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Hydrologic properties and water balance of the forest floor of a Canadian west coast watershed Plamondon, André P. 1972

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THE CINNAMIC ACID PATHWAY AND HISPIDIN BIOSYNTHESIS IN CULTURES OF POLYPORUS HISPIDUS FRIES by PETER WILLIAM PERRIN B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1968  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of Botany  We a c c e p t t h i s t h e s i s as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1972  In p r e s e n t i n g an  this thesis  advanced degree at  the  Library  in p a r t i a l  the U n i v e r s i t y  f o r s c h o l a r l y purposes may representatives.  be granted by  his  of  t h i s t h e s i s f o r f i n a n c i a l gain  for  requirements  Columbia, reference  the Head o f my  It i s u n d e r s t o o d that  permission.  of  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  British  the  I agree and  Columbia  shall  not  that  thesis  Department  c o p y i n g or  for  study.  for extensive copying'of t h i s  by  Department  of  s h a l l make i t f r e e l y a v a i l a b l e  I f u r t h e r agree t h a t p e r m i s s i o n  written  f u l f i l m e n t of  or  publication  be a l l o w e d without  my  i ABSTRACT The  biosynthesis of hispidin,  6-(3,4-dihydroxystyryl)-  4-hydroxy-2-pyrone, was examined i n c u l t u r e s o f Polyporus hispidus F r .  C u l t u r a l s t u d i e s were undertaken t o determine  the most s u i t a b l e medium o f t h i s pigment.  f o r i n v e s t i g a t i n g the b i o s y n t h e s i s  These s t u d i e s showed t h a t l i g h t was nec-  e s s a r y f o r h i s p i d i n formation  and t h a t the development o f  b a s i d i o c a r p s w i t h v i a b l e spores media.  c o u l d be a c h i e v e d  on agar  On the l i q u i d medium employed f o r b i o c h e m i c a l  studies,  the maximum r a t e o f h i s p i d i n p r o d u c t i o n was observed t o l a g the maximum r a t e o f growth by about f i v e days. Trimethylhispidin, 4-methylhispidin synthesized  and yangonin were  f o r comparative purposes and f o r d i l u t i o n i n t r a c e r  experiments.  These and numerous o t h e r p h e n o l i c and aromatic  compounds were employed as r e f e r e n c e s nonradioactive  culture extracts.  i n examinations o f  P-coumaric, c a f f e i c , £ -  hydroxybenzoic, p r o t o c a t e c h u i c and o- and p_-hydroxyphenyla c e t i c a c i d s were d e t e c t e d Bis-noryangonin  i n e x t r a c t s o f the c u l t u r e medium.  (6-(4-hydroxystyryl)-4-hydroxy-2-pyrone)  o t h e r p o s s i b l e s t y r y l p y r o n e s were d e t e c t e d  and  i n extracts of  the mycelium. T r a c e r experiments e s t a b l i s h e d t h a t was m e t a b o l i z e d  t o cinnamic,  protocatechuic acids.  phenylalanine  b e n z o i c , p_-hydroxybenzoic and  The i n c o r p o r a t i o n o f r a d i o a c t i v i t ; '  11  into phenyllactic, phenylacetic aric ity  acid,  phenylpyruvic,  a c i d s a l s o was  caffeic  acid  aric  radioactivity  acid,  caffeic  Degradation cursors  Hispidin  Furthermore, incorporated  a l s o was  shown  from t y r o s i n e , cinnamic  acid,  malonic  acid  o f the l a b e l l e d h i s p i d i n  confirms  synthesized  observed.  and h i s p i d i n  from p h e n y l a l a n i n e .  porate  p h e n y l a c e t i c and rj-hydroxy-  the hypothesis  obtained  that this  from a p h e n y l p r o p a n o i d  radioactivto incor-  acid,  and sodium  p_-coum-  p_-coum-  acetate.  from these  molecule  moiety with  pre-  is bio-  the a d d i t i o n  o f two e q u i v a l e n t s o f a c e t a t e . Crude and p a r t i a l l y enzymes  related  ylalanine ed  preparations  of several  m e t a b o l i s m were o b t a i n e d .  a n d t y r o s i n e a m m o n i a - l y a s e a c t i v i t y were  in cell-free  lyase  to aromatic  purified  preparations.  a c t i v i t y was  obtained  i t h m i c phase o f growth. cinnamic  acid,  benzoic  obtained  in vitro.  demonstrat-  Maximum p h e n y l a l a n i n e  ammonia-  from c u l t u r e s d u r i n g the l o g a r -  Enzymes acid  Phen-  capable  of hydroxylating  and b i s - n o r y a n g o n i n  a l s o were  iii TABLE OF CONTENTS PAGE ABSTRACT  i  TABLE OF CONTENTS  i i i  LIST OF TABLES  ,  vi  LIST OF FIGURES  v i i  ACKNOWLEDGMENT  ix  INTRODUCTION  1  LITERATURE REVIEW  4  I . The a c e t a t e - p o l y m a l o n a t e pathway i n f u n g i  4  I I . The s h i k i m i c a c i d pathway i n f u n g i . . . . . . . . . . . .  6  I I I . Aromatic amino a c i d metabolism i n B a s i d i o 11  mycetes IV. Pigment p r o d u c t i o n  16  i n fungi i n Basidiomycetes.........  17  CHAPTER ONE. CULTURAL STUDIES OF POLYPORUS HISPIDUS...  21  V.  Sporophore formation  Introduction  21  M a t e r i a l s and Methods  22  I . Sources o f c u l t u r e s  22  I I . Sources o f medium c o n s t i t u e n t s  22  I I I . Media employed  23  IV. C u l t u r i n g techniques  24  V. Measurements o f h i s p i d i n p r o d u c t i o n  24  VI. Dry weight d e t e r m i n a t i o n s  25  Results  and D i s c u s s i o n . .  26  I . Comparison o f P_. h i s p i d u s  and P. schwein-  itzii  26  I I . Growth and pigment p r o d u c t i o n  i n P. h i s -  pidus I I I . Sporophore formation P. h i s p i d u s .  30 i n agar c u l t u r e s o f 45  iv TABLE OF CONTENTS (cont'd) PAGE CHAPTER TWO. CHEMICAL STUDIES OF PHENOLIC ACIDS, STYRYLPYRONES AND RELATED COMPOUNDS  49  Introduction  49  M a t e r i a l s and Methods  49  I . Chemicals  49  I I . - Chromatography.  50  I I I . Spectroscopy  51  IV. M e l t i n g p o i n t s  51  V. Chemical p r e p a r a t i o n s  51  T r i a c e t i c lactone.  •  51  6-methyl-4-methoxy-2-pyrone.  52  Trimethylhispidin  53  4-methylhispidin  53  Alkaline hydrolysis of trimethylhispidin...  54  3,4-dimethoxycinnamic a c i d . . . . . . . . . . . . . . . . .  55  3,4-bis-(methoxymethoxy)benzaldehyde  55  • Vera t r i e a c i d from t r i m e t h y l h i s p i d i n  56  V I . Radioautography R e s u l t s and D i s c u s s i o n  57 58  CHAPTER THREE. RADIOACTIVE FEEDING EXPERIMENTS WITH CULTURES OF POLYPORUS HISPIDUS  70  Introduction  70  M a t e r i a l s and Methods  70  I. A n a l y s i s o f phenolic acids I I . Preparation  70  and a d m i n i s t r a t i o n o f r a d i o a c -  t i v e compounds III. Detection of r a d i o a c t i v i t y IV. Recovery o f f r e e amino a c i d s 14 V. M e t h y l a t i o n o f C-labelled hispidin R e s u l t s and D i s c u s s i o n  70 71 72 72 73  V  TABLE OF CONTENTS  (cont'd) PAGE  CHAPTER FOUR. PRELIMINARY STUDIES OF ENZYMES  ASSO-  CIATED WITH AROMATIC METABOLISM  92  Introduction  92  Materials  93  and Methods  I. P h e n y l a l a n i n e ammonia-lyase...........  93  II.  94  III.  T y r o s i n e ammonia-lyase.... Benzoic and cinnamic a c i d - 4 - h y d r o x y l a s e . . .  IV. B i s - n o r y a n g o n i n - 3 - h y d r o x y l a s e Results  and D i s c u s s i o n  94 95 96  GENERAL SUMMARY AND CONCLUSIONS  100  BIBLIOGRAPHY  104  APPENDICES  113  A.  Characteristic  B. S p r a y  Reagents  o f Wrattan  filters  113 114  vi LIST OF TABLES TABLE I.  PAGE M y c e l i a l d r y weight and pH o f medium o f c u l t u r e s o f P. h i s p i d u s a f t e r s i x t e e n days when grown on DM#1 a t v a r i o u s i n i t i a l pH* s  II.  Colours violet  34  o f p h e n o l i c a c i d s i n l o n g wave u l t r a l i g h t and i n v i s i b l e  light after  spray-  i n g w i t h d i a z o t i z e d - p _ - n i t r o a n i l i n e reagent III.  „  60  C h a r a c t e r i s t i c s o f some s t y r y l p y r o n e s and r e l a ted  p h e n o l i c compounds chromatographed on c e l l u -  l o s e TLC p l a t e s i n s o l v e n t system C and sprayed w i t h v a r i o u s reagents IV.  Metabolic products pounds a d m i n i s t e r e d  V.  (Appendix B)  66  from v a r i o u s aromatic  com-  t o P. h i s p i d u s  Incorporation o f various precursors  82 into hispid-  i n by 17-day-old c u l t u r e s o f P. h i s p i d u s  87  vii  LIST OF FIGURES FIGURE  PAGE  1*  Naturally-occurring styrylpyrones  2  2.  Acetate-polymalonate-derived  5  3.  Complex p o l y k e t i d e s from Basidiomycetes  7  4*  S h i k i m i c a c i d pathway t o aromatic  amino a c i d s . . .  8  5.  Shikimic-acid-derived metabolites  of Basidio-  fungal products....  mycetes  10  6.  Fungal metabolites  7.  Transformations  o f mixed b i o g e n e s i s  o f cinnamyl compounds i n L. l e p o  ideus 8.  12  14  Comparison o f the growth o f P. h i s p i d u s and  P.  s c h w e i n i t z i i on l i q u i d MYP 9.  27  O p t i c a l d e n s i t y measurements o f e t h e r e x t r a c t s o f the mycelium o f P. s c h w e i n i t z i i  28  10.  pH  32  11.  V a r i a t i o n i n c o l o n y diameter w i t h  change d u r i n g growth o f P_. h i s p i d u s on DM#1..  o f P. h i s p i d u s c u l t u r e s on MYP 12.  temperature  agar  35  E f f e c t o f wavelength o f l i g h t source  on growth  and pigment development i n agar c u l t u r e s 13.  E f f e c t o f malt e x t r a c t and Soytone  37  concentration  on c o l o n y diameter a f t e r f i f t e e n days.. 14.  40  Growth and h i s p i d i n p r o d u c t i o n o f P_. h i s p i d u s on GYSS  15.  44  Pigment development i n the mycelium o f P. pidus  a f t e r t e n and  his-  f i f t e e n days i n c u b a t i o n  on  GYSS 16.  46  Sporocarp o f P. h i s p i d u s t e n days a f t e r t i o n on MYP  17.  initia-  agar  46  Diagrammatic r e p r e s e n t a t i o n o f two-dimensional chromatogram o f a u t h e n t i c samples o f p h e n o l i c and  cinnamic a c i d d e r i v a t i v e s  59  viii LIST OF FIGURES  (cont'd)  FIGURE  PAGE  18.  Standard absorbance  curve o f p_-coumaric  acid.....  19.  Standard absorbance  curve o f c a f f e i c a c i d  62  20.  Standard absorbance  curve o f t r i m e t h y l h i s p i d i n . . .  63  21.  Standard absorbance  curve o f v e r a t r i c a c i d  64  22.  U l t r a v i o l e t s p e c t r a o f y e l l o w , f l u o r e s c e n t bands from chromatograms o f P_. h i s p i d u s e x t r a c t s  23.  Diagrammatic  61  68  r e p r e s e n t a t i o n o f the compounds de-  t e c t e d i n chromatographed e x t r a c t s o f the medium u s i n g 48 h r i n d u c t i o n by replacement medium 24.  75  Probable pathways o f L - p h e n y l a l a n i n e d e g r a d a t i o n i n P. h i s p i d u s  25  76  Probable r e l a t i o n s h i p s o f r a d i o a c t i v e  cinnamic  a c i d d e r i v a t i v e s d e t e c t e d i n c u l t u r e s o f P_. h i s pidus 26.  78  Aromatic amino a c i d metabolism  v i a the cinnamate  pathway i n P. h i s p i d u s 27.  85  B i o s y n t h e s i s and d e g r a d a t i o n o f r a d i o a c t i v e  his-  pidin. 28.  91  P h e n y l a l a n i n e ammonia-lyase a c t i v i t y i n c u l t u r e s o f P. h i s p i d u s  29.  A l t e r n a t e routes proposed  97 f o r the b i o s y n t h e s i s  o f h i s p i d i n i n P. h i s p i d u s  102  ix ACKNOWLEDGMENT  I wish t o express my s i n c e r e g r a t i t u d e t o Dr. R.J. Bandoni and Dr. G.H.N. Towers w i t h whose f a c i l i t i e s and under whose guidance t h i s work was c a r r i e d o u t .  T h e i r en-  couragement, a d v i c e and c r i t i c i s m o f t h i s manuscript a r e g r a t e f u l l y acknowledged.  The a s s i s t a n c e and a d v i c e o f Dr.  C.K. Wat and t h e f a c i l i t i e s p r o v i d e d by Dr. T. Money were greatly appreciated.  To my c o l l e g u e s , f o r h e l p f u l  discus-  s i o n s , and t o t h e members o f my committee, f o r t h e i r comments on t h i s manuscript, I g i v e my thanks.  The f i n a n c i a l  support o f t h e N a t i o n a l Research C o u n c i l o f Canada and o f the  H.R. M a c M i l l a n f a m i l y i s acknowledged. A l s o , I would l i k e t o thank my w i f e , Janne, f o r h e r  encouragement throughout t h i s work.  INTRODUCTION  1  N a t u r a l l y o c c u r r i n g sporophores o f Polyporus h i s p i d u s F r . , P_. s c h w e i n i t z i i F r . and  s e v e r a l s p e c i e s o f Gymnopilus  c o n t a i n the s t y r y l p y r o n e pigment h i s p i d i n ( F i g . and  Smith 1961,  f i e l d and  Edwards e t a l 1961,  Brady 1971).  pyrone b i s - n o r y a n g o n i n  l.)(Bu'Lock  Ueno e t a l 1964,  Gymnopilus a l s o c o n t a i n s ( F i g . 1 . ) ( H a t f i e l d and  the  ysticum  Forster.  kawain and  (Nees e t Mart.) Mez.  and  plants,  P i p e r meth-  From these have been i s o l a t e d 5,6-dehydro-  yangonin  (Fig.1.)(Borsche  l i e b and Mors 1959). ura e t a l 1966)  styryl-  Brady 1968).  Compounds o f t h i s type were f i r s t known from h i g h e r e s p e c i a l l y Aniba f i r m u l a  Hat-  and  and Gerhardt 1914,  Gott-  More r e c e n t l y s p e c i e s o f A l p i n i a Ranunculus  (Shibata e t a l 1972)  (Kim-  a l s o have  been shown t o c o n t a i n these m o l e c u l e s . In h i g h e r p l a n t s t h e r e i s evidence t h a t pyrones are s y n t h e s i z e d v i a the s h i k i m i c and pathways w i t h the a d d i t i o n o f two same manner as the k i n e t a l 1957, z a k i 1957, i t has  'A'  a c e t a t e u n i t s much i n the  Swain 1957,  Donovan 1953).  (Wat-  S h i b a t a and Yama-  A l s o , i n P. s c h w e i n i t z i i  been demonstrated t h a t p h e n y l a l a n i n e  incorporated  cinnamic a c i d  r i n g o f f l a v o n o i d s i s produced  Geissman and  B i r c h and  styryl-  i n t o the h i s p i d i n molecule  and  acetate  ( H a t f i e l d 1970).  t h e r e i s p r e l i m i n a r y evidence t h a t f u n g i are capable o f a t i n g upon p h e n y l p r o p a n o i d m o l e c u l e s .  are Thus elabor-  2  YANGONIN (Piper, Ranunculus)  OH HO-  b i s-NORYANGONIN (Gymnopilus)  OCH,  5,6-DEHYDROKAWAIN (Aniba, A l p i n i a )  OH  HISPIDIN (Polyporus, FIG.  1.  Naturally-occurring  Gymnopilus)  styrylpyrones.  3  T h i s study w i l l p r e s e n t  a d d i t i o n a l evidence f o r the  biosynthesis of styrylpyrones i n fungi. a r o m a t i c a c i d metabolism o f P. h i s p i d u s r a d i o a c t i v e t r a c e r s and l a b e l l e d precursors  The  intermediary  i s examined w i t h  the i n c o r p o r a t i o n o f  specifically  i n t o the h i s p i d i n molecule i s shown.  Also,  the presence o f c e r t a i n n e c e s s a r y enzymes i s demonstrated i n c e l l - f r e e e x t r a c t s o f the fungus.  Bu'Lock (1967) r e p o r t e d  t h a t P. s c h w e i n i t z i i produced h i s p i d i n when grown on c u l t u r e media, but  t h a t P. h i s p i d u s y i e l d e d t h i s compound  o n l y when grown on wood b l o c k s . o f e n v i r o n m e n t a l and p i d i n production ined.  usual  In t h i s study, the e f f e c t s  n u t r i t i o n a l f a c t o r s on growth and  his-  i n l i q u i d c u l t u r e s o f J?. h i s p i d u s i s exam-  LITERATURE REVIEW  4  I . The  a c e t a t e - p o l y m a l o n a t e pathway i n As  linic  early  a c i d was  as  Two  In  1953,  later,  6-methylsalicylic  Penicillium  manner o f Birch  et  involve  a l 1961, of  ( F i g . 2 . ) by  o c c u r by  the  Zwitkowits  which w i l l  of  1970).  et  P.  for  Penicillium  and  as  for  of a  the  acid  reported  a "building  unit"  Keil  by  subin  the  1961,  More r e c e n t l y ,  the  methyl t r i a c e t i c  stipitatum  lac-  h a v e b e e n shown (Bentley  to  and  a l 1969).  Cell-free  enzyme  p a t u l u m now  have been  obtained  and  m a l o n y l Coenzyme A  triacetic  e x t e n d an  styrylpyrone  synthesis  Evidence  a l 1962).  Tanenbaum e t  1953).  griseofulvum via  t e t r a c e t i c l a c t o n e and  acid  the  of  indicated  Donovan  b i o s y n t h e s i s ( B e n t l e y and  Penicillium  orsel-  6-methylsalicylic  malonic acid  However, t h e r e a r e  or  and  a l 1955). and  of  condensation  g r i s e o f u l v u m was  Bu*Lock e t  parations which w i l l flavonoid  obained  acid  condense a c e t y l  methylsalicylic al  (Birch  a c e t a t e - p o l y m a l o n a t e pathway  1967,  preparations  and  the  Penicillium  (Birch  orsellinic  fatty acid  biosynthesis  correct  a c i d by  urticae  sequently to  o c c u r by  e v i d e n c e was  p o l y a c e t a t e pathway production of  to  biosynthesis  experimental evidence  C o l l i e ' s t h e o r y was years  tone  ( C o l l i e ) the  considered  acetate units. that  1907  fungi  no  acid  reports  lactone of  into  6-  (Dimroth  cell-free  pre-  a r o m a t i c a c i d p r e c u r s o r as  biosynthesis.  et  in  5  O H  H  3  C  ^ 0 ^ 0  METHYL TRIACETIC LACTONE  O H  TETRACETIC LACTONE  FIG. 2.  Acetate-polymalonate-derived  f u n g a l products,  6 In a d d i t i o n t o simple p o l y k e t i d e s , many complex p o l y k e t i d e s a r e produced as secondary m e t a b o l i t e s o f f u n g i , a l t h o u g h these compounds a r e r e l a t i v e l y r a r e i n Basidiomycetes  (Turner 1971).  C o r t i n a r i u s sanguineus has y i e l d e d  the r e l a t e d anthraquinones, emodin, dermoglaucin and dermocybin  ( F i g . 3 . ) ( K o g l and Postowsky  A u s t e l 1966).  1925, S t e g l i c h and  These o c t a k e t i d e s a r e among the most complex  p o l y k e t i d e s known from B a s i d i o m y c e t e s .  