°C i n c u b a t o r .  t r e a t e d i n t h i s manner. low f r e q u e n c y As recorded shiny  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 h e l o w 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  o f successful fusions, approximately  the fusant (Table  with  Approximately  colonies  grew,  a  number  1/10 . 6  o f common  morphologies  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 ,  smooth  edges.  The f i n a l  selected  colonies  c o l o u r e d c o l o n i e s t h a t had undergone e i t h e r o f t h e f u s i o n Those presumed fusants  a  were  were  convex,  taken  from  techniques.  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  produce a p p r e c i a b l y orange pigments, were c u l l e d .  The c e l l  l i n e s t h a t were  e s t a b l i s h e d i n c l u d e d #3, 1 0 , 2 2 , 2 3 , 2 6 , 35 a n d 4 0 . C u l t u r e #26 was s e l e c t e d as  t h e most d e s i r a b l e f o r f u r t h e r study  possessed  t h e most pigment i n t e n s i t y .  s i n c e t h e c e l l s grew r a p i d l y and  - 34 -  Table I I I . Colony morphology + KCL m e d i a .  Colony type  Colony  o f some p r e s u m e d  morphology  f u s a n t s on YNB - G a l a c t o s e  Representative fusants  1  r e d d i s h b r o w n , l a r g e ( 3 mm d i a m . ) , convex, s h i n y w i t h smooth edges f a s t growing  #1  2  p i n k / o r a n g e , s m a l l (1 mm d i a m . ) , convex, smooth edges, slow growing, shiny  #3-9, 2 0 , 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 , convex, smooth edges  #11, 12  4  o r a n g e , l a r g e ( 4 mm d i a m . ) , convex, jagged edges  #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 , convex, smooth edges  #23, 2 6 , 3 0 , 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 were round or s l i g h t l y oblong demonstrating m u l t i l a t e r a l budding ( F i g . 2 ) . 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 "  There  o b s e r v e d i n the f u s a n t as  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 in colour intensity of fusants.  D u r i n g t r a n s f e r s , the 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 frequent.  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  rate optimal  temperature  or carbon source u t i l i z a t i o n  less growth  demonstrated  by  the  fusant. Fusant  cell  lines  in liquid  g a l a c t o s e c o n t a i n i n g media  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 ) .  grew  rapidly  C e l l s viewed using a microscope,  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 . Biochemical  t e s t s w e r e a l s o d o n e on #26  to determine which  parent i t  most resembled ( T a b l e I ) .  VI.  I d e n t i f i c a t i o n o f Pigment Thin  mass  spectrum  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 .  Pigments  i n #26 P.  layer  Produced  chromatography,  spectrophotometry,  and  c e l l s grown i n l i q u i d and s o l i d media were a n a l y z e d .  rhodozyma,  Rhodosporidium  toruloides,  and  authentic  Pigments  from  crystalline  a s t a x a n t h i n (Roche) were a l s o examined f o r a comparison.  a)  Thin Layer TLC  solvent  was system  Chromatography used  as  a technique f o r identification  u s e d was  70:30 h e x a n e : a c e t o n e  A n d r e w e s & S t a r r , 1976; A n d r e w e s ,  and  isolation.  a c c o r d i n g to the methods  S t a r r , & P h a f f , 1976.  The of  - 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 )  -  F i g . 5A  F i g . 5B  39  -  P. rhodozyma grown on MEA p l a t e s a t 22°C (600X m a g n i f i c a t i o n )  F\ rhodozyma grown on YNB-glucose b r o t h a t 22°C (1500X m a g n i f i c a t i o n )  - 40 -  T a b l e IV.  Pigments isolated from various r e s p e c t i v e TLC and a b s o r p t i o n d a t a .  