S e v e r a l p e n t a - and  lower m o l e c u l a r weight k e t i d e s a l s o have been i s o l a t e d Basidiomycetes. naphthoquinone 1951) ealis  from  Some examples o f t h e s e a r e 6-methyl-l,4from Marasmius graminum  and 8-hydroxy-3-methylisocoumarin ( F i g . 3.)(Bendz  ( F i g . 3.)(Bendz from Marasmius ram-  1959).  I I . The s h i k i m i c a c i d pathway i n f u n g i E v i d e n c e i n d i c a t e s t h a t i n f u n g i the s h i k i m i c  acid  pathway i s used much l e s s commonly than the a c e t a t e - p o l y m a l onate o r p o l y k e t i d e pathway f o r the b i o s y n t h e s i s o f a r o m a t i c secondary m e t a b o l i t e s (Bentley 1962). s h i k i m i c a c i d pathway  N e v e r t h e l e s s , the  ( F i g . 4.) i s b e l i e v e d t o be o p e r a t i v e  i n a r o m a t i c amino a c i d s y n t h e s i s i n most f u n g i Turner 1971).  (Burnett 1968,  A l t h o u g h much o f the r e s e a r c h i n e l u c i d a t i n g  the s h i k i m i c a c i d pathway has been c a r r i e d o u t u t i l i z i n g t e r i a l mutants  bac-  (Davis 1955, Gibson and G i b s o n 1964, S p r i n -  son I960), mutants o f Neurospora c r a s s a have been u t i l i z e d  7  R  Q  4  R5  DERMOGLAUCIN OH  OH  OCH  3  H  H  DERMOCYBIN  OH  OCH  3  OH  H  (Cortinarius  OH  sanguinius)  O  6-METHYL—l,4-NAPHTHOQUINONE (Marasmius graminum)  0  H  0  8-HYDROXY-3-METHYLISOCOUMARIN (Marasmius r a m e a l i s ) FIG.  3. Complex P o l y k e t i d e s from  Basidiomycetes  8  COOH  I  c=o  CHO I HCOH  I  HCOH CH OP0 H 2  I CHI HOCH I HCOH I HCOH CH OP0 H  COOH I COPO-H| 3 2 CH„ 3  2  2  E r y t h r o s e 4phosphate  Phosphoenolpyruvic acid  2  3  2  3-deoxy-2-keto-Darabino-heptulosonic a c i d 7-phosphate  'r  COOH  COOH  HO  COOH  HO OH 5-dehydroshikimic  Shikimic acid  COOH  5-phosphoshikimic acid NH CH -CH-COOH 2  2  Phenylalanine 5.  5-dehydroquinic acid  COOH  OH  FIG.  acid  COOH  H 0 P 2  3  OH Chorismic acid  3-enolpyruvylshikimic a c i d 5-phosphate  1  O CH _C-COOH  HOOC ' CH -C-COOH  2  Phenylpyruvic  acid  S h i k i m i c a c i d pathway t o aromatic  Prephenic amino  2  acid  acids.  9 e x t e n s i v e l y i n s t u d i e s o f t h i s pathway i n f u n g i .  A mutant  o f Neurospora has been shown t o possess the enzyme dehydros h i k i m i c dehydrase c a t a l y z i n g the c o n v e r s i o n o f shikimic a c i d to protocatechuic acid  5-dehydro-  (Gross 1958).  Another  mutant has been shown t o accumulate p r e p h e n i c a c i d  (Metzen-  berg and M i t c h e l l 1956).  The phosphate o f s h i k i m i c  acid  has been d e t e c t e d i n c u l t u r e s o f the Basidiomycete L e n t i n u s lepideus  (Eberhardt 1956).  The p r o d u c t i o n o f secondary m e t a b o l i t e s from the s h i k i m i c a c i d pathway e i t h e r d i r e c t l y , o r by way  of aromatic  amino a c i d s o r cinnamic a c i d d e r i v a t i v e s i s well-known i n the B a s i d i o m y c e t e s . from t h i s s o u r c e .  S e v e r a l diphenylbenzoquinones are known  One  Polyporus leucomelas  example i s leucomelone  (Akagi 1942).  from Peniophora sanguinea  ( F i g . 5.)  from  X y l e r y t h r i n (Fig..5.)  (Gripenberg 1965)  obviously  results  from c o n d e n s a t i o n o f p o l y p o r i c a c i d w i t h p-hydroxyphenylacetic acid.  The o n l y known f u n g a l f l a v o n e ,  chlorflavonin  ( F i g . 6.) which has been i s o l a t e d from c u l t u r e s o f A s p e r g i l l u s candidus  (Richards e t a l 1969)  i n the s h i k i m i c a c i d pathway. been unable t o v e r i f y t u r e s o f the fungus  p r o b a b l y has i t s o r i g i n  However, l a t e r s t u d i e s have  p r o d u c t i o n o f t h i s compound i n c u l -  (Towers, p e r s o n a l communication).  Many m e t a b o l i t e s o f h i g h e r p l a n t s such as the f l a v o n oids are derived  through  the s h i k i m i c a c i d pathway w i t h  10  LEUCOMELONE (Polyporus  leucomelas)  XYLERYTHRIN (Peniophora  FIG.  sanguinea)  5. S h i k i m i c a c i d - d e r i v e d M e t a b o l i t e s  of  Basidiomycetes  11 subsequent chain.  e l a b o r a t i o n by c o n d e n s a t i o n w i t h an a c e t a t e - d e r i v e d  In Basidiomycetes, a c e t a t e c h a i n s more t y p i c a l l y  form condensation p r o d u c t s w i t h t r i c a r b o x y l i c a c i d c y c l e mediates  or other acetate-derived molecules.  as a g a r i c i c a c i d and Vogelsang  ( F i g . 6.)  1907)  inter-  Such compounds  from Polyporus o f f i c i n a l i s  (Thorns  a r e d e r i v e d by condensation o f the c a r -  b o n y l group o f o x a l o a c e t a t e w i t h the a-methylene group o f a fatty acid.  Cortisalin  Cytidia salicina  ( F i g . 6.), which was  isolated  from  ( C o r t i c i u m s a l i c i n u m ) ( G r i p e n b e r g 1952,  Mar-  s h a l l and W h i t i n g 1957), and c h l o r f l a v o n i n r e p r e s e n t the o n l y f u n g a l p r o d u c t s reported, i n a d d i t i o n t o s t y r y l p y r o n e s , which appear t o be formed by p o l y k e t i d e e x t e n s i o n o f a s h i k i m i c a c i d - d e r i v e d phenylpropanoid  moiety.  I I I . Aromatic amino a c i d metabolism  i n Basidiomycetes  Many f u n g i have been shown t o produce  aromatic amino  a c i d s v i a the s h i k i m i c a c i d pathway, and the c o n v e r s i o n o f these aromatic compounds t o o t h e r a r o m a t i c compounds i s o f considerable i n t e r e s t . a l a n i n e ammonia-lyase  Many Basidiomycetes possess p h e n y l (PAL) a c t i v i t y ,  o f p h e n y l a l a n i n e t o cinnamic a c i d e t a l 1968). lyase  e f f e c t i n g the c o n v e r s i o n  (Power e t a l 1965,  In some o f these organisms  t y r o s i n e ammonia-  (TAL) i s a l s o p r e s e n t , y i e l d i n g p_-coumaric  tyrosine.  Bandoni  D e t a i l e d s t u d i e s on the metabolism  acid  from  of phenylala-  n i n e and t y r o s i n e have been r e p o r t e d and these have been r e viewed by Towers  (1969).  CHLORFLAVONIN ( A s p e r g i l l u s candidus)  HO~Y  7— ( C H = C H ) — COOH 7  CORTISALIN (Cytidia salicina)  HOOC  CH_,  \ CH (CH ) 3  2  /  1 ( )  3  CH—CH C H CCOH 2  AGARICIC ACID (Polyporus  FIG.  6 . Fungal m e t a b o l i t e s  officinalis)  o f mixed b i o g e n e s i s .  13  C u l t u r a l s t u d i e s w i t h L e n t i n u s l e p i d e u s employing r a d i o a c t i v e t r a c e r s showed t h a t carbon L - p h e n y l a l a n i n e was a c e t a t e was  from D-glucose  and  incorporated into i s o f e r u l i c acid,  a poor p r e c u r s o r  (Power e t a l 1 9 6 5 ) .  The  while data  f o r L - t y r o s i n e are q u e s t i o n a b l e i n l i g h t o f the d e t e c t e d  TAL  a c t i v i t y and unknown s p e c i f i c a c t i v i t y o f the t y r o s i n e f e d . E a r l i e r work by Shimazono  w i t h L. l e p i d e u s showed  (1959)  t h a t c a r b o x y l - l a b e l l e d methyl ja-coumarate was methyl p_-methoxycinnamate w i t h o u t  converted  s c r a m b l i n g o f the  The known t r a n s f o r m a t i o n s o f cinnamyl  to  label.  compounds i n t h i s  organism are summarized i n F i g . 7.(Towers  1969).  14  In L. l e p i d e u s , t y r o s i n e phenylacetic acid  C i s c o n v e r t e d t o p_-hydroxy-  (Power e t a l 1 9 6 5 ) w h i l e i n Sporobolomy-  ces roseus £-coumaric a c i d i s formed from L - t y r o s i n e and m-coumaric a c i d i s d e r i v e d from m-hydroxyphenylalanine f e e d ings  (Moore e t a l 1 9 6 8 ) . An i n v e s t i g a t i o n o f L - p h e n y l a l a n i n e metabolism by  Schizophyllum Towers 1 9 6 7 ) .  commune has  r e v e a l e d PAL a c t i v i t y  (Moore and  In a d d i t i o n , p h e n y l p y r u v i c a c i d and  L(-)-0-  p h e n y l l a c t i c a c i d became r a d i o a c t i v e l y l a b e l l e d when D l r 14  phenylalaninegus.  C was  a d m i n i s t e r e d t o c u l t u r e s o f the f u n -  However, the absence o f o t h e r l a b e l l e d  o i d molecules  i n the fungus suggests  phenylpropan-  t h a t , as i n many  14  COOH I H«NCH I CHo  COOH I CH  II  z  CH  OH  (I)  (III)  OCH.  (V)  FIG. 7 . T r a n s f o r m a t i o n s o f Cinnamyl Compounds i n L. l e p i d e u s (I)phenylalanine; (IV)phloretic acid;  (Il)cinnamic acid;  acid;  (VII)caffeic  (Ill)rj-coumaric  (V)rj-methoxycinnamic a c i d ; acid.  acid;  (VI) i s o f e r u l i c  15 Basidiomycetes, the enzymes PAL and TAL f u n c t i o n p r i m a r i l y i n p r o v i d i n g a d e g r a d a t i v e r o u t e t o carbon d i o x i d e . Benzofurans have been i d e n t i f i e d as m e t a b o l i t e s o f p h e n y l a l a n i n e and t y r o s i n e i n Stereum s u b p i l e a t u m e t a l 1971).  In t h i s organism,  (Bu'Lock  the p h e n y l p r o p a n o i d  moiety  t o undergo 8 - c l e a v a g e b e f o r e a c e t y l a t i o n and  appears  p o r a t i o n i n t o the benzofuran m o l e c u l e s .  In Polyporus  incortum-  u l o s u s , the p r i n c i p a l r a d i o a c t i v e p h e n o l i c compounds i d e n 14 t i f i e d a f t e r long-term f e e d i n g o f g l u c o s e - U - C, s h i k i m i c 14 14 a c i d - U - C and a c e t a t e - 1 - C i n d i c a t e d t h a t t y r o s i n e was degraded  v i a p_-hydroxyphenylpyruvic a c i d t o carbon d i o x i d e  (Crowden 1967).  No o t h e r p h e n o l i c p h e n y l p r o p a n o i d molecules  were o b s e r v e d . R e c e n t l y , i n our l a b o r a t o r i e s , a survey was  made o f  f l a v o n o i d and p h e n o l i c a c i d p r o d u c t i o n i n t h i r t y - o n e f e r e n t s p e c i e s o f Basidiomycetes p e r s o n a l communication).  dif-  (Saleh,Bandoni and Towers,  These organisms were grown i n Roux  b o t t l e s on a l i q u i d medium c o n t a i n i n g g l u c o s e as the major carbon source and were examined a f t e r two weeks i n c u b a t i o n a t 23°C.  F l a v o n o i d compounds were not d e t e c t e d i n any o f  the c u l t u r e s examined, w h i l e o - h y d r o x y p h e n y l a c e t i c a c i d was  produced by Fomes subroseus and P o r i a w e i r i i .  c u l t u r e s o f Polyporus f i b r i l l o s u s and P o r i a xantha caffeic  acid.  In a d d i t i o n / produced  16 IV. Pigment p r o d u c t i o n i n f u n g i Great v a r i a t i o n s i n the e f f e c t s o f pH, C/N  ratio,  n u t r i e n t s and  temperature,  l i g h t on pigment p r o d u c t i o n  found among d i f f e r e n t f u n g i .  These v a r i a t i o n s w i l l depend  on the nature o f the pigment and on the organism Carlile it  (1965) has  are  reviewed the p h o t o b i o l o g y  itself.  o f f u n g i , and  i s apparent t h a t the q u a l i t a t i v e e f f e c t o f l i g h t on  p r o d u c t i o n o f many d i f f e r e n t pigments may  the  be o f c o n s i d e r a b l e  importance. L i g h t s t i m u l a t i o n o f c a r o t e n o i d s y n t h e s i s has r e p o r t e d f o r Pyronema c o n f l u e n s Phycomyces b l a k e s l e e a n u s crassa  been  ( C a r l i l e and F r i e n d 1956),  (Garton e t a l 1951), Neurospora  (Zalokar 19 54) and Dacryopinax s p a t h u l a r i a ( V a i l  L i l l y 1968). fungi.  I t i s a l s o observed  i n o t h e r Dacrymycetaceous  However, l i g h t appears t o i n h i b i t  c a r o t e n o i d s i n B l a s t o c l a d i e l l a emersonii s t e i n 1956).  the s y n t h e s i s o f (Cantino and  Horen-  S i m i l a r l y , l i g h t has been shown t o s t i m u l a t e  melanin p r o d u c t i o n i n Aureobasidium p u l l u l a n s (Lingappa a l 1963)  and  and  inhibit  o f Neurospora c r a s s a spora p r o v i d e d an  et  the p r o d u c t i o n o f melanin i n a mutant ( S c h a e f f e r 1953). The mutant o f Neuro-  example i n which l i g h t s t i m u l a t e d c a r o -  t e n o i d s y n t h e s i s but i n h i b i t e d melanin p r o d u c t i o n . The major e f f e c t s o f l i g h t may photooxidation.  be a t t r i b u t a b l e t o  The p h o t o o x i d a t i o n o f one  pigment t o  another  1 7  has  been r e p o r t e d f o r P e n i c i l l i u m h e r q u e i  ter  1958).  The e x t i n c t i o n o f p h o t o i n d u c t i o n o f c a r o t e n o i d  synthesis by reducing substances hydrogen peroxide aqueductium it  for light  (Thiemer  and t h e s u b s t i t u t i o n o f  has been demonstrated  a n d Rau 1970).  pigment and p r e c u r s o r formation, light  effecting sueda  affects  i n Fusarium  In Cercosporina  has been demonstrated t h a t l i g h t  that  (Reidhart and Por-  i s necessary  and i t has been  kikuchii  f o r both suggested  t h e e n t i r e m e t a b o l i c pathway r a t h e r  conversion a t the l e v e l  than  o f t h e m e t a b o l i t e (Mat-  1969). In  some c a s e s  wavelengths o f l i g h t  i t has been demonstrated t h a t a r e more e f f e c t i v e  than  certain  others with r e -  spect  t o pigment p r o d u c t i o n .  360 -  380 nm i n s t i m u l a t i n g c a r o t e n o i d s y n t h e s i s i n P h y c o -  myces  (Delbruck  (Page 1965)  The e f f e c t i v e n e s s o f l i g h t  and S h r o p s h i r e  indicates  1960)  that a flavin  and P i l o b o l u s k l e i n i i o r f l a v o p r o t e i n may b e  t h e p h o t o s e n s i t i v e compound i n t h e s e V. S p o r o p h o r e  near  fungi.  formation i n Basidiomycetes  Basidiocarp production i n cultures o f wood-rotting Basidiomycetes  h a s b e e n i n v e s t i g a t e d many t i m e s  B a d c o c k 1943, Lohwag 1952, N o b l e s 1958).  In addition,  factors  affecting  out w i t h  more d e t a i l e d  sporophore  some B a s i d i o m y c e t e s .  (notably,  1948, T a m b l y n a n d D a c o s t a s t u d i e s o f morphogenetic  f o r m a t i o n have been A review  by Taber  carried (1966)  18 o u t l i n e s the s t u d i e s d e t e r m i n i n g the r o l e s o f l i g h t , a t u r e , pH, humidity, gases and v a r i o u s s u b s t r a t e uents.  temper-  constit-  I t has been suggested t h a t r e p r o d u c t i o n i s i n i t -  i a t e d by f a c t o r s t h a t check the growth o f an e s t a b l i s h e d mycelium w i t h o u t d r a s t i c a l l y p o i s o n i n g i t s metabolism rane 1958). er  (Coch-  I n g e n e r a l , r e p r o d u c t i o n occurs over a narrow-  range o f e n v i r o n m e n t a l c o n d i t i o n s than does growth, b u t  few o t h e r g e n e r a l i t i e s can be made c o n c e r n i n g s p o r o c a r p production i n Basidiomycetes. Cochrane ces  (1958) d e s c r i b e s the t h r e e p r i c i p a l respon-  t o l i g h t as i n d u c t i v e , i n h i b i t o r y and t r o p i c .  The r e -  quirement o f l i g h t f o r the i n d u c t i o n o f b a s i d i o c a r p s i n many Basidiomycetes i s w e l l e s t a b l i s h e d the  (Taber 1966), b u t  amount o f l i g h t , e f f e c t i v e wavelengths  ulus required are v a r i a b l e .  and time o f s t i m -  While Polyporus v e r s i c o l o r  r e q u i r e s 1000 l u x p e r day f o r s p o r o c a r p p r o d u c t i o n t o o c c u r (Koch 1958), P o r i a ambigua w i l l r e t a i n a l i g h t s t i m u l u s i n i t s mycelium  and form b a s i d i o c a r p s i n subsequent  (Robbins and Hervey 1960). ite  darkness  In some f u n g i , darkness i s r e q u i s -  f o r s p o r u l a t i o n t o o c c u r ( L i l l y and B a r n e t t 1951).  Research i n d i c a t e s t h a t the e f f e c t i v e wavelengths  f o r bas-  i d i o c a r p i n d u c t i o n a r e i n the b l u e r e g i o n o f the v i s i b l e spectrum  (Cochrane 1958).  While s h o r t wavelength  ultravi-  o l e t l i g h t shows i n h i b i t o r y o r l e t h a l e f f e c t s , near  ultra-  19 violet  i s often stimulatory to reproduction  P o s i t i v e phototropism reproduction. aJJLs,  i s a l s o important  Plunkett  (Leach  1962).  i n Basidiomycete  (1958)/ working w i t h Polyporus  brum-  found t h a t under normal c o n d i t i o n s , response t o l i g h t  b r i n g s the s t i p e t i p i n t o the a i r .  The s t i p e i s both pos-  i t i v e l y p h o t o t r o p i c and n e g a t i v e l y g e o t r o p i c , and when both s t i m u l i are present,  the p h o t o t r o p i c s t i m u l u s  takes  preced-  ence . In g e n e r a l , the temperature range p e r m i t t i n g d u c t i o n i s narrower than t h a t f o r v e g e t a t i v e growth.  reproWhile  e x p l o r i n g the r e l a t i o n s h i p o f temperature t o s p o r u l a t i o n in non-light s e n s i t i v e fungi i s r e l a t i v e l y  straighforward,  d e f i n i n g the r e l a t i o n s h i p o f l i g h t t o s p o r u l a t i o n and the i n t e r a c t i o n o f l i g h t and temperature i s c o n s i d e r a b l y more complex  (Leach  will partially formation  1967).  In some cases h i g h e r  temperatures  r e p l a c e a l i g h t requirement f o b a s i d i o c a r p  (Carlile  1965).  I t i s commonly observed t h a t b a s i d i o c a r p i s reduced i n c l o s e d c o n t a i n e r s  (Badcock 1943).  formation Although  t h i s may, i n p a r t , be caused by oxygen d e f i c i e n c y , the a c cumulation  o f carbon d i o x i d e and o t h e r v o l a t i l e  ucts i s a l s o involved.  Levels of v o l a t i l e s  waste p r o d -  tolerated or  r e q u i r e d v a r i e s c o n s i d e r a b l y and l i t t l e q u a n t i t a t i v e work has  been done.  