Pigment Astaxanthin  Rf  (Roche)  cells  (Amax) V i s i b l e A b s o r p t i o n  .24  470  .24  471  .30  470  .67  473  .72  447  .25  487  fraction)  .64  483  P h a f f i a rhodox.yma ( p l a t e )  .24  470  F u s a n t #26  (plate)  F u s a n t #26 ( l i q u i d ) A (acetone f r a c t i o n ) B (hexane f r a c t i o n ) 8-carotene  (Sigma)  R. t o r u l o i d e s ( p l a t e ) A (hexane:acetone) B (acetone  and  their  (nm)  - 41 -  Fig. 6  250 ml flask culture of Fusant #26 grown on Standard Media + galactose at 30°C 72 hours o l d . (A - blank; B - Fusant #26)  42  -  F  i  g  .7  T  L  f a  C o r  n  f  a  o dP  m e d i a  l  m i  l  F g  u  m  ( L ) .  t s  e  r  a  n  a n  t  n s  f  s  a  t  # r  s  t 2  o  a 6  m  F  x g  a  n  r u  t  h  o s  a  i  w n  n n  t  ( o  #  A  )  ,  n s 2  6g  P  o r  i  l o  g i  w  i  m  e  d  m  n  i  n e n  t  s d  i  a  (  G  )  -  a)  carotenoid  extracted  into  astaxanthin  (Roche)  solvents,  Rf  of  values  astaxanthin  0.24  to  taken  from  spotted  on  j \  all  three  (Fig.  6).  orange  in  fusants silica  rhodozyma pigments  grown gel  plates  extracts.  When  from  the  three  Co-chromatography of  a single  band  on  (data  not  grown  on  solid  media  and  compared  run  in  sources  the  the  had  were to  afore  identical  fusant  pigments  with  The  pigments  also  shown).  colour.  C u l t u r e s from L i q u i d Media The  silica  pigments  gel  (plates) being  taken  plates  and  extracts.  red in  0.67.  Neither  Silica  of  acetone:hexane  medium  to  astaxanthin  separated  .30 and the  media were  into  and  several  spotted  rhodozyma  j\  bands,  second being  on  the  first  o r a n g e w i t h an R f  co-chromatograph with  astaxanthin,  pigments.  the  ratios  from c u l t u r e s  pigment of the  pigments  were  pigment  were  100% a c e t o n e .  P h a f f i a and the  with  the  the  added  started  to  silica  hexane to  the  move  from  fusant  the  #26  grown  fraction  second f r a c t i o n  (A)  fusant gel  wash. hexane  as  t h e b a n d was a c h i e v e d w i t h  chromatographed,  acetone:hexane while  of  placed onto  bands  acetone  Recovery of  When  compared  these pigments would  s o l i d media were of  column.  above  liquid  Gel Column Chromatography  containing  volume  as  cultures  These pigments  When p i g m e n t s  movement  from  c o l o u r w i t h an Rf o f  o r Pi rhodozyma  using  and  and  resulted  had a s i m i l a r  2.  pigments  acetone  mentioned  b)  -  1. C u l t u r e s f r o m S o l i d M e d i a The  of  43  a  #26  column As  100% a c e t o n e liquid  (B)  was  there  the  band  eluted  no  with  1:24  through  the  (Table  galactose  was  increasing,  starting  single  in  from g a l a c t o s e  VI). containing  using  c o u l d o n l y be c o m p l e t e l y  1:12 removed  - 44 c)  Spectrophotometry  1. C u l t u r e s g r o w n on S o l i d Pigments silica  g e l column  range. ^max  These  a t  4 7 1  A  (Roche)  «  (A  toruloides  were d i s s o l v e d  g r o w n on s o l i d m e d i a  i n a c e t o n e and  single  T n i s  broad  s t a n d a r d and pigments  a t 470  m a x  f r o m f u s a n t #26  scanned  p u r i f i e d on a  over the  visible  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 peak  n m  astaxanthin had  isolated  Media  nm  (Fig. 8).  447  m a x  nm)  ( F i g . 10)  A  corresponds with  that of  the  i s o l a t e d from P h a f f i a s o l i d c u l t u r e t h a t This differs  ( F i g . 9) m a x  peak  with  that  has  from the 8-carotene standard 3  bands  and  Rhodosporidium  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 g r o w n 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 from t h e s i l i c a g e l column 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 hexane and t h e second w i t h a c e t o n e ( A ) . A  having A  pigment  m a x  a t 470  B having A  B-carotene.  m a x  nm  These pigments have a s p e c t r a w i t h pigment  but d i s p l a y i n g  a t 473  nm  (B)  a  l a r g e s h o u l d e r a t 535  nm  and  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  These pigments d i f f e r from those i s o l a t e d from  of  Rhodosporidium  t o r u l o i d e s ( F i g . 