Although the pH range f o r b a s i d i o c a r p f o r -  20 m a t i o n may b e n a r r o w e r t h a n can  be made.  tant  f o r growth,  There i s evidence,  to sporulation  i n some  i s most l i k e l y  celium low  exhausts  tion  nutrients  i n some  t o o c c u r when a v i g o r o u s l y  growing  (Klebs  levels  1900).  kett  1953).  In addition,  In  tion.  nitrogen  tle  source and i t s  indicates that  sive quantities w i l l duction  will  t h a n on v e g e t a t i v e  much g r e a t e r growth  sporula-  there  dif-  is lit-  e x i s t i n these r e -  i n limiting e f f e c t s on  (Hawker  t h i s op-  undoubtedly have  growth and r e p r o d u c t i o n .  c e r t a i n substances exert  i s obvious.  influence  qualitative differences  f o r vegetative  require-  concentration  and m i c r o e l e m e n t r e q u i r e m e n t s ,  evidence that  (Plun-  and i n p a r t i c u l a r , t h e  Although d i f f e r e n t Basidiomycetes  quirements also  to occur  a r e concerned,  with different conditions,  ferent vitamin  nutrient  of different nutrient  carbon source and i t s c o n c e n t r a t i o n timum.  Basidiomycetes,  l e v e l s tend t o suppress  The optimum n i t r o g e n  may v a r y  large  f o r sporulation  The s p e c i f i c i t y  high  concentra-  i s more d e c i s i v e t h a n t h a t o f  ments i n s o f a r a s d i f f e r e n t s p e c i e s In g e n e r a l ,  the  mass o f t h e s p o r o c a r p , h i g h e r  must be m a i n t a i n e d  my-  o r i s t r a n s f e r r e d t o a medium  (Cochrane 1958).  owing t o t h e g r e a t e r  t h a t pH i s i m p o r -  reproduction  o f the carbon source  other  that  i t s nutrients,  i n nutrients  however,  fungi.  E a r l y workers r e p o r t e d fungi  few g e n e r a l i z a t i o n s  1966).  Evidence o r excesrepro-  CHAPTER  ONE  CULTURAL STUDIES OF POLYPORUS HISPIDUS  21  Introduction A t t h e commencement o f t h i s study, h i s p i d i n had been i s o l a t e d o n l y from Polyporus h i s p i d u s and P. s c h w e i n i t z i i and  Bu'Lock  (1967) had r e p o r t e d t h a t w h i l e  P. s c h w e i n i t z i i  would produce h i s p i d i n on u s u a l c u l t u r e media, i t was necess a r y t o grow P. h i s p i d u s on wood b l o c k s h i s p i d i n production.  Therefore,  i n order t o achieve  before h i s p i d i n biosynthesis  c o u l d be i n v e s t i g a t e d i t was n e c e s s a r y t o e s t a b l i s h c u l t u r i n g c o n d i t i o n s i n which j?. h i s p i d u s would produce r e a d i l y i s o l a b l e q u a n t i t i e s o f h i s p i d i n on a l i q u i d medium. Initially,  comparisons o f growth and pigment  formation  a l s o were made w i t h an i s o l a t e o f P. s c h w e i n i t z i i . The growth o f t h e two s p e c i e s  (dry weight) was compared on sev-  e r a l d i f f e r e n t media and t h e i r pigment p r o d u c t i o n was comp a r e d on a n u t r i e n t - r i c h u n d e f i n e d production  medium by m o n i t o r i n g the  o f m y c e l i a l c o n s t i t u e n t s w i t h absorbance a t 370 nm.  A f t e r e v a l u a t i n g the comparative study, i t was  decided  t h a t o n l y P_. h i s p i d u s would be examined f u r t h e r i n c u l t u r a l investigations.  In these,  the e f f e c t s o f v a r i o u s c o n s t i t -  uents o f the medium and o f d i f f e r e n t e n v i r o n m e n t a l on growth and pigment p r o d u c t i o n way,  a medium  and l i g h t  were r e c o r d e d .  and temperature  conditions  In t h i s  conditions  22  a p p r o p r i a t e t o b i o c h e m i c a l s t u d i e s were e s t a b l i s h e d .  In  view o f the n a t u r a l o c c u r r e n c e o f h i s p i d i n i n the sporophores o f the fungus, the p r o d u c t i o n o f sporophores i n c u l t u r e  was  examined a l s o . M a t e r i a l s and Methods I . Sources o f C u l t u r e s ? The c u l t u r e o f Polyporus h i s p i d u s was Dr. M.K.  Nobles o f the Mycology S e c t i o n , C e n t r a l  a l Farm, Ottawa.  T h i s c u l t u r e , F2037, was  C u l t u r e C o l l e c t i o n number UBC itzii  o b t a i n e d from  (UBC 686) was  513.  Experiment-  g i v e n the  UBC  The c u l t u r e o f P.  schwein'  i s o l a t e d from a sporophore c o l l e c t e d  on  the U n i v e r s i t y o f B r i t i s h Columbia Endowment Lands. I I . Sources o f Medium C o n s t i t u e n t s : A l l chemicals employed  i n the media were o b t a i n e d  from F i s h e r S c i e n t i f i c Company o r J . T. Baker Chemical Company and were o f C. P. grade o r were d e s i g n a t e d as meeting ACS  o r USP  y s a t e was  standards.  V i t a m i n - and s a l t - f r e e c a s e i n h y d r o l -  o b t a i n e d from N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n ,  C l e v e l a n d , Ohio and agar was couver, B.C.  from K & S L a b o r a t o r i e s , Van-  M a l t e x t r a c t , y e a s t e x t r a c t , and  Soytone  (an enzymatic h y d r o l y s a t e o f soybean meal) were o b t a i n e d from D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n . i n the media was  glass-distilled.  A l l water used  23 I I I . Media Employed: A. Malt-Yeast-Soytone  (MYP): malt e x t r a c t 7g, y e a s t  e x t r a c t 0.75g, Soytone l.Og p e r l i t r e o f water. S o l i d medium was prepared  by adding  1.5% agar.  B. M o d i f i c a t i o n s o f Czapek's Medium: KF^PO^ l g , MgS0  4  . 7H 0 0.5g, KC1 0.5g, F e S 0 2  4  . 7H 0 O.Olg 2  p l u s 10 g o f g l u c o s e , s u c r o s e , maltose o r x y l o s e and one o f NaNO^ 2.0g, NH N0 4  1.55g  0.94g,  3  (NH ) S0 4  2  4  o r c a s e i n h y d r o l y s a t e 2.0g p e r l i t r e o f  water C. D e f i n e d Medium #1 (DM#1): K H P 0 2  0.25g, NaCl 0.05g, C a C l 10g,  2  4  l.Og, MgS0  4  . 7H 0 2  . 2H 0 0.2g, g l u c o s e 2  c a s e i n h y d r o l y s a t e 2.0g and water 1 l i t r e .  D. Four V i t a m i n Stock S o l u t i o n (VSS): b i o t i n 0.5 mg, thiamine  10 mg, p y r i d o x i n e 10 mg, i n o s i t o l 0.5g  p e r 100 ml o f 20% ( v o l . ) aqueous e t h a n o l .  The  s t o c k s o l u t i o n was employed a t a c o n c e n t r a t i o n o f 1 ml p e r l i t r e o f medium. E. G l u c o s e - Y e a s t - S o y t o n e - S a l t s  (GYSS): g l u c o s e 15g,  y e a s t e x t r a c t 0.5g, soytone l.Og,  s a l t stock  sol-  u t i o n 10 ml and water 1 l i t r e . F. S a l t Stock S o l u t i o n : K H P 0 2  NaCl 0.5g, C a C l  4  5g, MgSC>  4  „ 7H 0 2.5g, 2  . 2H O 0.2g and 100 ml o f water.  24  IV. C u l t u r i n g Liquid Each b o t t l e pipetting The  Techniques: c u l t u r i n g was  c o n t a i n e d 100  ml  Roux  o f medium a n d was the  bottles.  inoculated  ml o f s t e r i l e  w e r e m a i n t a i n e d on MYP  were p r e p a r e d by p i p e t t i n g e a c h s t a n d a r d 10 cm  Petri  o f a g a r medium.  d i s t i l l e d water.  agar, and  these  i n o c u l u m was  cultures  1 m l o f t h e homogenate  into  dish containing approximately  Agar  phal tips  a 2 mm  cultures  f o r studies  involving center.  cube c u t from t h e a g a r n e a r t h e  o f two-week-old c o l o n i e s .  mycelium-side  MYP The  c o l o n y d i a m e t e r m e a s u r e m e n t s were i n o c u l a t e d n e a r t h e The  by  bottle.  p r e p a r e d by b l e n d i n g a two-week-old  a g a r c u l t u r e w i t h 250  25 m l  using  10 m l o f c u l t u r e homogenate i n t o  homogenate was  organisms  performed  down o n t h e new  agar  The  cubes were  hy-  placed  surface.  V. M e a s u r e m e n t s o f H i s p i d i n P r o d u c t i o n : Hispidin, 370 ium.  nm  which has  i n alcoholic  The  a strong absorbance  solution,  medium and m y c e l i u m  was  extracted  mycelium  ding  diethyl  -  was  -  removed i n vacuo  95% e t h a n o l . was  employed An  and  and  the r e s i d u e  f o r the o p t i c a l  density  a l t e r n a t i v e p r o c e d u r e was  mycelHC1,  e x t r a c t e d by ether.  t a k e n up  A U n i c a m SP.800 r e c o r d i n g  near  from the  were . . a c i d i f i e d w i t h 6 N  t h e medium f i l t e r e d o f f , a n d ~ t h e i n a mortar w i t h s i l i c a  peak  The  grin-  ether  i n 5 ml  of  spectrophotometer  measurements. employed  f o r the  25  quantitative hispidus.  d e t e r m i n a t i o n o f h i s p i d i n p r o d u c t i o n i n P.  The medium was  removed b y  c e l i u m washed w i t h d i s t i l l e d The  frozen  f r o z e n mycelium  was  then l y o p h i l i z e d  the  dry weight o f t i s s u e  A  volume and banded  plates.  ^  n  a  the band  was  p e r f o r m e d by powdering  the  powder i n a 1 cm  The  tinction  column  thin  system  and  Elution  methanol.  s p e c t r u m o f t h e r e c o v e r e d m a t e r i a l was  coefficient  a t 366  nm  was  con-  placing  eluted with  to that of hispidin,  layer  (5/4/1 by v o l . )  eluted.  t h e r e c o v e r e d band w h i c h was  reduced  cellulose  formic acid  The  methanol  e x t r a c t was  c o r r e s p o n d i n g t o h i s p i d i n was  correspond c l o s e l y  of  ortar.  o n 500 m i c r o n A v i c e l  and  to  m  of toluene: ethylformate:  The u l t r a v i o l e t  and  determined before e x t r a c t i o n .  The p l a t e s w e r e d e v e l o p e d i n a s o l v e n t  sisting  my-  i n an a c e t o n e -  then exhaustively extracted with  by g r i n d i n g w i t h l 2 ° 3 in  the  and  i c e bath.  was  and  water  dry  d r i e d mycelium  filtration  and  observed  the Molar  used t o c a l c u l a t e  ex-  the  yield  hispidin.  VI.  Dry Weight D e t e r m i n a t i o n s : The medium was  removed from t h e m y c e l i u m  t h r o u g h c h e e s e c l o t h a n d w a s h i n g w i t h 100 m l If  the mycelium  after  were t o be  examined  for phenolic  o t h e r w i s e d r y i n g was  filtering  of d i s t i l l e d  d r y w e i g h t measurement t h e m a t e r i a l was  d e s c r i b e d above,  by  water.  compounds  lyophilized  performed  as  i n an oven  o 90 C.  A l l w e i g h i n g s were p e r f o r m e d a t room t e m p e r a t u r e  humidity.  and  at  26 Results I.  and D i s c u s s i o n  Comparison o f Polyporus The  at  h i s p i d u s a n d P. s c h w e i n i t z i i  g r o w t h o f P. h i s p i d u s a n d P. s c h w e i n i t z i i  2 2 - 2 4 ° C i n d a r k c u p b o a r d s were c o m p a r e d  u r e m e n t s o f t h e d r y w e i g h t p e r Roux b o t t l e cated  that while total  pidus .  I t was o b s e r v e d  lier  was s u s p e c t e d  extracts for  optical  than value  t h a t t h e y e l l o w c o l o u r a t i o n was  obtained  and as a crude  are presented  These measurements  (Fig. 9.).  f o r P. s c h w e i n i t z i i ,  fifty  The v a l u e s times  although  smaller  a maximum  f o r P. h i s p i d u s a f t e r  21 d a y s .  The maximum  b a n c e a t 370 nm f o r c u l t u r e s o f P. s c h w e i n i t z i i was  absorobtained  t e n days o f growth. The  pected  assess-  o f 0.6 a b s o r b a n c e u n i t s p e r gram o f d r y m y c e l i u m was  recorded  after  asso-  d e n s i t y m e a s u r e m e n t s a t 370 nm o f e t h e r  f o r P. h i s p i d u s were more t h a n  those  ear-  c u l t u r e s o f P. h i s p i d u s .  o f t h e m y c e l i u m were r e c o r d e d .  P. s c h w e i n i t z i i  obtained  a  and subsequent browning  t h e development o f h i s p i d i n ,  ment o f t h i s ,  indi-  a l s o t h a t t h e c u l t u r e s o f P. s c h w e i n -  a n d more s t r o n g l y t h a n  ciated with  (triplicate)  d r y w e i g h t o f m y c e l i u m was p r o d u c e d b y P. h i s -  showed y e l l o w p i g m e n t a t i o n  It  ( F i g . 8 . ) . Meas-  P. s c h w e i n i t z i i m a t u r e d more r a p i d l y ,  greater  itzii  o n MYP  optical  d e n s i t y m e a s u r e m e n t s r e p o r t e d c a n be e x -  o n l y t o g i v e an i n d i c a t i o n  since other acidic,  ether  of styrylpyrone synthesis  s o l u b l e compounds may a b s o r b  at this  27  wavelength.  However, the g r e a t d i f f e r e n c e i n o p t i c a l den-  s i t y readings  between P_. s c h w e i n i t z i i and  P. h i s p i d u s  a b l y confirms  the evidence  t h a t P.  itzii  (Bu Lock 1967) 1  schwein-  i s capable o f much b e t t e r h i s p i d i n p r o d u c t i o n  t u r e than i s P. h i s p i d u s .  prob-  in cul-  A l s o , under these c u l t u r a l con-  d i t i o n s , maximum h i s p i d i n p r o d u c t i o n might be expected l i e r i n P. s c h w e i n i t z i i (10-11 days) than i n P. (ca. 21 d a y s ) .  The  measurements and mycelium  observed decrease i n o p t i c a l  ear-  hispidus density  concommitant browning o f the medium and  i n c u l t u r e s o f P. s c h w e i n i t z i i a f t e r  ten days  might be the r e s u l t o f i n c r e a s e d o x i d a t i v e a c t i v i t y .  This  s p e c u l a t i o n i s f u r t h e r s u b s t a n t i a t e d by the decrease i n d r y weight observed a f t e r 18 days  ( F i g . 8.).  Attempts were made t o compare the growth o f the  two  organisms on simple m o d i f i c a t i o n s o f Czapek"s medium employi n g d i f f e r e n t carbon and n i t r o g e n sources d e s c r i b e d i n Mate r i a l s and Methods.  While P. h i s p i d u s grew w e l l on a l l d e f -  i n e d media examined, the dry weights per Roux b o t t l e f o r P. s c h w e i n i t z i i were l e s s than 0.05 I t was  g a f t e r two  weeks.  hoped t h a t the comparative experiments would  h e l p i n e s t a b l i s h i n g c u l t u r i n g c o n d i t i o n s f o r P.  hispidus  t h a t would r e s u l t i n s u i t a b l e h i s p i d i n p r o d u c t i o n . it  soon became apparent t h a t the two  However,  organisms were v e r y  30  d i f f e r e n t i n t h e i r growth.  A l t h o u g h both f u n g i produce  h i s p i d i n , many p h y s i o l o g i c a l d i f f e r e n c e s a r e well-known. P. h i s p i d u s i s a w h i t e - r o t  fungus, one which c h a r a c t e r i s t i c -  a l l y shows s t r o n g l i g n i n decomposition when growing on wood. P_. s c h w e i n i t z i i , on the o t h e r hand, i s a t y p i c a l brown-rot fungus, d e r i v i n g i t s energy l a r g e l y through enzymes.  cellulolytic  I t a l s o has been suggested t h a t h i s p i d i n  biosyn-  t h e s i s i n P. h i s p i d u s may proceed r e a d i l y o n l y i f p h e n y l propanoid  moieties  (Bu'Lock 1967). ment f o r m a t i o n  from l i g n i n  decomposition a r e p r e s e n t  Thus, c o n d i t i o n s most f a v o u r a b l e  for pig-  i n one organism a r e u n l i k e l y t o be e f f e c t i v e  i n the o t h e r . I I . Growth and pigment p r o d u c t i o n I n i t i a l l y , a simple,  i n Polyporus  hispidus  d e f i n e d l i q u i d c u l t u r e medium  was sought f o r the growth o f P_. h i s p i d u s .  The comparative  s t u d i e s w i t h P_. s c h w e i n i t z i i had a l r e a d y shown t h a t P_. h i s pidus would grow w e l l on such a medium.  A d e f i n e d medium  would f a c i l i t a t e the study o f h i s p i d i n b i o s y n t h e s i s by ens u r i n g t h a t , l a r g e l y , o n l y known k i n d s and q u a n t i t i e s o f p h e n o l i c compounds o r p o s s i b l e h i s p i d i n p r e c u r s o r s were being  i n t r o d u c e d i n t o the c u l t u r e medium. The  and  f o u r d i f f e r e n t carbon sources  f o u r d i f f e r e n t n i t r o g e n sources  (filter-sterilized)  t h a t were employed i n  the m o d i f i c a t i o n s o f Czapek's medium were supplemented  31 with  salt  stock  combinations ly  at a fixed  constant  C/N  were r a t e d on of  solution  daily  and  were e x a m i n e d i n a l l  c o n c e n t r a t i o n which maintained  ratio.  Each sugar  and  each n i t r o g e n  the b a s i s o f v i s u a l p i g m e n t a t i o n  t h e m y c e l i a l mat  incubation at  (10 ml/1)  in triplicate  cultures after  23°C i n n o m i n a l d a r k n e s s  f o r i n s p e c t i o n o f the  m a l t o s e were b o t h  (dark  cultures).  good carbon  and  sources  a  source  density  two  weeks  cupboards  While glucose f o r growth,  fair-  opened and  glucose  gave b e t t e r pigment p r o d u c t i o n .  Of  s e i n hydrolysate stimulated both  growth and  pigmentation  glucose,  casein hydroly-  best.  The  s a t e and #1  medium w h i c h c o n t a i n e d  salt  stock  s o l u t i o n was  the n i t r o g e n sources,  designated  as D e f i n e d  ca-  Medium  (DM#1). Since  pigmentation  i t seemed p o s s i b l e t h a t t h e with  c a s e i n h y d r o l y s a t e m i g h t be  to b u f f e r i n g action, (DM#1) was  the  c h a n g e i n pH  examined i n t r i p l i c a t e  p e r i o d o f g r o w t h o f P_. h i s p i d u s . 23°C i n d a r k e n e d c u p b o a r d s . idly  f r o m a b o u t pH  growth o f the stage,  7 t o pH  fungus  however,  enhanced growth  The  The pH  was  week  to f a l l  l o g a r i t h m i c phase  During  f o r the d u r a t i o n o f the  fering  not  and  a five  observed  increased rapidly  apparent,  c u l t u r e medium  c u l t u r e s were grown a t  where i t r e m a i n e d a c t i o n was  attributable  c u l t u r e s over  5 d u r i n g the  ( F i g . 10.).  t h e pH  o f the  and  rapof  the d e c e l e r a t i o n t o a b o u t pH experiment.  the d a t a were  6, Buf-  similar  0  5  10 AGE OF  15  20  CULTURE (Days)  25  30  t o those o f Crewther and  Lennox  (1953) f o r A s p e r g i l l u s  yzae.  In t h e i r experiment, the pH  o f the  t o t a l nitrogen,  but began t o i n c r e a s e gen  ium  phosphate and  and  ( F i g . 10.),  reduction  carbon i n the medium,  a g a i n as the amount o f p r o t e i n  i n the medium i n c r e a s e d .  hispidus  declined with  strong  In my and  or-  nitro-  pH-growth study o f  P.  r a p i d browning o f the med-  mycelium began i n the c u l t u r e s a f t e r f i f t e e n days.  At the same time, the dry weight o f the mycelium showed a n i f i c a n t decrease i n growth r a t e .  The  observations  i n c i d e n t decrease i n dry weight, i n c r e a s e  i n pH and  o f browning are c o n s i s t e n t w i t h the o b s e r v a t i o n and  Lennox  of  sig-  co-  onset  o f Crewther  (1953) t h a t the amount o f p r o t e i n n i t r o g e n  in  the medium i s i n c r e a s i n g , e s p e c i a l l y i f much o f t h i s p r o t e i n i s r e p r e s e n t e d by o x i d a t i v e enzymes.  I t seems t h a t  strong  o x i d a t i v e a c t i v i t y i n the c u l t u r e s s h o u l d be a v o i d e d i n o r der t o a c h i e v e h i g h e r y i e l d s o f h i s p i d i n from the In a second experiment employing DM#1, c u l t u r e s were a d j u s t e d pH  4,  5, 6,  t u r e s had  7 and  p r i o r to a u t o c l a v i n g  8 w i t h 1% ^ °4 S  o  r  %  *  KOH  quadruplicate to approximately A  f  t  e  r  t h e  c  u  l  _  grown f o r s i x t e e n days a t 23°C i n darkened cup-  boards the m y c e l i a l d r y weights and measured  1  2  fungus.  (Table  teen days was  I.).  pH o f the medium were  In every i n s t a n c e  lower than the  i n i t i a l pH.  the pH a f t e r s i x Although  only  s m a l l d i f f e r e n c e s i n m y c e l i a l dry weights were observed i n  34 c u l t u r e s i n i t i a t e d a t pH 4, 5, 6, and 7, the d r y weight mycelium was  of  more than 2 0 % g r e a t e r than these, i n c u l t u r e s  i n i t i a t e d a t pH 8.  Unfortunately, v i s i b l e pigmentation  was  not enhanced i n c u l t u r e s i n i t i a t e d a t pH 8 and browning  was  more n o t i c e a b l e .  TABLE I .  M y c e l i a l d r y weight and pH o f medium o f c u l t u r e s o f P. h i s p i d u s a f t e r s i x t e e n days when grown on DM#1 a t v a r i o u s i n i t i a l pH's.  Initial pH  M y c e l i a l Dry Wt. a f t e r 16 days (g)  pH o f Medium a f t e r 16 days  3~9  0.28  375  4.9  0.30  4.3  6.0  0.29  4.9  7.0  0.31  5.5  8.3  0.38  5.8  Higher dry weights o f mycelium  ( F i g . 8 . ) and more i n -  tense v i s i b l e p i g m e n t a t i o n were observed i n c u l t u r e s employi n g MYP  than were r e c o r d e d f o r growth on DM#1  F u r t h e r c u l t u r i n g s t u d i e s t o determine  ( F i g . 10.) .  the optimum tempera-  t u r e , e f f e c t i v e i l l u m i n a t i o n and the e f f e c t o f o x i d i z i n g and r e d u c i n g substances i n the medium were performed i n g MYP.  employ-  Subsequent m o d i f i c a t i o n o f t h i s medium p e r m i t t e d  the f o r m u l a t i o n o f the medium f o r b i o c h e m i c a l s t u d i e s . P.  h i s p i d u s was  grown on MYP  agar and the c o l o n y  diameters o f f o u r r e p l i c a t e s were measured a f t e r two weeks a t f i v e d i f f e r e n t temperatures mum  temperature  ( F i g . 1 1 . ) . While the o p t i -  f o r i n c r e a s e i n c o l o n y diameter was  near  35  FIG.  11.  Variation  i n colony diameter with  o f P_. h i s p i d u s  cultures  on MYP  temperature  agar.  36 26°C, t h e b r o w n i n g o f t h e c u l t u r e s , idative, and,  w h i c h was p r o b a b l y o x -  was g r e a t l y e n h a n c e d a t t e m p e r a t u r e s a b o v e  therefore,  23°C was c h o s e n f o r f u r t h e r  studies.  The e f f e c t o f t h e w a v e l e n g t h o f t h e l i g h t g r o w t h a n d p i g m e n t d e v e l o p m e n t was i n v e s t i g a t e d tures  o f P. h i s p i d u s .  spectral  from c u l t u r e s day.  A l l but f i l t e r e d  i n c u b a t e d a t 23°C w i t h  w i t h known  to the  was  excluded  G r o w t h was a s s e s s e d b y measurement o f t h e c o l o n y  this  study,  i t i s apparent that  p i g m e n t a t i o n was  growth and pigment development. light  stimulate  cultures  I t seemed p o s s i b l e  light  that  cultures  ly  arrest  further colour  is  probable  that  or  full  I t was a l s o n o t e d o r d a r k n e s s , dev-  24 h when e x p o s e d t o f u l l  light.  so exposed t o darkness d i d n o t immediatedevelopment  i n the mycelium.  p h o t o i n d u c e d enzymes r e m a i n a c t i v e  f o r several  fluorescent  i n both  photo-oxidation  grown f o r t e n d a y s i n r e d l i g h t  Returning  From  growth i s slower under  f o r both observed e f f e c t s .  eloped pigmentation within  mycelium  i s important  dia-  evalu-  (Fig. 12.).  While blue  pigment development,  these conditions. was r e s p o n s i b l e  light  days.  cul-  24 h i l l u m i n a t i o n p e r  a t e d e m p i r i c a l l y on a f i v e - p o i n t s c a l e  the  light  on  i n MYP a g a r  In t h i s study, Wrattan f i l t e r s  meter a f t e r t e n days, and m y c e l i a l  that  source  c h a r a c t e r i s t i c s ( A p p e n d i x A) were f i t t e d  tops o f P e t r i dishes.  23°C  As a r e s u l t o f t h i s  i l l u m i n a t i o n i n the incubators  It  i n the  study was  changed  H  o  g J y  oi—i  1  FULL LIGHT  BLUE  1  r  RED & YELLOW  RED  1 GREEN  COLOUR OF LIGHT RECEIVED  BY CULTURES  DARKNESS  38 from 40 watt C o o l White lamps t o 40 watt D a y l i g h t lamps t o p r o v i d e approximately  twice as much i l l u m i n a t i o n i n the  b l u e r e g i o n o f the v i s i b l e spectrum.  A l s o , i l l u m i n a t i o n was  p r o v i d e d f o r two hours p e r day f o r a l l f u t u r e s t u d i e s . In an attempt t o a s c e r t a i n i f the r o l e o f l i g h t was o x i d a t i v e , s m a l l q u a n t i t i e s o f hydrogen p e r o x i d e  o r hydrox-  ylamine h y d r o c h l o r i d e were added t o MYP agar c u l t u r e s . substances  Both  were added a s e p t i c a l l y a f t e r the medium had been  autoclaved.  Hydrogen p e r o x i d e  caused s l i g h t o x i d a t i v e  browning i n the dark when employed a t a c o n c e n t r a t i o n o f 10  -2 -2 M / l . A t 3*10 M / l no growth o f the fungus o c c u r r e d , _3  w h i l e a t a c o n c e n t r a t i o n o f 10 noticed.  No y e l l o w pigmentation  M / l no o x i d a t i v e e f f e c t was was observed  i n cultures  c o n t a i n i n g hydrogen p e r o x i d e when they remained i n darkness. The  e f f e c t o f hydroxylamine h y d r o c h l o r i d e a t a con_2  c e n t r a t i o n o f 10  M / l was almost n e g l i g i b l e , and the h i g h e r  c o n c e n t r a t i o n s employed produced unusual  or l e t h a l  effects,  and d i d not a p p r e c i a b l y a r r e s t pigment development i n the light. These r e s u l t s i n d i c a t e t h a t hydrogen p e r o x i d e a b l e t o r e p l a c e l i g h t i n pigment p r o d u c t i o n i n t h i s although  o x i d a t i v e e f f e c t s were observed.  i s unfungus,  Similarly,  hydrox-  ylamine h y d r o c h l o r i d e i s unable t o b r i n g about the e x t i n c t i o n o f p h o t o i n d u c t i o n as i t has been r e p o r t e d t o do i n  39 Fusarium  (Thiemer and Rau 1970).  Although p h o t o i n d u c t i o n  of  pigment development i s apparent i n P. h i s p i d u s , i t i s s t i l l u n c e r t a i n t h a t t h i s i s an o x i d a t i v e  process.  Having e s t a b l i s h e d s u i t a b l e i l l u m i n a t i o n c o n d i t i o n s and  temperature f o r growth and pigment p r o d u c t i o n  on MYP,  v a r i o u s m o d i f i c a t i o n s o f t h i s medium were e v a l u a t e d  i n order  t o o b t a i n , i f p o s s i b l e , a b e t t e r medium f o r s t u d y i n g pidin biosynthesis.  his-  V a r i a t i o n s i n the c o n c e n t r a t i o n o f  malt e x t r a c t , y e a s t e x t r a c t and soytone were examined. A l s o , the e f f e c t on c o l o n y diameter and p i g m e n t a t i o n was noted when v a r i o u s combinations o f d e f i n e d n u t r i e n t s were s u b s t i t uted  f o r these three undefined  medium  constituents.  T r i p l i c a t e agar c u l t u r e s o f P_. h i s p i d u s were i n c u bated f o r f i f t e e n days on a medium c o n t a i n i n g y e a s t  extract  0.5 g/1, Soytone 1.0 g/1 and v a r i a b l e amounts o f malt e x t r a c t . Increases  i n malt e x t r a c t c o n c e n t r a t i o n beyond 7 g/1 e f f e c t e d  much s m a l l e r r e l a t i v e i n c r e a s e s i n c o l o n y the same i n c r e a s e s concentrations  diameter than d i d  i n malt e x t r a c t below 7 g/1  ( F i g . 1 3 ) . However,  o f malt e x t r a c t o f 14 - 15 g/1 r e s u l t e d i n  much s t r o n g e r y e l l o w p i g m e n t a t i o n o f the mycelium than d i d lower  concentrations. In a s i m i l a r experiment, the c o n c e n t r a t i o n s  o f yeast  e x t r a c t , malt e x t r a c t and Soytone were v a r i e d , and i n some c u l t u r e s , d e f i n e d n u t r i e n t s were s u b s t i t u t e d f o r one o r more o f these i n g r e d i e n t s .  E x c l u s i o n o f y e a s t e x t r a c t had l i t t l e  40  4 0 -  CONCENTRATION OF MALT EXTRACT (g/l) FIG.  13. E f f e c t  o f malt e x t r a c t  and Soytone  on c o l o n y d i a m e t e r a f t e r  fifteen  concentration  days.  41 apparent extract pure  effect  on c o l o n y d i a m e t e r so l o n g as e i t h e r  o r Soytone  was  p r e s e n t i n t h e medium.  sugars and v i t a m i n - f r e e e x t r a c t and Soytone,  resulted  i n a further  yeast  less  1.0 g/1 o f S o y t o n e  1.0 g/1 S o y t o n e  pigmentation a f t e r  15 d a y s  the e f f e c t  fifteen  yeast  extract  tract  14 g/1 o r m a l t o s e  mm  d a y s was  0.5 g/1, S o y t o n e  s m a l l e r than  The a g a r medium  stock  Of t h e s e c o m b i n a t i o n s ,  extract  was  stock  the r e p l a selected  alone, the pigmentation  considered beneficial  ex-  solution  the colony diameter  i n t e n s e w i t h o u t showing browning  extract  contained  1.0 g/1 a n d e i t h e r m a l t  Although  those  extract.  o b s e r v e d when m a l t  cement o f m a l t e x t r a c t w i t h g l u c o s e p l u s s a l t s as t h e most d e s i r e a b l e .  Colony  10 g/1, g l u c o s e 4 g/1 a n d s a l t  10 m l / 1 o r g l u c o s e 14 g / 1 .  smaller than with malt  0.5  on c o l o n y d i a m e t e r and  10 m l / 1 o r g l u c o s e 14 g/1 a n d s a l t  s o u r c e was  either  (Fig. 13.).  were 1 - 5  replaced with other nutrients.  solution  containing  a t a l l concentrations o f malt  I n one e x p e r i m e n t ,  more  of  a t various concentrations o f malt  d i a m e t e r s a t 0.5 g/1 S o y t o n e  was  diameter.  extract.  e x t r a c t were r e c o r d e d a f t e r  at  i n colony  extract  i n t e n s e i n the absence  Colony diameters o f cultures or  to replace  the exclusion o f yeast  25% r e d u c t i o n  M y c e l i a l p i g m e n t a t i o n was  However, when  amino a c i d s were u s e d  the malt  malt  and t h e d e f i n e d  was was carbon  f o r biochemical studies.  42 In MYP,  malt e x t r a c t i s c l e a r l y the major source o f  n u t r i t i o n a l carfcxm, and when p r e s e n t i n g r e a t e r than  7-10  g/1, o t h e r components o f the medium become more l i m i t i n g than the carbon supply f o r growth as expressed by the c o l o n y diameter.  Components o f malt e x t r a c t which enhance pigment-  a t i o n o f the mycelium c o n t i n u e t o do so beyond c o n c e n t r a t i o n s o f 10 g/1 o f malt e x t r a c t . Yeast e x t r a c t i s r i c h i n amino a c i d s and low molecul a r weight pep tidies and a l s o s u p p l i e s a number o f s o l uble B vitamins. i n the absence undoubtedly  Although P. h i s p i d u s grows w e l l on  DM#1  of y e a s t e x t r a c t , t h i s complex v i t a m i n source  enhances growth and pigment development.  Soy-  tone s u p p l i e s carbohydrates i n a d d i t i o n t o v i t a m i n s , p r o t e i n s and amino a c i d s .  Because the replacement o f Soytone  c a s e i n h y d r o l y s a t e i n MYP  with  r e s u l t s i n a 50% loss i n colony  diameter, i t seems p r o b a b l e t h a t Soytone t a n t v i t a m i n s o r c o f a c t o r s t o the medium.  i s s u p p l y i n g imporSoytone  concen-  t r a t i o n a f f e c t e d pigment p r o d u c t i o n v a r i o u s l y w i t h d i f f e r e n t l e v e l s o f malt e x t r a c t and d i f f e r e n t carbon s o u r c e s , augmenting pigmentation i n one i n s t a n c e , and d i m i n i s h i n g i t i n another. C/N  These e f f e c t s d i d not appear  r a t i o s and an e x p l a n a t i o n was  not  to c o r r e l a t e with  found.  The use o f g l u c o s e p l u s s a l t s f o r replacement o f malt e x t r a c t o r as employed i n DM#1  had the d e s i r e a b l e e f f e c t o f  43  augmenting the y e l l o w c o l o r a t i o n enhancing uation,  the browning  o f the mycelium  activity.  t h e medium, GYSS, was  With  without  respect to this  developed  eval-  for biochemical stud-  ies . In o r d e r to s u b s t a n t i a t e the v i s u a l ment p r o d u c t i o n and on  t o e v a l u a t e the growth o f the  the newly-developed  forty  day  hispidin  for five  a s s a y was  ( F i g . 14.).  different  performed  m e a s u r e m e n t s were made a f t e r H i s p i d i n p r o d u c t i o n was trophotometry The duction five was  sets  The  on o n l y two  by  b e t w e e n 0.5  the  weights and  t h e maximum r a t e of h i s p i d i n  maximum d r y w e i g h t and  0.6  g after  idly  o f the mycelium.  first after  The  t e n days o f g r o w t h . this  Dry  weight  lyophilization.  21  a t t a i n e d p e r Roux  ( F i g . 15.)  bottle  days i n c u b a t i o n .  a t 24  The  approximately days.  mycelium remained Yellow coloration  w i t h browning  pro-  approximately  The  for hispidin production correlate well with visual  the  the  technique described.  p e r gram o f d r y m y c e l i u m o r 0.5%  vations  a  chromatographic-spec-  maximum h i s p i d i n p r o d u c t i o n r e c o r d e d was mg  dry  of these.  oven-drying or  assayed  d a t a show t h a t  The  fungus  of cultures  l a g g e d t h e maximum r a t e o f g r o w t h b y  days.  pig-  GYSS, c u l t u r e s were e x a m i n e d o v e r  incubation period  were a v e r a g e d  evidence o f  5  data  obser-  white  during  developed  appearing slowly  rap-  'SSAO uo snpTdsxu;  "a jo u o x ^ o n p o a d  DRY  uTpxdsTu; put? q ^ o a o  WEIGHT  co  HISPIDIN PRODUCTION  'pj ' O I J  PER BOTTLE ( g )  A  cji  (mg/g dry wt)  J  45 a f t e r two weeks.  A f t e r t h r e e weeks o f growth the c u l t u r e s  began t o darken more r a p i d l y but by f o u r weeks l i t t l e change was noted.  I t i s p r o b a b l e t h a t the s t r o n g  o x i d a s e a c t i v i t y which r e s u l t e d i n d a r k e n i n g  further  catechol  o f the  fruit-  body and p o l y m e r i z a t i o n and c e l l - w a l l - b i n d i n g o f the phenols  (Bu Lock and Smith 1961) 1  o f the fungus.  i s a l s o p r e s e n t i n the c u l t u r e s  T h i s would account f o r the sharp decrease  i n e x t r a c t a b l e h i s p i d i n and the s t r o n g browning observed i n c u l t u r e s o f the fungus a f t e r 24 days.  I I I . Sporophore Formation i n Agar C u l t u r e s o f P. h i s p i d u s Sporophore week-old  i n i t i a t i o n had been observed i n 3 - 4  agar c u l t u r e s grown f o r p r e v i o u s experiments.  employing GYSS and MYP  By  with various i n o c u l a t i o n techniques  and i n c u b a t i o n c o n d i t i o n s , sporophore f o r m a t i o n was  a-  chieved. I n o c u l a t i o n was from 2-week-old MYP  made w i t h 2mm  agar cubes  transferred  agar c u l t u r e s o r w i t h one ml o f the homo-  genate p r e p a r e d by b l e n d i n g a two-week-old MYP w i t h 100 ml o f s t e r i l e d i s t i l l e d water.  In one  agar c u l t u r e experiment,  1, 2, 3, 5 o r 10 agar cubes were e v e n l y spaced mycelium-side down on the agar s u r f a c e o f GYSS and MYP.  In one s e t , these  agar cubes were taken from the r e g i o n o f h y p h a l t i p s o f the inoculum c u l t u r e and i n another they were taken from about 1 cm away from the c e n t e r o f the c u l t u r e .  In the second  FIG.  16.  S p o r o c a r p o f P. on MYP  hispidus agar.  t e n days  after  initiation  47  experiment  1 ml o f homogenate was e v e n l y spread on the s u r -  f a c e o f MYP and GYSS agar.  C u l t u r e s i n both  experiments  were i n c u b a t e d under each o f t h r e e d i f f e r e n t  conditions:  20°C w i t h 8 h r i l l u m i n a t i o n p e r day from C o o l White e s c e n t lamps;  fluor-  o o 23 C w i t h the same i l l u m i n a t i o n and 22 - 24 C  w i t h f l u o r e s c e n t room l i g h t i n g  (ambient).  B a s i d i o c a r p i n i t i a t i o n w i t h f o r m a t i o n o f b a s i d i a and viable  b a s i d i o s p o r e s was observed t h r e e times i n these  cultures.  