10).  d)  Mass S p e c t r o m e t r y Carotenoid pigments  Pi rhodozyma  f r o m f u s a n t #26  cultured  in liquid & solid  media,  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) chromatography,  column.  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 b y t h i n  i t produced a s i n g l e band.  shown i n F i g s . 1 1 - 1 4 .  The M a s s S p e c t r a o b s e r v e d  layer are  - 45 -  Fig. 8  V i s i b l e Absorption Spectra of pigments from rhodozyma, a l l - t r a n s astaxanthin and Fusant #26 grown on sol id media.  o-astaxanthin ;»-P. rhodozyma ; * - f u s a n t 2 6  - 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 and pigments 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 g r o w n o n l i q u i d media. • -ft-carotene ; • -fraction A; A-fraction B  -  47 -  0.6-f  wavelength  (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 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  from  48 -  0  IS  I I I I I I I  Fig.  11  Mass S p e c t r u m o f  I  I  I  I I I I I I I I I  Astaxanthin.  - 49 -  0 0  iiss IS  I II I I I ! I I I  II  I I II  I I I I  >(ead a s e q j.o  rr  T T  I II II I II II  %  F i g . 12 Mass Spectrum of pigments isolated from P. grown on MEA at 22°C.  rhodozyma  50  ii  SI  oo  II  i i i i i i  T T  Tl  t t•s» s» <s *ao co  (TTT  *s es <s  F i g . 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 II I I  >|P9d a s e q j o %  Fig.  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 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 - p r o b e t e m p e r a t u r e 280°C -  - 52 One  obvious  conclusion  from  mass s p e c t r o m e t r y  s p e c t r a obtained from the c e l l e x t r a c t s corresponds Astaxanthin  (Roche)  ( F i g . 11).  The  all-trans  i s t h a t none o f  to t h a t of the astaxanthin  does  the  all-trans give  a  c h a r a c t e r i s t i c m a s s 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 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-207 a r e m i s s i n g a n d two  a n d M-233 do n o t h a v e e x p e c t e d  intensities.  The 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 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 high molecular weight  m a t e r i a l , and  same ( E i g e n d o r f f , p e r s o n a l  e)  with  of  respect to  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  grown their the  communication).  Colour reactions A s t a x a n t h i n and pigments  from both l i q u i d and 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 addition of concentrated sulphuric acid. immediately  colourless (Table V).  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  - 53  T a b l e V.  -  C o l o u r change 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 concentrated sulphuric a c i d to acetone s o l u t i o n s .  Colour of soln. before H S0  Colour of soln. a f t e r H S 0 added  astaxanthin  dark  dark  B-carotene  yellow  light blue  f u s a n t #26 p l a t e  orange  medium-dark blue  P. r h o d o z y m a  orange  medium-dark blue  Rhodosporidium sp. (acetone:hexane f r a c t i o n )  pink/orange  colourless  (acetone  pink  colourless  Carotenoid  2  fraction)  4  orange/red  2  4  blue  - 54 -  Table VI.  E l u t i o n c h a r a c t e r i s t i c s of pigments r h o d o z y m a F u s a n t #26, a n d R^. t o r u l o i d e s column.  Carotenoid  Elution  astaxanthin  acetone  B-carotene  hexane  f u s a n t #26  (plate)  solvent  acetone  P. r h o d o z y m a  acetone  F u s a n t #26 ( l i q u i d ) fraction B fraction A  hexane:acetone acetone  (12:1)  hexane:acetone hexaneracetone  (14:1) (1:9)  R.  toruloides fraction A fraction B  i s o l a t e d from f\ on a s i l i c a g e l 60  - 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 characteristics favouring containing  medium,  the production  while  also  e x t r a c t i o n of the pigments.  