T h i s o c c u r r e d on both media under ambient  con-  d i t i o n s , where the inoculum was 1 o r 2 agar cubes, and the c u l t u r e age a t the onset o f s p o r u l a t i o n was 3 - 4 The appearance  o f a t e n - d a y - o l d s p o r o c a r p which  on MYP agar i s shown i n F i g . 16.  weeks. developed  Spores from t h i s  basidio-  c a r p germinated w i t h o u t treatment a f t e r s i x months on MYP agar i n s e a l e d P e t r i d i s h e s .  However, w i t h i n t h r e e days  o f germ tube i n i t i a t i o n the agar on which the spores germinated showed s i g n s o f r a p i d l y d r y i n g and i t was suspected t h a t slow d e s i c c a t i o n t o g e t h e r w i t h a c r i t i c a l water t e n s i o n o f the s u b s t r a t e were important i n spore g e r m i n a t i o n . When o l d mycelium  o r homogenate was — employed as i n -  oculum f o r the c u l t u r e s , o x i d a t i v e browning was observed much e a r l i e r than i n c u l t u r e s i n o c u l a t e d w i t h m y c e l i a l  tips.  Sporocarp f o r m a t i o n took p l a c e o n l y i n c u l t u r e s which r e mained l i g h t - c o l o u r e d u n t i l almost t h r e e weeks  incubation.  48 The u l t r a v i o l e t  a b s o r b a n c e s p e c t r u m o f an e t h a n o l i c s o l u t i o n  of a d i e t h y l ether clearly fairly tural are  showed  the presence o f h i s p i d i n ,  low c a t e c h o l conditions  also  formation  extract o f a six-week-old  oxidase  activity.  that are favourable  favourable  probably  enzymes.  indicating  I t appears t h a t t o sporophore  to h i s p i d i n biosynthesis  of oxidative  basidiocarp  cul-  formation  and r e t a r d t h e  CHAPTER TWO  CHEMICAL STUDIES OF PHENOLIC ACIDS, STYRYLPYRONES AND RELATED COMPOUNDS  49  Introduction The s y n t h e s i s o f h i s p i d i n and r e l a t e d s t y r y l p y r o n e s has been performed many times by d i f f e r e n t workers. and h i s coworkers  (1914 - 1933)  Borsche  prepared yangonin and s e v e r -  a l o t h e r s t y r y l p y r o n e s i n t h e i r s t u d i e s on the components o f the r o o t s o f P i p e r methysticum,  however, they a s s i g n e d  a gamma-pyrone s t r u c t u r e t o the m o l e c u l e . c h e m i s t r y o f s t y r y l p y r o n e s Macierewicz  In a study o f the  (1939,  1950)  dated the c o r r e c t a-pyrone s t r u c t u r e f o r yangonin, hydrokawain  and a number o f o t h e r r e l a t e d pyrones.  r e c e n t l y , employing onin 1961)  (Bu'Lock  5,6-deMore  u n e q u i v o c a l s y n t h e t i c p r o c e d u r e s , yang-  and Smith 1960), h i s p i d i n  (Edwards and W i l s o n  and twenty-three analogues o f h i s p i d i n  M i r 1967)  eluci-  (Edwards and  have been s y n t h e s i z e d .  To f a c i l i t a t e the i n v e s t i g a t i o n o f h i s p i d i n b i o s y n t h e s i s i n P. h i s p i d u s , the c h e m i s t r y o f a number o f pheno l i c compounds has been examined.  Also, t r i m e t h y l h i s p i d i n  and r e l a t e d molecules were s y n t h e s i z e d f o r comparative  pur-  poses and f o r d i l u t i o n i n d e g r a d a t i v e s t u d i e s w i t h r a d i o a c t i v e compounds. M a t e r i a l s and Methods I. Chemicals: A l l chemicals employed i n s y n t h e t i c work were o f  C.P.  50  grade.  P h e n o l i c compounds and  t a l l i z e d b e f o r e use.  critical  reagents were r e c r y s -  E t h a n o l and methanol were d r i e d , where  necessary, by r e f l u x i n g w i t h magnesium and subsequently t i l l i n g o f f the d r i e d s o l v e n t .  Acetone was  dis-  d r i e d by p a s s i n g  through a 2 X 20 cm column o f a c t i v i t y grade I Woelm Alumina. Protocatechualdehyde white was  was  r e c r y s t a l l i z e d from t o l u e n e  c r y s t a l s were o b t a i n e d .  Chloromethyl-methyl  until  ether  p r e p a r e d by s a t u r a t i n g a mixture o f 210 ml o f 36% aqueous  formaldehyde and 100 ml o f methanol w i t h HCl gas a t 10°C. chloromethyl-methyl  e t h e r was  d r i e d over anhydrous C a C l ^  and the d i s t i l l a t e b o i l i n g between 55 - 62°C was (Marvel and P o r t e r 1929). HCl gas, ^SO^  the gas was  The  employed  In the absense o f a c y l i n d e r o f  generated by dropping  i n t o c o n c e n t r a t e d aqueous H C l .  concentrated  C a f f e i c a c i d was  re-  c r y s t a l l i z e d from water f o r p r e p a r a t i o n o f the s t a n d a r d absorbance curve.  V e r a t r i e a c i d was  p u r i f i e d by vacuum  s u b l i m a t i o n and r e c r y s t a l l i z a t i o n from water f o r p r e p a r a t i o n o f the s t a n d a r d absorbance c u r v e . I I . Chromatography Paper chromatography employing b e n z e n e / a c e t i c a c i d / water i n the f i r s t dimension mic a c i d  s o l v e n t system A:  (10:7:3 by. v o l . (upper phase))  and s o l v e n t system B: 2% aqueous f o r -  (by v o l . ) i n the second dimension was  separation of phenolic acids.  used f o r the  T h i n l a y e r chromatography  51 (TLC) employing 500 micron A v i c e l c e l l u l o s e l a y e r s and s o l vent system C: t o l u e n e / e t h y l f o r m a t e / f o r m i c a c i d vol.)  was used f o r the s e p a r a t i o n o f s t y r y l pyrones.  o l i c compounds were d e t e c t e d by s p r a y i n g w i t h p _ - n i t r o a n i l i n e reagent gent  (Appendix B ) .  namaldehyde reagent Bromocresol  Phen-  diazotized  (Appendix B) o r f e r r i c c h l o r i d e r e a -  S t y r y l p y r o n e s were d e t e c t e d w i t h modi-  f i e d E h r l i c h ' s reagent  III.  (5:4:1 by  (Appendix B) o r  (Appendix B ) .  green reagent  p_-dimethylaminocin-  A c i d s were d e t e c t e d w i t h  (Appendix B ) .  Spectroscopy: U l t r a v i o l e t s p e c t r a were o b t a i n e d u s i n g a Unicam  model  SP.8O0  r e c o r d i n g spectrophotometer.  s p e c t r a were taken i n a Unicam  SP.200G  employing n u j o l mulls o r KBr d i s c s . onance  I n f r a r e d (IR) spectrophotometer  N u c l e a r magnetic r e s -  (NMR) s p e c t r a were o b t a i n e d on a V a r i a n 60 mHz model  spectrophotometer. IV. M e l t i n g P o i n t s : M e l t i n g p o i n t s were taken on a h o t - s t a g e m e l t i n g p o i n t apparatus  o r i n a Thomas, Hoover c a p i l l a r y m e l t i n g p o i n t ap-  paratus.  The v a l u e s o b t a i n e d were u n c o r r e c t e d .  V. Chemical  Preparations:  P r e p a r a t i o n o f t r i a c e t i c l a c t o n e (Berson  1952)  T h i r t y grams o f d e h y d r o a c e t i c a c i d was d i s s o l v e d i n 50 ml o f 90% H_SO.  i n a 100 ml round bottom f l a s k .  The f l a s k  52 and contents were immersed i n a 150 °C o i l bath w h i l e the f l a s k was  f l u s h e d c o n t i n u o u s l y w i t h a stream o f n i t r o g e n .  The  r e a c t i o n mixture was r a i s e d t o and maintained a t 130 - 140 °C f o r f i v e minutes and t h e r e s u l t i n g deep  reddish-brown  s o l u t i o n was c o o l e d r a p i d l y i n an i c e b a t h .  The s o l u t i o n  was  then added t o 200 ml o f i c e water.  was  c o n s i d e r e d t o be complete  Crystallization  i n f i v e minutes.  The o f f -  white p r e c i p i t a t e was c o l l e c t e d i n a Buchner f u n n e l and washed w i t h 100 ml o f i c e water. The y i e l d was 15 g o f crude t r i a c e t i c l a c t o n e which upon r e c r y s t a l l i z a t i o n from e t h y l a c e t a t e gave M.P. 191-192.5 ° C A  M  e  0  /  H  283 nm, NMR  spectrum  max S MeOH-d  4  2.25 (s, 3H,-CH ), 5.34 (d, IH, H ,.J=2.4 c p s ) , 5.63 3  (m, IH, H , coupled t o H  3  3  and -CH ), 5.98 (s, IH, -OH). 3  P r e p a r a t i o n o f 6-methyl-4-methoxy-2-pyrone  (Bu Lock 1  and Smith 1960)  Twenty grams o f d i m e t h y l s u l p h a t e was added t o a 500 ml round bottom  f l a s k c o n t a i n i n g 70 g o f anhydrous  potassium  carbonate and 20 g o f t r i a c e t i c l a c t o n e i n 300 ml o f d r y a c e t one.  The mixture was r e f l u x e d i n a h e a t i n g mantle w i t h  magnetic  s t i r r i n g f o r s i x hours w h i l e m a i n t a i n i n g anhydrous  conditions.  The r e a c t i o n mixture was then allowed t o c o o l ,  f i l t e r e d on a Buchner f u n n e l and evaporated under pressure.  reduced  R e c r y s t a l l i z a t i o n from petroleum e t h e r (60 - 110 °C)  gave a t o t a l y i e l d o f 17.3 grams o f white c r y s t a l s M.P. 89 - 90 °C, X 280 nm, NMR spectrum & C C l . 2.20 (s, 3H, max 4 M e 0 H  -CH ) 3  3.81  /  (d, 1H, H. , J=2.4 cps),'.5.65  (s, 3H, -OCH ) , 5.29  3  3  (m, 1H, H , . , coupled t o H  and - C H ) .  3  3  Preparation of trimethylhispidin  (Edwards  e t a l 1961)  4g o f magnesium t u r n i n g s were d i s s o l v e d i n 100 ml o f dry methanol w i t h magnetic s t i r r i n g a t room temperature under anhydrous c o n d i t i o n s . oxy-2-pyrone  Ten grams o f 6-methyl-4-meth-  and 10 g o f v e r a t r a l d e h y d e were i n t r o d u c e d ,  and the r e a c t i o n mixture was sixteen hours.  2-3  s t i r r e d a t room temperature f o r  ml o f a c e t i c a c i d were then added to  d i s s o l v e the magnesium h y d r o x i d e p r e s e n t , and the p r e c i p i t a t e d p r o d u c t was  c o l l e c t e d i n a Buchner  w i t h a few ml o f methanol and d r i e d .  On  f u n n e l , washed recrystallization  from methanol 6.5 grams o f lemon y e l l o w c r y s t a l s were obt a i n e d , M.P. ' (VC=0),  164 - 166  o  Me OH  C, A ' max  362 nm,  1555 and 1643 cm~ (Vpyrone C=C), 1  3.82  (s, 3H, pyrone-OCH ), 3.95  5.46  (d, IH, H ,  J=2.4 c p s ) , 5.91  6.44  (d, IH, H  J=16  3  3  Q /  c p s ) , 6.73  (d, 6H,  IR spectrum * NMR  1704  spectrum SCDC1  3  aromatic-0CH ), 3  (d, IH, Hg, J=2.4 c p s ) , - 7.23  (m, 3H,  aromatic),  o  7.45  (d, IH, H , ?  J=16cps).  Preparation of 4-methylhispidin  (Benton and D i l l o n  0.5 g o f t r i m e t h y l h i s p i d i n was  1942)  d i s s o l v e d i n 50 ml o f  methylene c h l o r i d e i n a round bottom f l a s k .  F l a s k and con-  t e n t s were then c o o l e d i n a b a t h o f acetone and d r y i c e a f t e r which 0.3 ml o f boron t r i b r o m i d e was  p i p e t t e d i n t o the  54 reaction vessel.  The  r e a c t i o n mixture was  warm to room temperature w i t h o c c a s i o n a l  then a l l o w e d  to  a g i t a t i o n by hand.  A f t e r s t a n d i n g a t room temperature o v e r n i g h t , 50 ml o f water was  added to the  t h o r o u g h l y and The  mixture was  reaction flask.  The  mixture was  a l l o w e d t o stand an a d d i t i o n a l h a l f hour. then f i l t e r e d on a Buchner f u n n e l  greenish-yellow p r e c i p i t a t e c o l l e c t e d . z a t i o n from 6 0 % e t h a n o l , point nm,  252 - 255  stirred  o  C as  0.22  g was  After  c o l l e c t e d with melting  25 nm  1  the  recrystalli-  fine pale yellow c r y s t a l s , ^  IR spectrum 1682 c m " C = C ) .  and  MeOH  375  m a x  bathochromic s h i f t  u l t r a v i o l e t a b s o r p t i o n on a d d i t i o n o f sodium b o r a t e , no observed on a d d i t i o n o f sodium Alkaline hydrolysis F i f t y mg o f 1 0 % NaOH and f o r 1 hour. was  cooled  of t r i m e t h y l h i s p i d i n  s o l u t i o n was  At the end  Buchner f u n n e l .  The  (Bu*Lock e t a l d i s s o l v e d i n 12  the mixture f i l t e r e d i n a  f i l t r a t e was  then a c i d i f i e d by  first  Upon a c i d i f i c a t i o n a  white p r e c i p i t a t e formed which was  collected.  further extracted  and  the  residue  ml  r e a c t i o n mixture  adding i c e , then c o n c e n t r a t e d HCl.  was  1962)  r e f l u x e d on an o i l b a t h  o f t h i s time the  i n an i c e bath and  shift  acetate.  o f t r i m e t h y l h i s p i d i n was the  in  with e t h y l acetate,  the  The  solvent  combined w i t h the p r e c i p i t a t e .  removed  Recrystall-  i z a t i o n from benzene y i e l d e d a p p r o x i m a t e l y 6 mg methoxy cinnamic a c i d as white c r y s t a l s , M.P.  filtrate  of  3,4-di-  181 - 183°C.  P r e p a r a t i o n o f 3,4-dimethoxycinnamic a c i d 0.5 g o f f e r u l i c a c i d ,  3.5 g o f anhydrous p o t a s s i u m  carbonate and 1 ml o f d i m e t h y l s u l p h a t e were p l a c e d i n 15 ml o f anhydrous acetone and r e f l u x e d  with s t i r r i n g  under  anhydrous c o n d i t i o n s i n a 50 ml round bottom f l a s k . t i o n mixture was allowed t o c o o l , under reduced p r e s s u r e . was  The r e a c -  f i l t e r e d and evaporated  An orange o i l was o b t a i n e d t o which  added 2 ml o f IN NaOH. T h i s mixture was a l l o w e d t o stand  f o r 16 h r and an a d d i t i o n a l the pH t o n e u t r a l i t y .  0.2 g o f NaOH was added t o b r i n g  R e a c i d i f i c a t i o n w i t h 6 drops o f 4N  HC1 caused i n t e n s e p r e c i p i t a t i o n . T h i s p r e c i p i t a t e was c o l lected  (M.P. 70 - 71 °C) and the f i l t r a t e washed w i t h 3 X  10 ml o f d i e t h y l e t h e r . The e t h e r e x t r a c t was d r i e d o v e r anhydrous sodium s u l p h a t e and taken t o dryness under reduced pressure.  T h i s was combined w i t h the p r e c i p i t a t e t o g i v e  0. 45 g o f o f f - white c r y s t a l s .  These c r y s t a l s were then  heated under r e f l u x i n a s o l u t i o n o f 50% aqueous NaOH f o r 2 hr.  The s o l u t i o n was a l l o w e d t o c o o l and a c i d i f i e d w i t h  c o n c e n t r a t e d HC1 r e s u l t i n g i n a white p r e c i p i t a t e which was c o l l e c t e d , washed w i t h water and d r i e d .  T h i s m a t e r i a l was  r e c r y s t a l l i z e d from water t o a f f o r d white c r y s t a l s M.P. 181 - 183 °C, > y max  M e 0 H  293, 319; y i e l d 0.25 g. 3  P r e p a r a t i o n o f 3,4-bis-(methoxymethoxy)-benzaldehyde and W i l s o n 1961)  (Edwards  56  To p r o t o c a t e c h u a l d e h y d e toluene 120  ml  (250 m l ) ,  (20 g)  sodium e t h o x i d e  e t h a n o l ) was  ( f r o m 10  to  0°C  a l c o h o l was and  distilled  t h i r t y minutes,  hr  s h a k e n w i t h 40 m l  l a y e r was  a t 2 mm  collected. was  o f f and  stirred o f 2N  The  added u n t i l  and  the  fraction  p r o d u c t was an  then  c o o l e d t o -20°C whereupon w h i t e  M.P.  58 - 6 0 ° C ; y i e l d  dry  acetone.  potassium at  mg  by hand.  p e r m a n g a n a t e and  At  under reduced technical to  the acetone  room t e m p e r a t u r e  up  over  grade  the r e a c t i o n  The  and  anhydrous  was  water  mixture  was  c r y s t a l s were o b t a i n e d ,  to v e r a t r i c  s o l u t i o n was  time  1.0  ml  sodium b i s u l p h i t e flask  toluene  t o 152°C  acid  agitated  i n 5 ml  a d d e d 50 mg allowed  hr with occasional  of this  p r e s s u r e and  for 4  i n e t h a n o l and  t h e m i x t u r e was  f o r two  t h e end  ml)  The  o f t r i m e t h y l h i s p i d i n were d i s s o l v e d To  (27  g.  Oxidation of trimethylhispidin Two  cooled  r e s i d u e brown o i l was  o i l began t o s e p a r a t e .  6.4  ether  dried  boiling  dissolved  re-  t h e r e s i d u e was  sodium h y d r o x i d e .  The  alcohol  The  a t room t e m p e r a t u r e  evaporated.  and  the  of nitrogen.  s e p a r a t e d , washed w i t h w a t e r ,  sodium s u l p h a t e and distilled  g sodium  treated with chloromethyl-methyl  during and  boiling  added d u r i n g t h i r t y , minutes,  m e a n w h i l e b e i n g removed i n a s t r e a m maining  in stirred  the acetone o f IN H C l  to  of react  agitation was  and  removed  1.0  ml  of  ( 1 0 % a q u e o u s ) were a d d e d to obtain a  of  colourless  solution.  T h i s s o l u t i o n was  e x t r a c t e d w i t h 3 X 2 ml o f  d i e t h y l e t h e r and the e t h e r s o l u t i o n t r a n s f e r r e d t o the l i m a t i o n v e s s e l where i t was warming and sublimed a t 0.3  taken t o dryness w i t h g e n t l e mm  r e c o v e r y o f v e r a t r i c a c i d was absorbance  VI.  sub-  o f mercury a t 110°C.  the  c a l c u l a t e d from the s t a n d a r d  curve t o be a p p r o x i m a t e l y  40%.  Radioautography Two-dimensional  chromatograms were p l a c e d i n l i g h t -  t i g h t X-ray f i l m exposure h o l d e r s w i t h a 14 X 19 i n c h sheet o f Kodak Blue Brand M e d i c a l X-ray f i l m t h r e e t o f o u r weeks.  ( E s t a r Base) f o r  A t the end o f the exposure  period  the f i l m s were removed and developed under a r e d s a f e l i g h t .  58 R e s u l t s and D i s c u s s i o n In o r d e r t o become f a m i l i a r w i t h p h e n o l i c and namic a c i d d e r i v a t i v e s l i k e l y t o be encountered, i t e two-dimensional was  prepared  cin-  a compos-  paper chromatogram o f a u t h e n t i c compounds  ( F i g . 17.).  T h i s was  developed  i n solvent  systems A and B and the spots were v i s u a l i z e d under l o n g wave u l t r a v i o l e t l i g h t , by s p r a y i n g w i t h d i a z o t i z e d p_-nitroa n i l i n e reagent o r by r a d i o a u t o g r a p h y .  The c o l o u r s observed  f o r the p h e n o l i c spots are r e c o r d e d i n Table I I .  Radio-  14 active  C - l a b e l l e d samples were employed f o r those compounds  not r e a d i l y d e t e c t e d w i t h spray r e a g e n t s .  I t was  observed  t h a t l e s s t a i l i n g o f spots i n the f i r s t dimension was c o u n t e r e d when the chromatogram was  en-  developed o p p o s i t e t o  the machine d i r e c t i o n i n manufacture o f the paper,, To f a c i l i t a t e the d e t e r m i n a t i o n o f s p e c i f i c a c t i v i t i e s 14 14 o f samples o f p_-coumaric a c i d - 2 - C and c a f f e i c a c i d - 2 - C which were t o be employed i n f e e d i n g experiments,  standard  absorbance  curves f o r these two a c i d s were p r e p a r e d .  absorbance  o f p_-coumaric  18.)  and the absorbance  324 nm  ( F i g . 19.).  a c i d was  determined  o f c a f f e i c a c i d was  The  a t 310 nm ( F i g . determined  at  Both a c i d s were d i s s o l v e d i n methanol  f o r p r e p a r a t i o n o f the c u r v e s . prepare s t a n d a r d absorbance  i t was  necessary a l s o to  curves f o r t r i m e t h y l h i s p i d i n  and v e r a t r i c a c i d t o be used i n the d e t e r m i n a t i o n o f  specific  m  FIG. 17. Diagrammatic  r e p r e s e n t a t i o n o f two-dimensional  chromatogram o f a u t h e n t i c samples o f p h e n o l i c and c i n n amic a c i d  derivatives.  I m-hydroxymandelic a c i d , I I p_-hydroxymandelic a c i d , I I I p_-hydroxybenzyl a l c o h o l , IV p h e n y l l a c t i c a c i d , V p_-hydroxy p h e n y l a c e t i c a c i d , VI o - h y d r o x y p h e n y l a c e t i c a c i d , VII p_-hydroxybenzaldehyde, V I I I c i s p_-coumaric a c i d , IX c i s c a f f e i c a c i d , X p_-hyroxybenzoic a c i d , XI p r o t o c a t e c h u i c a c i d , X I I t r a n s p_-coumaric a c i d , X I I I t r a n s c a f f e i c a c i d , XIV p h e n y l a c e t i c a c i d , XV c i s cinnamic a c i d , XVI b e n z o i c a c i d , XVII t r a n s cinnamic a c i d .  60 TABLE I I . C o l o u r s  of phenolic  a c i d s i n l o n g wave  olet  light  and i n v i s i b l e  with  diazotized p_-nitroaniline  Compound  light  after  ultravispraying  reagent. Colour a f t e r Spraying  Colour i n l o n g wave UV  m-hydroxymandelic  acid  red  p_-hydroxymandelic  "  pink  alcohol  pink  rj-hydroxybenzyl  p_-hydroxyphenylacetic  acid  o-hydroxyphenylacetic  "  purple wine-red brown  p_-hydr o x y b e n z a l d e h y d e p_-coumaric a c i d  caffeic  acid  ( c i s and trans)  ( c i s and trans)  protocatechuic  and  blue  green  (fades t o brown)  green  (  fluorescence  acid  o f these  degradation  blue  acid  p_-hydroxybenzoic  activities  blue fluorescence a f t e r NH^OH v a p o u r s  "  pink  compounds  of hispidin  obtained  isolated  from the m e t h y l a t i o n  from c u l t u r e s o f the  fungus t h a t had been f e d r a d i o a c t i v e p r e c u r s o r s .  The  absor-  b a n c e o f t r i m e t h y l h i s p i d i n was  d e t e r m i n e d a t 365 nm ( F i g .  20.) a n d t h a t o f v e r a t r i c  a t 253 nm  determinations The chieved  acid  s y n t h e s i s o f t r i m e t h y l h i s p i d i n was  employing a standard  the product  its  importance  These  a l s o w e r e made i n m e t h a n o l .  chemical  and  (Fig. 21.).  was  method  critically  for dilution  a-  (Edwards e t a l 1961)  c h a r a c t e r i z e d because o f  i n tracer studies.  The p h y s i c a l  )  •pfoe oxaeumoo-cT 3 0 e/ano eoueqaosqp paepue^s * 8 T  T9  9IJ  •p-uoe o T a j g e o 3 0 sAano a o u e q a o s q e pjtepuet+s * 6 T 'Did  Z9  • UTPTCTSTU_TAU_H\9UITJI^  J O  5AJ.no  Z9  a o u e q j o s q n  p a e p u e ^ s  '02  'Did  •p-cop D T J ^ P I S A  30  a A a n o  aoueqaosqe paepue^s  fr9  "IZ  'Did  65 constants closely al  determined  to those  1967).  and  spectral  reported  data obtained  (Edwards and  Y a n g o n i n a l s o was  prepared  and  6 - m e t h y l - 4 - m e t h o x y - 2 - p y r o n e by  use  as  a r e f e r e n c e compound.  °C), u l t r a v i o l e t ' spectrum  1725  the product  The  (Bu*Lock a n d  obtained  through  same p r o c e d u r e  )\  et  for  (155  358 nm  M e O H  max  (VC-O) c o n f i r m t h e  S m i t h 1960,  Bartle  anisaldehyde  melting point  A sample o f a u t h e n t i c b i s - n o r y a n g o n i n p o s e s was  1967,  from  the  absorbance spectrum  (vC=0), 1258  Mir  conformed  -  and  157 IR  identity  Edwards a n d  Mir  for reference  the c o u r t e s y o f Dr.  G.M.  of  1967). purHat-  field. The hispidin  f o u r r e f e r e n c e s t y r y l p y r o n e s and  obtained  from  a sample  t h e m y c e l i u m o f P_. h i s p i d u s were  c o m p a r e d c h r o m a t o g r a p h i c a l l y i n s o l v e n t s y s t e m C on lulose  TLC  plates.  Caffeic  veratric  a c i d were a l s o  system.  The  examined i n t h i s  reactions with  the spray  (Table  Veratric  green  reagent  Rf o f t h i s  a c i d was  (Appendix  their  detected with  t h e R f v a l u e o f 0.19  recorded  The  0.82.  spray  f o r the  colour  bromocresol  a blue background.  the c o l o u r r e a c t i o n s w i t h  ultraviolet  and  B) were  compound i n s o l v e n t s y s t e m C was  I I I . ) and  tractable,  reagents  cel-  chromatographic  compounds and  as a y e l l o w s p o t on  Although (Table  a c i d , p_-coumaric a c i d  Rf v a l u e s o f t h e s e  III.).  of  reagents  methanol-ex-  f l u o r e s c e n t (yellow-green)  component  66 TABLE I I I . C h a r a c t e r i s t i c s o f some s t y r y l p y r o n e s and p h e n o l i c compounds chromatographed on  related  cellulose  TLC p l a t e s i n s o l v e n t system C and sprayed w i t h v a r i o u s reagents Compound  Rf X  (Appendix  100  B).  Reagent 1  Reagent 3  Reagent 4  trimethylhispidin  97  yellow  yel-gr  yel-gr  4-methylhispidin  44  orange  yel-gr  green  hispidin  19  pink-brown  blue-gr  gr-blue  bis-noryangonin  44  wine r e d  blue-gr  gr-blue  p a l e brown  lt-blue  pale gr  purp-blue  bleached  lt-blue  bleached  yangonin  100  p_-coumaric caffeic  acid  acid  60  blue  36  green  o f P. h i s p i d u s p r o v i d e d p r e l i m i n a r y evidence t h a t t h i s stance was  h i s p i d i n , a d d i t i o n a l c h a r a c t e r i z a t i o n was  H i s p i d i n was  sub-  obtained.  i s o l a t e d a c c o r d i n g t o the procedure g i v e n i n  P a r t V., M a t e r i a l s and Methods, Chapter one.  T h i s substance  MeOH gave UV spectrum 1661  N  C=0),  1669,  (^ aromatic C=C).  366, 1564  251,  221 and IR  (V pyrone C=C),  spectrum  1608  and 1530  cm"  1  A f t e r m e t h y l a t i o n o f the pigment w i t h  d i m e t h y l s u l p h a t e (Edwards e t a l 1961),  the product was  found  t o correspond t o the a u t h e n t i c sample upon chromatography and examination o f the UV spectrum.  After  recrystallizing  h i s p i d i n t h r e e times from ethanol-water, M.P. o b t a i n e d ( L i t . M.P.  259°C).  Chromatographic the fungus  254 - 257°C  examination o f methanol e x t r a c t s o f  r e v e a l e d f i v e y e l l o w - c o l o u r e d components i n  was  67 a d d i t i o n to h i s p i d i n .  Under long wave UV  stances f l u o r e s c e d shades o f yellow, green.  The  y e l l o w - g r e e n and  (Band I ) , 0.37  (Band II)  (Band IV) were removed from the p l a t e s and  f u t u r e examination.  Their u l t r a v i o l e t spectra  suggest t h a t they are a l s o  t r a c e amounts a l s o was green i n long wave UV I t was  in  and  saved f o r  (Fig.  22.)  styrylpyrones.  Another o f these components which was  system C.  blue-  three most prominent o f these bands o b t a i n e d  s o l v e n t system C a t Rf 0.62 Rf 0.00  l i g h t these sub-  examined.  present  only i n  T h i s band f l u o r e s c e d  l i g h t and moved a t Rf 0.44  scraped from the TLC  p l a t e s and  blue-  i n solvent eluted  from the powdered c e l l u l o s e w i t h methanol i n a 1 cm  column.  This substance cochromatographed w i t h b i s - n o r y a n g o n i n i n s o l v e n t system C on both c e l l u l o s e and plates.  TLC  I t gave i d e n t i c a l c o l o u r r e a c t i o n s t o b i s - n o r y a n g -  o n i n w i t h spray reagents 1,3 considered  and  4  (Table I I I . ) and  t o be the same substance.  noryangonin i n P. h i s p i d u s precursor  s i l i c a gel G  i s very  The  was  presence o f b i s -  important as a p o s s i b l e  i n hispidin biosynthesis.  Very i n t e r e s t i n g  the a d d i t i o n a l f a c t t h a t b i s - n o r y a n g o n i n c o u l d not be t e c t e d i n Polyporus s c h w e i n i t z i i ( H a t f i e l d 1970).  i n P. h i s p i d u s .  Band I o r another o f the y e l l o w  The  de-  This  c o u l d suggest t h a t an a l t e r n a t e pathway or more than pathway i s o p e r a t i v e  is  possibility  one that  f l u o r e s c e n t components observed  68  FIG.  22. U l t r a v i o l e t s p e c t r a o f y e l l o w , f l u o r e s c e n t from chromatograms o f P. h i s p i d u s e x t r a c t s .  bands  69 is  6-styryl-4-hydroxy-2-pyrone  to  this  suggestion.  provides  further  credibility  CHAPTER THREE  RADIOACTIVE FEEDING EXPERIMENTS WITH CULTURES OF POLYPORUS HISPIDUS  70 Introduction P r e l i m i n a r y examinations  o f P_. h i s p i d u s c u l t u r e s grown  on GYSS e s t a b l i s h e d t h a t numerous p h e n o l i c a c i d s were p r e s e n t i n the medium a f t e r two weeks i n c u b a t i o n . 14  Phenylalanine-3-  . . . C was then f e d t o these c u l t u r e s under v a r i o u s c o n d i t i o n s  t o e s t a b l i s h the best procedure  f o r e l u c i d a t i n g the metabol14  ism o f t h i s and o t h e r aromatic  compounds.  A variety of  re-  l a b e l l e d compounds was f e d t o the fungus and the m e t a b o l i c products  examined w i t h radioautography  and s c i n t i l l a t i o n  techniques. M a t e r i a l s and Methods I. Analysis of phenolic a c i d s : The medium was removed w i t h f i l t r a t i o n , pH 2 w i t h 2N H C l and e x t r a c t e d t h r e e times  a c i d i f i e d to  i n a separatory  f u n n e l w i t h a t o t a l o f 200 ml o f d i e t h y l e t h e r .  The e t h e r  e x t r a c t was a n a l y s e d by paper chromatography employing aut h e n t i c samples as r e f e r e n c e s ( F i g . 1 7 . ) . I I . P r e p a r a t i o n and a d m i n i s t r a t i o n o f r a d i o a c t i v e compounds: 14 14 Phenylalanine-3C, p h e n y l a l a n i n e - 2 C, 3,4-dihydroxy14 14 phenylalanine-2C, p h e n y l a c e t i c a c i d - 2 - C, sodium a c e t a t e -2-  14  C and malonic  clear Corporation.  acid-2-  14 C were o b t a i n e d from New England  Cinnamic a c i d - 2 -  o b t a i n e d from I n t e r n a t i o n a l Chemical  14  . 14 C and t y r o s i n e - 3 - C were  and Nuclear C o r p o r a t i o n and  14 benzoic a c i d - r i n g .  Nu-  C (U) was o b t a i n e d from the  Radiochemical  71  Center, Amersham, England. 14 acid-2-  w i t h malonic a c i d - 2 -  piperidine  C and  caffeic  C i n pyridine with a trace  ( A u s t i n and Meyers 1965).  the r a d i o a c t i v e compound was day,  14  . . C were prepared by condensation o f the a p p r o p r i a t e 14  benzaldehyde of  P-coumaric a c i d - 2 -  U s u a l l y , 2 ^uCi o f  a d m i n i s t e r e d on the a p p r o p r i a t e  d i r e c t l y i n t o the c u l t u r e medium o f each Roux b o t t l e .  The compound was  d i s p e r s e d by b r i e f a g i t a t i o n and the  cul-  t u r e s i n c u b a t e d w i t h the r a d i o i s o t o p e s f o r 24 hours. Ill,  Detection of r a d i o a c t i v i t y : Sprayed  chromatograms were used f o r radioautography.  Unsprayed d u p l i c a t e chromatograms were examined q u a n t i t a t i v e l y by c u t t i n g t'ae p h e n o l i c a c i d spots from the chromatograms and c o u n t i n g them d i r e c t l y by s c i n t i l l a t i o n ing  a Nuclear-Chicago  employ-  720 s e r i e s o r U n i l u x I I l i q u i d  t i l l a t i o n spectrometer.  scin-  Dual channel c o u n t i n g p e r m i t t e d  c a l c u l a t i o n o f e f f i c i e n c y from a quench curve prepared f o r each instrument employing samples.  a s e r i e s o f v a r i a b l y quenched  The s c i n t i l l a t i o n f l u i d employed f o r most samples  c o n s i s t e d o f 4 g 2,5-diphenyloxazole bis  2-(5-phenyloxazolyl)-benzene  toluene. for  Aquasol  (New  (PPO)  and 30 mg p_-  (POPOP) i n one  l i t r e of  England N u c l e a r C o r p o r a t i o n ) was  used  aqueous samples and methanolic samples o f t r i m e t h y l h i s -  p i d i n and v e r a t r i c  acid.  72 IV. Recovery o f f r e e amino a c i d s : Free amino a c i d s i n the c u l t u r e medium were r e c o v e r e d by removing the mycelium w i t h f i l t r a t i o n and p a s s i n g the medium onto a column o f Dowex 50W exchange r e s i n .  - X8 s t r o n g l y c a t i o n i c  The amino a c i d s were e l u t e d w i t h 5% aqueous  NH^OH and the ammonia was  removed by e v a p o r a t i o n i n vacuo.  A f t e r r e d u c i n g i t s volume by h a l f , was  f r o z e n and l y o p h i l i z e d .  The  the amino a c i d  r e s i d u e was  solution  taken up i n  70% e t h a n o l and chromatographed o n e - d i m e n s i o n a l l y on Whatman  No.  water  1 paper i n the upper phase o f n - b u t a n o l / a c e t i c a c i d /  (4:1:5 by volume).  The t y r o s i n e band was  rechromatographed on 500 micron A v i c e l l a y e r p l a t e s employing  a c t i v i t y was  (cellulose)  the same s o l v e n t system.  s i n e band r e c o v e r e d from TLC was determined  thin  The  tyro-  d i s t i n c t and the r a d i o -  by s c i n t i l l a t i o n .  the c e l l u l o s e scraped from the p l a t e s was scintillation  e l u t e d and  For t h i s  purpose,  suspended i n the  f l u i d w i t h C a b o s i l (Cabot C o r p . ) . 14  V. M e t h y l a t i o n o f  C-labelled  hispidin:  H i s p i d i n o b t a i n e d a f t e r chromatography o f f u n g a l extracts  was  d r i e d under vacuum.  The t o t a l amount r e c o v e r e d  from each r a d i o a c t i v e f e e d i n g was acetone assium  and t o the s o l u t i o n was carbonate  and 0.1  d i s s o v e d i n ten ml  added 200 mg  dry  anhydrous p o t -  ml o f d i m e t h y l s u l p h a t e . The  solution  was  r e f l u x e d f o r 24 hr over a steam bath employing a condenser  f i t t e d w i t h d r y i n g tube.  The  r e a c t i o n mixture was  l y reduced i n volume i n vacuo and 500  micron A v i c e l TLC  plates  0.97)  was  banded d i r e c t l y onto  (20 X 20 cm).  eloped i n s o l v e n t system C and  p l a t e s were dev(Rf  r e c o v e r e d from the p l a t e s i n the same manner as  the  The  r e c o v e r e d t r i m e t h y l h i s p i d i n showed i d e n t -  c a l chromatographic behaviour and t o spray reagents and  gave the same c o l o u r  (Table I I I . ) as the a u t h e n t i c  reaction  material.  Discussion  Cultures  o f P_. h i s p i d u s were examined a f t e r 14  21 days i n c u b a t i o n . 14 3-  The  four  the t r i m e t h y l h i s p i d i n band  h i s p i d i n band.  Results  subsequent-  These c u l t u r e s were f e d  and  phenylalanine-  . C and were examined a f t e r 6,  w i t h the r a d i o a c t i v e compound. medium was  r e p l a c e d w i t h 0.5%  p r i o r t o the  12 and In one  24 h r  set of cultures,  phenylalanine  f e e d i n g o f 2.5 ;uCi  incubation  (0.21 wM) o f /  s o l u t i o n 48  the hr  phenylalanine-  14 3-  C per  100  ml o f c u l t u r e medium.  In the second s e t o f  t u r e s , the same amount o f r a d i o a c t i v e p h e n y l a l a n i n e  was  cul-  added  to the medium without replacement. Two-week-old c u l t u r e s which were incubated alanine  s o l u t i o n f o r 48 hr p r i o r t o f e e d i n g  poration of r a d i o a c t i v i t y i n t o phenylpyruvic, phenylacetic,  on  phenyl-  showed i n c o r phenyllactic,  cinnamic, b e n z o i c and p_-hydroxybenzoic a c i d s .  74 Two o t h e r u n i d e n t i f i e d compounds were a l s o r a d i o a c t i v e . a d d i t i o n , p_-hydroxyphenylacetic, p_-coumaric,  In  c a f f e i c and  p r o t o c a t e c h u i c a c i d s and one o t h e r u n i d e n t i f i e d  compound  were p r e s e n t on the chromatograms without  Figure  label.  23. shows a diagrammatic r e p r e s e n t a t i o n o f the d e t e c t e d compounds on these chromatograms.  Spot number 1. which  gave a v i o l e t c o l o u r r e a c t i o n w i t h spray reagent 1. (p_-NA) and spot number 2. (brownish w i t h p_-NA) c o u l d n o t be d e t e r mined from a v a i l a b l e r e f e r e n c e compounds.  F i g u r e 24. shows  the p r o b a b l e i n t e r r e l a t i o n s h i p s o f the determined  radioactive  compounds. Examination  o f three-week-old  c u l t u r e s which a l s o  were i n c u b a t e d w i t h n o n r a d i o a c t i v e p h e n y l a l a n i n e p r i o r t o f e e d i n g showed l e s s c o n s i s t e n t r e s u l t s , a l t h o u g h the p a t t e r n o f compounds was s i m i l a r t o t h a t observed week-old c u l t u r e s .  P-hydroxyphenylacetic  f o r two-  a c i d and p r o t o -  c a t e c h u i c a c i d showed i n c o r p o r a t i o n o f l a b e l a f t e r 6, 12 and  24 hours i n c u b a t i o n w i t h r a d i o a c t i v e p h e n y l a l a n i n e .  A l s o , l a b e l i n p h e n y l a c e t i c a c i d was not d e t e c t e d and r a d i o a c t i v i t y i n two spots which chromatographed v e r y c l o s e t o c i s and t r a n s c a f f e i c a c i d s was d e t e c t e d a f t e r 6 and 12 hour f e e d i n g p e r i o d s .  