of carotenoids  possessing  a  thin  from  wall  having  galactose  to  promote  T o 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 to produce astaxanthin,  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  g r o w on g a l a c t o s e c o n t a i n i n g m e d i u m . Enzymatic normal  removal  conditions  wall  ( 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  there  success  are multiple  i t r e s i s t a n t t o most l y t i c enzymes. forced to occur  little  as  under  i n the cell  that  very  o f j \ rhodozyma grown  The  suggests  with  wall  expected.  literature  met  of the cell  layers  When m o r p h o l o g i c a l  due t o t h e e f f e c t o f t h e AEC, a r e a s  d i f f e r e n c e s were of the cell  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 l y t i c enzymes. and  Often  i t appeared that  were a t t a c k e d . resulting  these susceptible c e l l s resembled the "germination  When t h e s e  tube" of these  structures  ( F i g . 1) a l s o showed a  t h a t t h e c e l l s were s e v e r e l y a f f e c t e d by t h e AEC.  other  cell  t h e c e l l s g r o w i n AEC b u t , u p o n r e t u r n t o MEA  the  rhodozyma r e t u r n s .  amino a c i d analogues t e s t e d  such d e s i r a b l e c e l l s .  in regulation  (Table  II) d i d not produce  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 change  suggesting  This particular  c h a r a c t e r i s t i c wall and shape o f The  that  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  pseudomycelium and pleomorphic c e l l s  remains while  by t h e  chlamydophores,  lack of resistance to enzymatic digestion of the cell wall  type  wall  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  of aspartokinase  (Toskata  e t al.. 1978).  It is  - 56 possible  that  in Phaff ia  components  t h a t make up  the  cell  wall  are  likewise affected. 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 g r o w n on m e d i a w i t h o u t a m i n o a c i d analogues,  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  g r o w n on AEC  was  q u i t e low.  blame f o r t h i s . in  fact  from J \ rhodozyma  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  over  a  long  period  of  time  possibly  i n c o n s i s t e n c i e s i n the c e l l wall s t r u c t u r e or composition. t h i s , the  concentration  high,  and  thus  most  likely  later  of the c e l l  protoplasts  destroyed.  Not  digestion  a l l of  period  were  to  Because of  formed e a r l y i n the d i g e s t i o n p e r i o d the  cells  exposed  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 .  i n the  due  w a l l d i g e s t i v e enzyme m i x t u r e  t h i n w a l l s and odd s h a p e s , t h u s a p e r c e n t a g e  l i k e l y may  to  The 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 and  occurred  developed  cells  chosen  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  to  of the  a  were  the  AEC  population  Protoplasts  because  was  formed  proportionally  stages.  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 JC f r a g i l i s simple.  The  young  hours denoting fusant.  one  of  positive qualities  to  be  1  transfered  1/2 to  in this  a  study  r e p o r t e d by M o r g a n e t a l _ . ( 1 9 8 0 ) a n d J o h a n n s e n e t  (1984). Two  were  the  to p r o t o p l a s t s w i t h i n  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  c o r r e l a t e d with those al-  c e l l s were converted  was  d i f f e r e n t approaches f o r the f u s i o n of the y e a s t  attempted.  The  conventional  PEG  method  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 ) . reported  to  conventional  h a v e an PEG  and  protoplasts the  dipole  E l e c t r o f u s i o n has  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  method  (Bates  et  a_l_. 1 9 8 6 ) .  In  the  to  end,  been the the  - 57 e l e c t r o f u s i o n was  very  successful  a number o f t h e  selected  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  selected  presumed f u s a n t s i n c l u d i n g fusant When one  considers  producing #26.  