No spots i n a d d i t i o n t o those ob-  served i n two-week-old c u l t u r e s were d e t e c t e d . The suspected r e l a t i o n s h i p o f p r o t o c a t e c h u i c a c i d i n the d e g r a d a t i o n o f  75  Solvent Front  FIG.  (system A)  23. Diagrammatic r e p r e s e n t a t i o n o f the compounds d e t e c t e d i n chromatographed e x t r a c t s o f the medium u s i n g 48 h r i n d u c t i o n by replacement medium.  « •  - d e t e c t e d by  radioautography  .JtfL  - were r a d i o a c t i v e w i t h three-week-old c u l t u r e s o n l y  7 6  FIG.  24. Probable pathways o f L - p h e n y l a l a n i n e  degradation  i n P.hispidus. I L - p h e n y l a l a n i n e , I I cinnamic a c i d , I I I Benzoic a c i d , IV p_-hydroxybenzoic a c i d , V p h e n y l l a c t i c a c i d , VI p h e n y l p y r u v i c a c i d , VII p h e n y l a c e t i c a c i d , V I I I p_-hydroxypheny l a c e t i c a c i d , IX p r o t o c a t e c h u i c a c i d .  phenylalanine i s included i n Figure  24.  In c u l t u r e s which were f e d r a d i o a c t i v e p h e n y l a l a n i n e without p r i o r i n c u b a t i o n on replacement e l l i n g p a t t e r n emerged.  Although  medium, a new  lab-  the r e c o v e r a b l e q u a n t i t i e s  o f p h e n o l i c compounds from three-week o l d c u l t u r e s was s i d e r a b l y s m a l l e r than those  con-  from two-week-old c u l t u r e s ,  r a d i o a c t i v e l y - l a b e l l e d p_-coumaric and c a f f e i c a c i d s were p r e s e n t i n both.  E x t r a c t s o f two-week-old c u l t u r e s con-  t a i n e d r a d i o a c t i v e c a f f e i c and p r o t o c a t e c h u i c a c i d s o n l y a f t e r 24 h r i n c u b a t i o n w i t h the r a d i o a c t i v e p h e n y l a l a n i n e . While no r a d i o a c t i v e p_-hydroxyphenylacetic  a c i d was  i n e x t r a c t s o f two-week-old c u l t u r e s , i t was 24 h r f e e d i n g o f three-week-old the compounds and  cultures.  detected  after  In o t h e r r e s p e c t s ,  l a b e l l i n g d e t e c t e d i n these c u l t u r e s was  s i m i l a r to those which were grown on replacement The probable  detected  medium.  r e l a t i o n s h i p s o f the newly-detected r a d i o a c t i v e  compounds are shown ( F i g . 25.). The  r e s u l t s o f t h i s experiment i n d i c a t e t h a t degrad-  a t i v e pathways t o carbon d i o x i d e as w e l l as the cinnamate pathway are o p e r a t i v e i n p h e n y l a l a n i n e metabolism i n P_. h i s pidus c u l t u r e s . to those commune. found  The  d e g r a d a t i v e products  found by Moore and  Towers  No h y d r o x y l a t e d cinnamic  i n t h a t fungus,  e t a l 1965)  are v e r y  (1967) i n  similar  Schizophyllum  a c i d d e r i v a t i v e s were  however, i n L e n t i n u s l e p i d e u s  (Power  s i m i l a r h y d r o x y l a t i o n s have been shown t o occur  78  COOH  L- PHENYLALANINE  CINNAMIC ACID  FIG. 25. Probable r e l a t i o n s h i p o f r a d i o a c t i v e cinnamic a c i d d e r i v a t i v e s d e t e c t e d i n c u l t u r e s o f P_. h i s p i d u s .  79 ( F i g . 7.).  When c u l t u r e s were i n c u b a t e d on p h e n y l a l a n i n e  medium p r i o r t o r a d i o a c t i v e f e e d i n g ,  the h y d r o x y l a t e d  cin-  namic a c i d d e r i v a t i v e s , although e a s i l y d e t e c t e d on the chromatograms, were n o n - r a d i o a c t i v e .  However, when the r e -  placement medium was not used, these compounds were r a d i o actively labelled.  T h i s has been i n t e r p r e t e d as i n d i c a t i n g  t h a t when replacement  medium i s used the cinnamate pathway  becomes s a t u r a t e d and the p h e n o l i c products the pathway, p r o b a b l y a t cinnamic  a c t to repress  acid-4-hydroxylase.  These  same products may a l s o derepress  the d e g r a d a t i v e pathways  t o carbon d i o x i d e .  more  Another  u n l i k e l y . p o s -  s i b i l i t y i s t h a t the h y d r o x y l a t e d cinnamic from p h e n y l a l a n i n e through 4-hydroxylase  acids are a r i s i n g  t y r o s i n e and t h a t p h e n y l a l a n i n e -  o r t y r o s i n e ammonia-lyase a r e b e i n g  repressed.  In any case, i t appeared t h a t i n o r d e r t o f o l l o w the c i n namate pathway and examine h i s p i d i n b i o s y n t h e s i s , c u l t u r e s s h o u l d not be i n c u b a t e d on replacement  medium p r i o r t o r a -  d i o a c t i v e f e e d i n g and c o n c e n t r a t i o n s o f l a b e l l e d  compounds  f e d t o the c u l t u r e s s h o u l d be kept as s m a l l as p o s s i b l e t o avoid repressive e f f e c t s .  Two-week-old c u l t u r e s were s e l e c t e d  as the most s u i t a b l e f o r f e e d i n g r a d i o a c t i v e compounds, s i n c e l a r g e r q u a n t i t i e s o f p h e n o l i c compounds were r e c o v e r a b l e from e x t r a c t s o f the medium.  The lower  recovery  t h r e e weeks i s l i k e l y the r e s u l t o f o x i d a t i v e enzyme  after activity.  80 Because r a d i o a c t i v e dihydroxy-compounds n o r m a l l y were not recovered  from the medium except a f t e r 24 h r f e e d i n g s ,  this  l e n g t h o f time was chosen f o r f u t u r e f e e d i n g s t u d i e s . C u l t u r e s o f P. h i s p i d u s grown under the c o n d i t i o n s e s t a b l i s h e d i n Chapter  One , were f e d 5^Ci (0.42 ^ M ) o f DL-  14 phenylalanine-3-  C i n a d d i t i o n t o 1 mg n o n - l a b e l l e d p h e n y l -  a l a n i n e p e r Roux b o t t l e . T e h - d a y - o l d c u l t u r e s were examined a f t e r 18 hours t o determine i f r a d i o a c t i v i t y had become i n c o r p o r a t e d i n t o the f r e e t y r o s i n e p o o l i n the medium.  The f r e e  amino a c i d s were r e c o v e r e d and the t y r o s i n e p u r i f i e d by the method d e s c r i b e d .  S c i n t i l l a t i o n c o u n t i n g o f the r e c o v e r e d  t y r o s i n e d e t e c t e d 3,800 d i s i n t e g r a t i o n s p e r minute the t o t a l t y r o s i n e o b t a i n e d  (dpm) i n  from one Roux b o t t l e .  v a l u e was c o n s i d e r e d s i g n i f i c a n t l y h i g h t o conclude  This that  enzymes produced by the fungus were capable o f h y d r o x y l a t i n g p h e n y l a l a n i n e t o t y r o s i n e and t h a t h y d r o x y l a t e d  cinnamic  a c i d d e r i v a t i v e s c o u l d a r i s e from p h e n y l a l a n i n e  through  tyrosine. To o b t a i n more q u a n t i t a t i v e and more e x t e n s i v e  evi-  dence o f aromatic metabolism i n P. h i s p i d u s , s e v e r a l r a d i o a c t i v e compounds were f e d t o c u l t u r e s a t v a r i o u s  stages  o f growth and the i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o pheno l i c a c i d s and t h e i r p r e c u r s o r s was examined w i t h s c i n t i l 14 14 l a t i o n techniques. DL-phenylalanine-3C, D L - t y r o s i n e - 3 C,  81 cinnamic  acid-2-  14 14 C, p_-coumaric a c i d - 2 - C, 3,4-dihydroxy14  phenylalanme-2-  14  C and b e n z o i c a c i d - r i n g  cubated w i t h the c u l t u r e s f o r 24 h r .  C (U) were i n -  Two m i c r o c u r i e s o f  each compound was a d m i n i s t e r e d t o each Roux b o t t l e  except  14 f o r p_-coumaric a c i d - 2 The  C, o f which 0.5 yuCi was  administered.  r a d i o a c t i v i t y d e t e c t e d i n m e t a b o l i c products was  recorded  (Table I V . ) . In t h i s experiment, as i n most t r a c e r experiments which y i e l d q u a n t i t a t i v e data, g r e a t c a u t i o n must be exerc i s e d i n i n t e r p r e t i n g the r e s u l t s . and d i f f i c u l t  t o determine.  Many f a c t o r s are unknown  The most s i g n i f i c a n t o f these  are, perhaps, turnover r a t e , p o o l s i z e and p e r m e a b i l i t y o f the  fungus  t o the compound  i n question.  Turnover  r a t e o f any compound w i l l depend p a r t i a l l y on the p o o l  size  as w e l l as on the number o f pathways l e a d i n g t o and from t h i s compound and the a c t i v i t y o f the enzymes r e g u l a t i n g these pathways.  The p e r m e a b i l i t y o f the hyphae may v a r y  w i t h the age o f the organism, the c o n c e n t r a t i o n o f the compound b e i n g c o n s i d e r e d , the c o n c e n t r a t i o n o f another or f o r many o t h e r reasons.  In an examination  compound  such as t h i s ,  both the i n t e r n a l and e x t e r n a l p o o l s w i l l a f f e c t the data o b t a i n e d . The p o o l s i z e may a l s o v a r y w i t h the age o f the organism.  However, s c i n t i l l a t i o n counting has proved  s e n s i t i v e technique  than radioautography  t o be a more  for detection of  82 o  w p S3  P  « W W J o EH W o <m o u  o  rH  TSL  > •H u O ft  o  •—  o  m-H  U  o  o CN  ro < CJ ^—. ON CO  CJ  £ ft -cs  -H  SH  —'  ro TJ S -H OO 3 o o <c O  EH  oi ,  H  >  H  EH CJ  rH —^  Ti  •  Q  «  u  cu  f  \  VO ON —•  o o NO ^-^  £A  X cn O  vp  '—^  ^—  ON CO  cn  o o N*  o o  O O  rH  rH  CN  cn  -—V NP  NO ON  o o  cn  NO  QN  ON cn  CO  CO  o  o  o  CN  CN  o CM CN  o N0  CN  o LO  o f>  Nt  in  CN  N*  H  rH  o o N*  N  Nt  o rH  CO  CN  o  O  o  o  O  O  O  cn  ». cn  CO  O  o in  , ^  cn  N*  rH  o rH  cn  H  N*  O  o  o O  6N  o o  o o  00  CO N?  NO ON  NO ON  o LO  CN  dN  o co  in  cn  NO ON  o il  CO  CN  0"N  NO ON  t> ID  NO ON.  o o  N*  '  o  N C CU  " Q . co  NO ON  N? ^-^  o  ro ^  CD  N? CN  O  ffl  0 •H  o \0  0)  s—v  0-  a CJN •H  >H  , ,  s  O o < -H e o fO -H fl o  o  H  NP ON cn  c TS CO^ -H cy •H rH •H ft I  o CN  rH  •—v  CJ •H  NO ON  >1 ro  •  •H  53 H  <3  CO  TJ  ^ NP O N CO  o  IH  -H  A  /  •*  o  o  •H 4->  CJ •H  -H •p  o ro  o  O O  o o  in  co  tr;  EH W cn S O >,  o OS  rH  <d EH  rH  CN  rH  1  o  CN  W  a H  53 <  o u  w  TABLE I V . M e t a b o l i c  products  administered i n brackets  radioactivity the p r e c e d i n g  U  s  H  m  I W  55 H  o  H £  I  I P  CN  I  H  <  CJ <  o  CJ  p  H  g H u  cu  Percentages  u  u H  >;  H  CJ  u <  H  •NT  rH  H  1  55  Q w  CO  1  cn 1  p  rH  CJ  U  rH  rH  recovered figure.  o  53 W  from v a r i o u s a r o m a t i c  compounds  t o P_. h i s p i d u s . indicate  the f r a c t i o n  on t h e c h r o m a t o g r a m  o f the t o t a l  represented  by  83 radioactive  substances  on chromatograms and i t i s f o r t h i s  reason t h a t  i t i s employed here i n a q u a l i t a t i v e manner.  The data o b t a i n e d from f e e d i n g p h e n y l a l a n i n e were v e r y s i m i l a r t o those from the experiment hydroxybenzoic  a c i d was  radioactive  just described.  P_-  on chromatograms o f a l l  t h r e e growth stages examined, r e p r e s e n t i n g 22% o f the r e covered r a d i o a c t i v i t y a f t e r 11 days, 5% a f t e r 20 days.  7% a f t e r 14 days and  P r o t o c a t e c h u i c a c i d showed no  radioactivity  a f t e r 11 days and r e p r e s e n t e d l e s s than 1% o f the r e c o v e r e d r a d i o a c t i v i t y a f t e r 14 and  20  days.  Cinnamic a c i d showed the a n t i c i p a t e d p_-coumaric a c i d and c a f f e i c a c i d ,  conversion to  indicating  that  cinnamic  a c i d - 4 - h y d r o x y l a s e and p-coumaric a c i d - 3 - h y d r o x y l a s e are erative  i n the fungus.  a c i d was  observed  A much h i g h e r r e c o v e r y o f c a f f e i c  i n cultures  which were f e d a f t e r 18 days  growth compared t o the r e c o v e r y from younger c u l t u r e s . i l a r l y , p_-coumaric a c i d metabolized to c a f f e i c The cates that  version of tyrosine  tumulosus  was  acid. t o p_-coumaric a c i d  In a d d i t i o n ,  the s i g n i f i c a n t  t o p_-hydroxyphenylacetic  a d e g r a d a t i v e pathway s i m i l a r t o t h a t  Polyporus  Sim-  indi-  ammonia-lyase a c t i v i t y i s p r e s e n t i n  two-week-old c u l t u r e s .  that  f e d t o two-week-old c u l t u r e s  conversion of tyrosine tyrosine  op-  (Crowden 1967)  acid  con-  suggests  reported f o r  might a l s o be o p e r a t i v e  84 i n t h i s organism.  While r a d i o a c t i v e p_-hydroxyphenylacetic  a c i d was a l s o p r e v i o u s l y d e t e c t e d i n three-week-old f e d r a d i o a c t i v e p h e n y l a l a n i n e a f t e r 48 h r on  cultures  replacement  medium, i t might have been formed v i a t y r o s i n e s i n c e t h e c o n v e r s i o n o f p h e n y l a l a n i n e t o t y r o s i n e a l s o has been observed. The nine  detected conversion o f  3,4-dihydroxyphenylala-  (DOPA) t o c a f f e i c a c i d r e q u i r e s a more thorough exam-  ination.  The background r a d i o a c t i v i t y d e t e c t e d on the chrom-  atograms o f t h e medium e x t r a c t s was s i g n i f i c a n t l y h i g h e r than t h a t d e t e c t e d i n the c a f f e i c a c i d s p o t s , thus the a c c u r a c y o f the method.  lowering  I n a d d i t i o n , DOPA ammonia-lyase  a c t i v i t y i s n o t known i n f u n g i .  Nevertheless, there could  be some b i o l o g i c a l importance f o r t h i s a c t i v i t y i n a p a r a s i t e o f h i g h e r p l a n t s , as a number o f h i g h e r p l a n t s a r e known t o produce DOPA as an i n t e r m e d i a t e i n the f o r m a t i o n o f DOPA-melanin. When b e n z o i c a c i d was f e d t o two-week-old c u l t u r e s o f t h e fungus,  the o n l y i d e n t i f i a b l e product was p_-hydroxy-  benzoic a c i d .  However, the absense o f r a d i o a c t i v e p r o t o -  c a t e c h u i c a c i d on the chromatograms does not i n d i c a t e t h a t r i n g cleavage does n o t o c c u r .  L a t e r s t u d i e s showed t h a t  l a r g e q u a n t i t i e s o f r a d i o a c t i v e carbon d i o x i d e were r e l e a s e d when b e n z o i c a c i d was f e d t o the organism  (Nambudiri,  personal  85  COOH H NCH 2  COOH  COOH H NCH 2  I  CH  II  CH  COOH  OH  OH  COOH  FIG.  26. Aromatic amino a c i d metabolism v i a the cinnamate pathway i n P_. h i s p i d u s .  Dashed l i n e s r e f e r t o i n t e r c o n v e r s i o n s f o r which no d i r e c t evidence has been o b t a i n e d . -^jr has been shown b u t r e q u i r e s a d d i t i o n a l examination (see t e x t ) .  86 communication). The d e t a i l s o f a r o m a t i c amino a c i d metabolism by these s t u d i e s a r e shown i n F i g u r e 26.  elucidated  A "grid-like"  net-  work o f i n t e r c o n v e r s i o n s o f these a r o m a t i c compounds i s p o s t u l a t e d ; u l t i m a t e l y l e a d i n g t o the f o r m a t i o n o f carbon dioxide.  In the next f e e d i n g experiment  the c o n v e r s i o n o f  many o f the same compounds and o t h e r s t o h i s p i d i n was mined.  The numbers  (1 - 5) i n f i g u r e 26. r e f e r t o some o f  the enzymes examined i n P. h i s p i d u s . i n Chapter  exa-  These w i l l be d i s c u s s e d  Four.  The r a t e o f h i s p i d i n p r o d u c t i o n i s h i g h e s t i n 14to 24-day-old c u l t u r e s  ( F i g . 14).  T h e r e f o r e , 17-day-old  c u l t u r e s were s e l e c t e d f o r examination o f i n c o r p o r a t i o n o f v a r i o u s p r e c u r s o r s i n t o the h i s p i d i n m o l e c u l e .  Two m i c r o -  14 c u r i e s o f each o f D L - p h e n y l a l a n i n e - 2 C, D L - p h e n y l a l a n i n e 14 14 . . . 14 . 3- C, D L - t y r o s i n e - 3 - C, cinnamic a c i d - 2 - C, m a l o n i c a c i d 14 14 . . . 14 2- C and sodium a c e t a t e - 2 - C, 1 JACI o f c a f f e i c a c i d - 2 - C 14 and 0.5 ^ C i o f p_-coumaric eight cultures.  acid-2-  C were f e d t o each o f  A f t e r 24 h r the h i s p i d i n was r e c o v e r e d as  d e s c r i b e d i n Chapter One, the t r i m e t h y l e t h e r was p r e p a r e d and i t s s p e c i f i c a c t i v i t y determined. h i s p i d i n from each f e e d i n g was degraded  In t u r n , the t r i m e t h y l to v e r a t r i c  acid  and the s p e c i f i c a c t i v i t y o f t h i s compound a l s o was d e t e r mined.  In t h i s way i t was p o s s i b l e t o a s c e r t a i n i f these  87 ft H  >H  CO  H • >CHEH ^ H O W U  1 1 1  H  o •H  1 1 1  H > E n <xN  C J H r f -H  o  D H Q  CJ  r^H  H H•  o VO  O O m  CN  m  jfnH . i H >EH I U H H -H W E-tg O ft U H CO < « 5s —  o rH  l  o  rH  X o  cn • cn  O O cn  o o  cn  o rH  l  o rH  X  X  X  rH  rH  •  rH  •  rH  m  •  i rH o X  H•  00 •  CN  o  T 1 O H  •  •  CO  CJ  Q  ON P O  ft  2 O  CJ  -K 9 W  CO H H  2 Q <  1  CN | CD C -H  rcj  CJ  1  o rH  rH  1 cn  l  CD •H C  ro  U rH  1 cn  I a  CD  rH  H  •H CO  >1  a» Xi  ft  rH  Io  rH  X o  • cn  O o CN  o rH  1  X  ro  >i C CD  Xi  ft  0 u  1 1  VO  VO•  o O O  o o rH  cn  1  VO  rH  I o  I o  rH  rH  cn  I o  I o  rH  H  X  X  X  X  o  CO  in  VO  00•  00  in  o  CO rH  vo rH  o•  o•  •  •  o  rH  rH  C  m  lo  CN•  X  CJ  rH  ro  in  X  <N  rH  1  1  CN  cn —  •  CN  •  m •  cn  i rH o X  o  CO  CO  m•  1  co<>  H EH  CN  •  rH (Ti  O  cn CJ>HCJ  o  I  CN | -H CJ  ro  O •H  e  ro  G -H O  •  -H O  ro  O •H  U ro  3 o CJ i, ft  •  •  o  •  •  CN  cj  CJ  1 <tf  CN rH |  H  u rH  rH  1  CN CD +>  •H CJ  -P <D O  rfl CJ •H CD  ro  CJ •H  P  3  CJ  CO  TABLE V. I n c o r p o r a t i o n o f v a r i o u s p r e c u r s o r s  1  TJ •H CJ  ro  •H  1  CN  ro  m m  ro  rH  1  CN |  TS  CJ  1  c o  rH  o  ra  into hispidin  by 17-day-old c u l t u r e s o f P. h i s p i d u s .  88 compounds were l a b e l l i n g the h i s p i d i n , and i f so, whether the phenylpropanoid  moiety was b e i n g i n c o r p o r a t e d i n t a c t .  