as compared t o the  number o f p r o t o p l a s t s ,  (see  One  Table  III).  the  m u s t remember t h a t  number seems q u i t e  the  fusants  selected  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 and formation.  O t h e r f u s i o n s t h a t may  characteristics  are  also  p r e s u m e d f u s a n t s may  were  pigment  not have produced the s t a b l e d e s i r e d  likely  to  have  occurred.  Low  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  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 their cell  low  not  numbers  of  poor v i a b i l i t y  have been a b l e  to  regenerate  walls.  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  the  voltages used to induce f u s i o n .  The v o l t a g e s e l e c t e d and a p p l i e d a t  the  electrodes  fuser  of  the  dipole  cell  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 mm.  T h i s h i g h v o l t a g e may  was  2700 v o l t s .  The  distance  the experiment ranged from 1 to  have a f f e c t e d the r e g e n e r a t i o n  a b i l i t y of  2 the  protoplasts. PEG  in  fusion. the  contrast  The PEG  cells  f u s i o n may accomplished  a s s o c i a t i o n and  (Peberdy, 1980).  much  slower  upon d i l u t i o n  method  for  r a t h e r i t draws of  chemical,  m o r e r a p i d l y and 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  the  similar  f u s i o n by t h e e l e c t r i c p u l s e .  u s i n g PEG  q u a l i t i e s such as to  This drawing together  the  was  fusants were obtained  pigmentation,  is a  of c e l l s  added b e n e f i t of inducing  possess  electrofusion  i t s e l f does not cause f u s i o n to occur,  into close occur  to  cell those  s u c h a s f u s a n t #22. morphology, galactose obtained  by  Some p r e s u m e d  These fusants  did  assimilation  and  electrofusion  (data  not  - 58  shown). be  This suggests  used  with  electrofusion  some  -  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 can success.  include  The  advantages  associated  with  the synchrony of f u s i o n making s e l e c t i o n e a s i e r ,  and 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 the p o t e n t i a l l y harmful  e f f e c t s o f PEG  3 0 ° C , 3°C  discouraged above the  avoiding  addition.  Fusants were s e l e c t e d with the p r o j e c t e d goal P h a f f i a was  the  in mind.  The  parent  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  reported  growth temperature  limit  at  ( M i l l e r e t aj_.  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 galactose.  The 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  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 and on g a l a c t o s e . achieve  n i a c i n r e q u i r e m e n t o f IC  with  very  Often  IC  the  presumed  e x c e l l e n t both at 30°C  Other s e l e c t i v e media were a l s o used s u c c e s s f u l l y to  d i f f e r e n t i a t i o n of  The  fusant  the  fusant  from  the  parent  culture  colonies  often  started  f r a g i l i s would overgrow the p l a t e  carotenoid  rate  of  the  IC  pigments  is  considered  m e t a b o l i s m and  usually occurs  fragilis  out  relatively  to  be  t r a n s f e r of coloured  fusants  obtained.  There  The a  evidence  product  of  of growth.  for  control  presumed  fusants  have  a  cellular  On  repeated  l i n e s of  Phaffia  d i f f e r e n t than t h a t of P h a f f i a ( F i g . 2 & 4 ) .  morphology  of  secondary  being  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 JC f r a g i l i s a s shown i n T a b l e The  III).  accumulation  colonies, stable cell is  small  (Table  (hence the need to  parent.  in l a t e stages  s e l e c t i o n and were  e.g.  fragilis.  l i t t l e c o l o u r though morphology v a r i e d s l i g h t l y  growth  selected  that  the the  1. is  These d i f f e r e n c e s  quite include  - 59  -  t h e c i r c u l a r shape 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 as 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 cytoplasm  are also evident  l i p i d s s i m i l a r to those biochemical  produced by JC f r a g i l i s .  test results of the red yeast  d i f f e r e n t from producing  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  that o f the parent  The morphology and  toruloides are uniquely  Phaffia or the resultant  carotenoid  f u s a n t #26 ( F i g s . 2, 3 & 4 ) .  The b i o c h e m i c a l  t e s t s t h a t were done were chosen f o r t h e i r a b i l i t y  to d i f f e r e n t i a t e between 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 from 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  yeast contaminants  as  appear  R^.  toruloides.  characteristics  that  The  presumed  fusants  c a n be c o n s i d e r e d  to  demonstrate  t y p i c a l t o each parental  ( 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 r a n d o m 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 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 is  is difficult  which i s the r e c i p i e n t . urease  activity  suggest  that  Viewing  and c e l l Phaffia  evidence  i s the  o f chromosomes.  n i a c i n , o n e may  recipient.  production, venture  Characteristics  such  g a l a c t o s e a s s i m i l a t i o n and heat t o l e r a n c e were t r a n s f e r r e d p o s s i b l y JC  that  s t r a i n i s t h e donor and  such a s c a r o t e n o i d  growth without  type  procedure  t e s t s , Table I suggests  t o determine which parental  such  to as from  fragilis. 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 J C 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 use n i t r a t e as a source also  unable  to utilize  nitrate.  This  of nitrogen. d i f f e r s from  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 .  Fusant  #26 i s  R^ t o r u l o i d e s a  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 Ri  toruloides.  These very  same c e l l s  develop a shape t h a t appears  when g r o w n  t o be t h e same a s  in glucose  broth,  rhodozyma grown under  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  JC 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 4B).  or  broth  (see F i g s . 3 &  T h i s again supports the idea of P h a f f i a being the r e c i p i e n t . The  l i t e r a t u r e suggests  t h a t 85% o f the c a r o t e n o i d s  f o r m e d by  Pi  rhodoyzma under most ambient growth c o n d i t i o n s are a s t a x a n t h i n  (Andrewes  et  authentic  a l _ . 1976,  all-trans  Johnson  astaxanthin  & was  Lewis  1979).  chosen  to  Because be  the  of  this,  carotenoid  to  use  for  comparisons. Carotenoid  analyses  from  TLC,  column  chromatography,  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 and mass s p e c t r o p h o t o m e t r y the pigments obtained media are pigments  the in  astaxanthin The  from j \ rhodozyma and  same turn,  ( F i g s . 7,  8,  tend  correlate  (Roche) with  results also  to  12,  14  the exception  indicate that  f u s a n t #26  and  Tables  closely  chemical  g r o w n on IV,  with  solid  V).  the  These  all-trans  o f t h e mass s p e c t r o m e t r y  indicate that  the  pigments  from  media) and j \ rhodozyma a r e not  the  same a s t h o s e  fusant  #26  data. (solid  i s o l a t e d from  the  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 IV, V ) . Oxo-carotenoids B-carotenes.  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  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  the  molecule  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  (Davies 1965).  f r o m f_i  g r o w n on s o l i d m e d i a h a v e t h i s b r o a d  rhodozyma and  f u s a n t #26  c h a r a c t e r i s t i c s i n g l e peak o f the x a n t h o p h y l l s the a b s o r p t i o n  curve plus t h e i r A  m a x  The p i g m e n t s i s o l a t e d  (Fig. 8).  The  correlates c l o s e l y with  shape of authentic  - 61 astaxanthin;  in  superimposed  on  xanthophyl1ic-1ike from  fact,  the  that  spectra from  from  fusant  astaxanthin  #26  can  (Fig.  8).  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  almost  be Other  i n t h e f r a c t i o n (A)  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)  from  toruloides. The  l i q u i d c u l t u r e s o f f u s a n t #26  that resembles  were unable  to produce  t h o s e o f F\ r h o d o z y m a g r o w n on s o l i d m e d i a .  pigments  H o w e v e r , on  l i q u i d medium under l i m i t e d oxygen 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 synthesis, produced  producing by  conditions.  these  B-carotene  in  c u l t u r e s appears  excess. t o be  in a d d i t i o n to a decrease  the  carotenoids  a f u n c t i o n of the  Johnson & Lewis (1979) suggest  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  Thus  culture  that under fermentative  o f a s t a x a n t h i n p r e c u r s o r s may  in astaxanthin levels.  or  occur  It is possible that 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 i n s u f f i c i e n t a e r a t i o n or o t h e r such undiscovered  limitations.  The  to 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 w o r k 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 . The anomaly o f t h e mass s p e c t r o m e t r y not l e s s than c o n f u s i n g . t h e m a s s p e a k a t 596 according  to  the  One  d a t a c a n be c o n s i d e r e d t o be  h i g h l y i r r e g u l a r r e s u l t was  the lack  i n the pigment i s o l a t e d from j \ rhodozyma which  literature  ( A n d r e w e s e t a l _ . 1976;  Johnson  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  of astaxanthin  in  grown  the  cells  ( F i g . 12).  i s o l a t e d under apparently e t aj.. (1976).) encouraging  (These  cells  missing (see F i g . 11).  since  were  and  &  Lewis  reported  the  pigment  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  Even the a u t h e n t i c  results  of  three  of  astaxanthin the  used  gave  less  c h a r a c t e r i s t i c peaks  than were  -  The r e s u l t s were  run  at  from the two  between  problems  mass  of  Irregularities temperatures, Again, from  from J \  one  mass  runs.  when In  subjected  the  derivative,  also  the  presence of  astacene  CH,  or  to  distinguished  6).  is  suggest  by  colour  bases  CH,  to  CH,  of  Jucker  oxidized of  CH,  C  C ^ , C - C H = CH-C=CHCH-CH-C=CHCH=CHCH=C-CH=.CHCH-C-CH-CH-(f  /  H.C-I  C-CH,  &  its  X  OC  pigments  (Karrer  converted  1976).  reactions  A s t a c e n e upon t h e a d d i t i o n  CH,  probe  that  CH, CH,  (1976).  astaxanthin,  similar.  CH,  C  inherent  & Weedon  are  and is  the  impurities,  (Moss  do  large  Weedon  carotenoid  tests  astaxanthin  &  samples  quite  of  Moss  sample  the  acids  are  one  ionization  that  strong  (formula  by  ( s o l i d media)  be  air  there  denotes  due  other  #26  to  show t h a t when t h e  suggested  conclude  rhodozyma and f u s a n t  pigments  as  conditions  data,  can  This  occurred  cannot  spectra  Carotenoids  1950).  have  data  temperatures,  spectrometry  insertion  though  the  these  may  -  mass s p e c t r a l  different  differences  62  CO  CH, CO-  CO  astacene Formula 6 concentrated 1950).  sulphuric  Results obtained  differences  between  concentration. is  acid  compared t o  dark  will  turn  a  from Table V i n d i c a t e blue  and  medium  Again a reaction difference R^  deep  toruloides.  blue this  blue  (Karrer  &  occurrance.  may  be  Jucker Colour  attributed  c a n be n o t e d when f u s a n t  to #26  - 63 -  CONCLUSIONS During fragilis  and  the  course  J\  of  t h i s study,  r h o d o z y m a was  an  intergeneric cross  c a r r i e d out.  The  Morphologically  the  types  t o l e r a n c e and c a r o t e n o i d p i g m e n t  presumed  fusants  are  most  IC  r e s u l t i n g presumed  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 g r o w t h on g a l a c t o s e , h e a t  of  like  such  as  production.  j\  rhodozyma  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 . Though the  carotenoid  rhodozyma cannot spectrometry other  tests.  pigments  i s o l a t e d from  be c a l l e d a s t a x a n t h i n due  data, TLC,  they  are  visible  s i m i l a r to  to the anomally each  other  wall  that i s easy  promptly.  #26  and  l i n e #26  to rupture thus  J\  o f the mass  according  to a l l  s p e c t r a , column c h r o m a t o g r a p h y and  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 The p r e s u m e d f u s a n t c e l l  fusant  colour  astaxanthin.  has a m i c r o s c o p i c a l l y t h i n c e l l  l i b e r a t i n g the c e l l u l a r  carotenoids  - 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 by Kluveromyces f r a g i l i s ; t h e e f f e c t on c e l l lipids. Can. I n s t . Food 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. 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P l a n t Tissue & Cel1 Culture. S i n c k , K.C. 1984. Protoplast H o r t S c i e n c e , V o l . 19, p . 33.  Fusion  f o r Plant  Improvement.  T o s a k a , 0., K. T a k i n a m i a n d Y. H i r o s e . 1 9 7 8 . L - l y s i n e P r o d u c t i o n b y s-(2-aninomethyl) L-cysteine and a-Amino-B-hydroxyvarelic Acid R e s i s t a n t Mutents o f Brevibacterium lactofermantum. Agric. Biol. Chem. V o l . 4 2 , p . 7 4 5 . T o s a k u , 0., H. H i r a k a w a a n d K. T a k i n a m i . 1978. R e g u l a t i o n o f L y s i n e B i o s y n t h e s i s by Leucine i n B r e v i b a c t e r i u m lactofermentum. Agric. B i o l . Chem. V o l . 4 2 , p . 1 5 0 1 . V i e n n e , P. a n d U. v o n S t o c k a r . 1 9 8 5 . An I n v e s t i g a t i o n o f E t h a n o l I n h i b i t i o n and other L i m i t a t i o n s o c c u r r i n g during the fermentation of concentrated whey permeate by Kluveromyces fragilis. B i o t e c h n o l o g y L e t t e r s , V o l . 7, p . 5 2 1 . W i e r z b i c k i , L . E . a n d F.V. K o s i k o w s k i . 1972. Lactase Potential of 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 . 5 6 , p. 26.  - 68 -  APPENDIX I  - 69 -  12i  i  i  i ii  i n|  1i  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 r a n g e s o f  individual  unbudded y e a s t  from  cells.  P o h l e t a l . , 1982  - 70  Fig.  18.  A model  for  -  t h e d i p o l e moments  in a yeast  from Pohl et  cells.  al.,  1982  - 71 -  APPENDIX II  Fig.  19.  Fusion  apparatus  - 73 -  APPENDIX I I I  - 74 -  The growth medium was 50 m l 0 1 M-phthalate-bufTered yeast nitrogen base medium (see Methods), containing 0-6 ° (w/v) Bacto-peptone and 200 mg carbon (supplied as the various sugars). The values represent the mean o f two determinations. C a r b o n sources were sterilized separately f r o m the basal medium. N o growth occurred i n the basal medium without the addition o f a carbon source. Growth Yeast Astaxanthin Astaxanthin rate, fi yield Yeast yield yield yield C a r b o n source Or') (mg m l ) [mg (mg c a r b o n ) " ] * (Mg m l " ) l/ig (g yeast)" ] D-Maltose 014 3-63 0 91 1 86 512 D-Cellobiose 0 10 3-48 0 87 2-27 652 0 19 Sucrose 3-72 093 1 89 508 0-67 Succinate 009 2-66 1 33 500 D-Maruiitol 0 16 3-68 092 1 80 489 D-Xylose 004 1-21 0 30 0 58 479 L-Arabinose 005 3 30 083 1 25 379 Glucono-<S-lactone 0 10 1 48 0-37 0 80 541 D-Glucose 0 2C 3 85 096 1 62 421 D-Glucose 021 646 0 81 111 171 0  - 1  1  1  1  +  • 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 o n 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 . from Johnson & Lewis,  1979.  -  75  -  APPENDIX IV  - 76 -  F i g . 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 rhodozy  Ash Total carbohydrate Total nitrogen Protein ( N x 6.25) Protein ( F o l i n reagent) RNA Total lipid Astaxanthin  Table  VIII  Proximate brewers  ma  5.6 40.3 4.82 30.1 25.0 8.2 17.0 .06  composition  yeast  Brewer's yeast 6.5 33.4 8.71 54.4 — 9.2 4.03 0  of  Phaffia  rhodozyma  and  (dwb).  f r o m J o h n s o n et  a],.,  1980.  

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