The data  o b t a i n e d from these f e e d i n g s a r e r e p o r t e d i n Table V. R a d i o a c t i v i t y was i n c o r p o r a t e d i n t o h i s p i d i n from a l l the compounds a d m i n i s t e r e d and, i n g e n e r a l , the data s u b s t a n t i a t e d the h y p o t h e s i s  t h a t the phenylpropanoid  p o r a t e d without  p r e c u r s o r s were i n c o r -  scrambling o f the l a b e l and t h a t a c e t a t e  primar-  i l y was i n c o r p o r a t e d i n t o the pyrone p o r t i o n o f the molecule. In view o f the s t a t i s t i c a l nature o f r a d i o a c t i v e decay, the 95% c o n f i d e n c e l i m i t s  f o r the data were c a l c u l a t e d and these  showed t h a t t h e s p e c i f i c a c t i v i t i e s r e p o r t e d i n Table V. might v a r y as much as + 7% and t h a t the f r a c t i o n s r e p o r t e d i n the l a s t column o f Table V. might v a r y by twice t h i s amount.  This var-  i a t i o n r e p r e s e n t s the maximum found i n any sample. No v a l u e s were o b t a i n e d f o r the s p e c i f i c a c t i v i t y o f 14 v e r a t r i c a c i d from the p h e n y l a l a n i n e - 2 -  C o r the malonic  acid-  14 2-  C feedings.  These samples were l o s t by o v e r o x i d a t i o n o f t h e  trimethylhispidin.  A maximum o x i d a t i o n time o f two hours s h o u l d  be s t r i c t l y adhered t o w i t h t h e o x i d a t i o n c o n d i t i o n s employed. Nevertheless,  i n view o f the data o b t a i n e d from feedings o f o t h e r  compounds, the l a b e l l i n g p a t t e r n i s apparent. D i l u t i o n values f o r the v a r i o u s p r e c u r s o r s must be i n t e r preted c a r e f u l l y .  As e x p l a i n e d p r e v i o u s l y , p o o l s i z e s ,  r a t e s and p e r m e a b i l i t i e s w i l l a f f e c t t h i s d a t a .  turnover  The d i l u t i o n  89 14 14 v a l u e s f o r p h e n y l a l a n i n e - 2 - C and p h e n y l a l a n i n e - 3 - C i n d i c a t e the v a r i a t i o n t h a t might be expected f o r the same compound.  How-  ever, t h e d i l u t i o n f o r cinnamic a c i d i s s i g n i f i c a n t l y h i g h e r than t h a t o b t a i n e d f o r p_-coumaric and c a f f e i c a c i d s . t h i s might seem u n l i k e l y a t f i r s t ,  Although  i t appears more a c c e p t a b l e  i n l i g h t o f the p r e v i o u s experiments which suggested t h a t  cinna-  mic a c i d 4-hydroxylase was a r e g u l a t o r y enzyme and a l t h o u g h 14d a y - o l d c u l t u r e s r e a d i l y c o n v e r t e d cinnamic a c i d t o p_-coumaric acid  (Table IV.) the r e c o v e r y o f h y d r o x y l a t e d cinnamic a c i d s  from 18-day-old c u l t u r e s was a p p r e c i a b l y lower.  Also, a larger  amount o f unconverted cinnamic a c i d was r e c o v e r e d from the medium o f 18-day-old c u l t u r e s  (Table I V . ) .  In addition,  i t has been  shown t h a t when cinnamic a c i d i s p r e s e n t i n t h e c u l t u r e medium o f P_. h i s p i d u s a t a c o n c e n t r a t i o n o f 0.1%, no growth i s observed i n the c u l t u r e s . The data a r e c o n s i s t e n t w i t h a p h e n y l p r o p a n o i d - a c e t a t e (malonate) b i o g e n e s i s o f h i s p i d i n .  Both types o f molecules a r e  r e a d i l y i n c o r p o r a t e d i n t o h i s p i d i n and the r a d i o a c t i v i t y i s l o c a t e d i n the a p p r o p r i a t e p o r t i o n o f the molecule When p h e n y l a l a n i n e (I) o r t y r o s i n e carbon  ( I I ) l a b e l l e d i n the t h i r d  (b*) a r e a d m i n i s t e r e d t o t h e fungus, both t h e t r i m e t h y -  hispidin  (VII) and the v e r a t r i c a c i d  (VIII) o b t a i n e d by o x i d a t i o n  show a p p r o x i m a t e l y the same s p e c i f i c a c t i v i t y ever, when cinnamic a c i d acid  ( F i g . 28.).  (Table V . ) .  ( I I I ) , p_-coumaric a c i d  How-  (IV), c a f f e i c  (V) o r a c e t a t e l a b e l l e d i n the second carbon  (a*, c*) a r e  90 a d m i n i s t e r e d almost no  r a d i o a c t i v i t y i s found i n the  a c i d while t r i m e t h y l h i s p i d i n activity  (Table  V.).  shows good i n c o r p o r a t i o n  veratric of  radio-  91  COOH  FIG.  27. B i o s y n t h e s i s  COOH  and d e g r a d a t i o n o f r a d i o a c t i v e  hispidin.  CHAPTER FOUR  PRELIMINARY STUDIES OF ENZYMES ASSOCIATED WITH AROMATIC  METABOLISM  Introduction The  conversion  of phenylalanine  t o t y r o s i n e and  cinnamic  and p_-coumaric a c i d s i s an a b i l i t y l a r g e l y r e s t r i c t e d t o p l a n t s and  c e r t a i n fungi  (Camm and  Towers 1969).  The  higher  non-oxida-  t i v e deamination o f p h e n y l a l a n i n e  a l s o has  tomyces  i s i m p l i c a t e d i n some b a c t e r i a .  Of the  (Bezanson e t a l 1970)  and  been d e t e c t e d  in  Strep-  f u n g i , p r i m a r i l y Basidiomycetes have been shown to possess  phenylalanine activity  ammonia-lyase  (PAL)  (Power et a l 1965,  and  t y r o s i n e ammonia-lyase  Bandoni e t a l 1968).  (TAL)  I t i s these  en-  zymes i n Polyporus h i s p i d u s which i n i t i a t e the sequence o f enzyma t i c conversions  l e a d i n g t o h i s p i d i n from the aromatic amino a c i d s .  Those enzymes which e f f e c t the h y d r o x y l a t i o n  o f the arom-  a t i c r i n g are a l s o c r i t i c a l t o h i s p i d i n b i o s y n t h e s i s . l i k e l y that hydroxylation  i n P. h i s p i d u s may  l e v e l o f cinnamic a c i d and styrylpyrone,  occur both a t  i t s d e r i v a t i v e s and  b i s - n o r y a n g o n i n and h i s p i d i n .  I t seems the  a t the l e v e l  This i s i n d i c a t e d  by the presence o f t r a c e amounts o f b i s - n o r y a n g o n i n and  strong  b i s - n o r y a n g o n i n 4-hydroxylase a c t i v i t y i n f u n g a l e x t r a c t s . work has  No  been r e p o r t e d on enzymes which are capable o f m e d i a t i n g  hydroxylation  a t the  l e v e l o f the s t y r y l p y r o n e s ,  a l t h o u g h exam-  i n a t i o n s o f enzymes which a c c o m p l i s h h y d r o x y l a t i o n mic  of  a c i d s have been r e p o r t e d  Maier 1972, b u d i r i 1971,  f o r higher  Hahlbrock e t a l 1971) and  references  many f u n g i t o h y d r o x y l a t e  plants  of  (Hasegawa  and micro-organisms  cited therein).  cinna-  The  and  (Nam-  ability  of  b e n z o i c a c i d t o p_-hydroxybenzoic  93 a c i d and p r o t o c a t e c h u i c a c i d i s well-known H a l s a l l e t a l 1969).  (Cain e t a l  1968,  T h i s a c t i v i t y t y p i c a l l y leads t o r i n g  cleavage. C e l l - f r e e p r e p a r a t i o n s o f Polyporus h i s p i d u s were examined f o r PAL and TAL a c t i v i t y .  PAL a c t i v i t y was  investiga-  t e d i n c u l t u r e s o f d i f f e r e n t ages and these data are r e p o r t e d . In a d d i t i o n , a number o f c e l l - f r e e p r e p a r a t i o n s were o b t a i n e d which e f f e c t e d h y d r o x y l a t i o n o f b e n z o i c a c i d , cinnamic a c i d s and the s t y r y l p y r o n e , b i s - n o r y a n g o n i n . M a t e r i a l s and Methods I . P h e n y l a l a n i n e ammonia-lyase C u l t u r e s were examined a t 2 - 4 day i n t e r v a l s f o r pheny l a l a n i n e ammonia-lyase a c t i v i t y .  The c u l t u r e s were  filtered,  the mycelium b l o t t e d w i t h paper towels and the e x t r a c t e d by g r i n d i n g w i t h an e q u a l weight  (equal t o the wet  weight  o f mycelium) o f aluminum oxide, and twice t h i s weight o f 0.05  N T r i s - H C l b u f f e r , pH 8.6.  fuged a t 18,00  The  centri-  g f o r ten minutes i n a r e f r i g e r a t e d  fuge and 5 ml o f the supernatent was 20 cm column o f Sepadex G - 25 den)  s l u r r y was  centri-  passed onto a 2 cm X  (Pharmacia,  Uppsala,  Swe-  p r e p a r e d w i t h the same b u f f e r , f o r removal o f the  low m o l e c u l a r weight m o l e c u l e s . c o l l e c t e d and assayed combining  F i v e ml o f e l u a t e  was  s p e c t r o p h o t o m e t r i c a l l y a t 290 nm  a p o r t i o n w i t h 1 ml o f 2.5 yJA  phenylalanine  after  94 i n b u f f e r s o l u t i o n and s u f f i c i e n t T r i s - H C l b u f f e r t o make a t o t a l o f t h r e e ml i n the spectrophotometer i n c u b a t i o n i n the c u v e t t e was  performed  cuvette.  a t 30°C,  and  The changes  i n o p t i c a l d e n s i t y were r e c o r d e d a t 5 min i n t e r v a l s f o r 20 min.  A l l procedures were performed  assay was  begun and each assay was  a t 0 - 5°C u n t i l  performed w i t h a t l e a s t  two d i f f e r e n t q u a n t i t i e s o f the enzyme p r e p a r a t i o n . was  not determined,  the  Protein  and s p e c i f i c a c t i v i t i e s were r e p o r t e d  w i t h r e s p e c t t o dry weight of  fungus.  I I . T y r o s i n e ammonia-lyase Three ml o f the enzyme p r e p a r a t i o n employed f o r the 14-day p h e n y l a l a n i n e ammonia-lyase assay was f o r t y r o s i n e ammonia-lyase a c t i v i t y .  a l s o examined  To t h i s was  added an  a d d i t i o n a l 5 ml of the supernatant which'had not been passed through the Sephadex G-25  column.  To t h i s t o t a l o f e i g h t  ml  14 was  added 1yaCi o f t y r o s i n e - 3 -  2-mercaptoethanol  C p l u s t h r e e drops o f  to prevent o x i d a t i o n .  cubated f o r 14 h r a t room temperature, with d i e t h y l ether.  5%  The mixture was i n a c i d i f i e d , and e x t r a c t e d  The e t h e r e x t r a c t was  chromatographed  2 - d i m e n s i o n a l l y on c e l l u l o s e TLC p l a t e s i n s o l v e n t systems A and  B.  I I I . Benzoic and cinnamic a c i d - 4 - h y d r o x y l a s e The fungus was above, employing r y was  h a r v e s t e d and macerated  0.1 M phosphate  b u f f e r , pH  as d e s c r i b e d  7.5.  c e n t r i f u g e d a t 27,000 g f o r ten minutes  The  and the  slursup-  95 e r n a t a n t a t 40,000 g f o r a f u r t h e r 10 min.  T h i s supernatant  was then c e n t r i f u g e d a t 105,000 g f o r 90 min t o o b t a i n a microsomal  pellet.  Both the p e l l e t and the supernatant were  examined f o r cinnamic a c i d - 4 - h y d r o x y l a s e a c t i v i t y . The microsomal  p e l l e t was suspended i n two 2 ml a l i -  quots o f 0.1 M phosphate b u f f e r pH 7.5.  To one p e l l e t s u s 14  p e n s i o n and the supernatant were added cinnamic a c i d - 2 -  C  O.S^uCi, n o n - l a b e l l e d cinnamic a c i d 0.15 mg, 2-mercaptoetha n o l 0.24 mg and t h r e e ml o f the NADPH + H tem.  +  generating sys-  To the second p e l l e t suspension was added b e n z o i c a c i d -  (UL) (RL)-  14 C 2/^.Ci,  2-mercaptoethanol  0.24 mg and t h r e e ml  + o f the NADPH + H  + g e n e r a t i n g system.  To p r o v i d e NADPH + H  f o r the enzymes, a mixture o f 1.6 mg NADP , 1.4 mg g l u c o s e +  6-phosphate and 0.4 u n i t s glucose-6-phosphate  dehydrogenase  i n t h r e e ml 0.1 M phosphate b u f f e r , pH 7.5, was prepared and i n c u b a t e d a t 30°C f o r t e n minutes b e f o r e a d d i t i o n t o the assay m i x t u r e . The assay mixture was i n c u b a t e d w i t h a g i t o a t i o n f o r 90 min a t 30 C.  The r e a c t i o n was t e r m i n a t e d by the  addition of hydrochloric acid. p_-hydroxybenzoic atography  R a d i o a c t i v e p_-coumaric and  a c i d s were examined for, employing  paper chrom-  i n s o l v e n t systems A and B.  IV. Bis-noryangonin-3-hydroxylase C u l t u r e s grown f o r 14 days on GYSS supplemented w i t h cinnamic a c i d  (3yxg/ml f o r 13 days and 0.1% f o r l a s t 24 h r )  96 were e x t r a c t e d  i n a mortar w i t h powdered g l a s s and  citrate-phosphate  b u f f e r , pH  7.0.  The  as the crude enzyme p r e p a r a t i o n .  phosphate b u f f e r , pH  7.0  t e d a t 30°C f o r 1 hour. t u r e was  and  Two  O^uMoles) a s c o r b a t e i n b u f f e r and  ether  t r e a t e d w i t h 0.5  (15 ml  passed onto a 2  total).  The  c o l l e c t e d and  0.2  ml  A t the end ml  5 N HCl  ether  and  M  citrateincuba-  extracted with d i e t h y l  e x t r a c t was p l a t e and  reduced i n volume  developed i n  A c o n t r o l t o which the enzyme was  b a t i o n was  s i m i l a r l y examined.  and  0.1  solvent  added a f t e r i n c u -  Discussion  In the assay f o r p h e n y l a l a n i n e ammonia-lyase, an i n absorbance i s r e c o r d e d a t 290 t o cinnamic a c i d .  ure o f the PAL  activity.  The  rate with respect  nm  rate of increase  i s a meas-  a l i q u o t s o f crude  employing the d i f f e r e n c e i n  t o the d i f f e r e n c e i n a l i q u o t s i z e as  measure o f a c t i v i t y , e r r o r s are minimized. ent s e t s o f c u l t u r e s were a n a l y z e d f o r PAL s e t s showed maximum PAL  increase  as p h e n y l a l a n i n e i s con-  By examining two  enzyme o f d i f f e r e n t s i z e s and  all  1.5  o f the r e a c t i o n time the mix-  system C.  verted  cm  used  ml o f crude enzyme,  were mixed i n a t e s t tube and  banded onto a c e l l u l o s e TLC  Results  cen-  column o f Sephadex G - 25 p r e p a r e d i n the same b u f f e r .  Twice the volume a p p l i e d t o the column was  ml  M  crude e x t r a c t was  t r i f u g e d a t 10,000 g f o r twenty minutes and X 20 cm  0.1  the  Three d i f f e r activity  and  a c t i v i t y a t the same c u l t u r e  age.  97 B T l l O a U3d  ( I M Ajp  FIG.  28.  B/u!iu/063 v)  Phenylalanine of  P_.  a O  1HOI3M  A1IAI10V  ammonia-lyase  h i s p i d u s .  AUG  a c t i v i t y  "IVd  i n  cultures  98 Maximum PAL a c t i v i t y was observed i n 14-day-old  cultures  ( F i g . 29.) which compares f a v o u r a b l y w i t h the observed v e r s i o n o f p h e n y l a l a n i n e t o cinnamic a c i d i n v i v o However, P. h i s p i d u s d i f f e r s  con-  (Table I V . ) .  from Rhodotorula g l u t i n u s and  Sporobolomyces roseus i n the time o f maximum PAL a c t i v i t y . While the l a t t e r organisms  showed maximum PAL a c t i v i t y as  the c u l t u r e s e n t e r e d the s t a t i o n a r y phase o f growth  (Ogata  e t a l 1967, Camm and Towers 1969), maximum PAL a c t i v i t y i n P. h i s p i d u s occurs  near the end o f the l o g a r i t h m i c phase  o f growth. R a d i o a c t i v e p_-coumaric  a c i d from the t y r o s i n e  ammonia-  l y a s e assay mixture was d e t e c t e d by a u t o r a d i o g r a p h y . t h i s confirms t h a t the enzyme i s a c t i v e i n c e l l - f r e e  While prep-  a r a t i o n s o f 14-day-old c u l t u r e s , no attempt was made t o d e t e r mine i f t h i s was the same enzyme which demonstrated  PAL  a c t i v i t y , o r i f two d i f f e r e n t enzymes were r e s p o n s i b l e f o r these a c t i v i t i e s as has been shown i n U s t i l a g o e t a l 1967) and i n the h i g h e r p l a n t Ipomoea Uritani  (Subba Rao  (Minimikawa and  1965). Both cinnamic a c i d and b e n z o i c a c i d were h y d r o x y l a t e d  i n the para p o s i t i o n by the microsomal  pellet preparation.  However, no cinnamic a c i d - 4 - h y d r o x y l a s e a c t i v i t y was observed i n the supernatant p r e p a r a t i o n from the 105,000 g c e n t r i fugation.  While b e n z o i c a c i d was r e a d i l y c o n v e r t e d t o  99 p_-hydroxy-benzoic a c i d  (12%), l i t t l e more than 1% c o n v e r s i o n t o  p_-coumaric a c i d from cinnamic a c i d was  effected.  Further  s t u d i e s o f cinnamic a c i d 4-hydroxylase i n crude enzyme p r e p a r a t i o n s from J>. h i s p i d u s have f a i l e d t o show more than c o n v e r s i o n i n a 90 minute i n c u b a t i o n p e r i o d communication).  1-2%  (Vance, p e r s o n a l  A c r i t i c a l study o f the f a c t o r s which con-  t r o l the a c t i v i t y o f t h i s enzyme i s r e q u i r e d . The b i s - n o r y a n g o n i n 4-hydroxylase p r e p a r a t i o n c o n v e r t e d a p p r o x i m a t e l y 2yUmoles o f b i s - n o r y a n g o n i n t o h i s p i d i n i n 1 hour. No h i s p i d i n was  d e t e c t e d i n the c o n t r o l .  T h i s suggests t h a t  h i s p i d i n can a l s o a r i s e from cinnamic a c i d through h y d r o x y l a tion of styrylpyrone.  The presence o f b i s - n o r y a n g o n i n i n crude  f u n g a l e x t r a c t s adds f u r t h e r support t o t h i s c l a i m .  I t has  not been determined whether the enzymes r e s p o n s i b l e f o r the h y d r o x y l a t i o n o f these v a r i o u s aromatic compounds a r e a c t u a l l y different.  S i n c e many f u n g i produce h y d r o x y l a t e d d e r i v a t i v e s  o f b e n z o i c a c i d w i t h o u t y i e l d i n g h y d r o x y l a t e d cinnamic a c i d s and s t i l l  o t h e r f u n g i w i l l h y d r o x y l a t e cinnamate,  l i k e l y t h a t d i f f e r e n t enzymes are i n v o l v e d .  i t seems  GENERAL SUMMARY AND CONCLUSIONS  100 General Summary and  Conclusions  C u l t u r a l c o n d i t i o n s amenable t o examining the  cinnamate  pathway and h i s p i d i n b i o s y n t h e s i s i n Polyporus h i s p i d u s were established.  A medium c o n t a i n i n g g l u c o s e ,  enzymatic h y d r o l y s a t e having  o f soybean meal and  an i n i t i a l pH o f 7 was  yeast extract, several salts  an  and  the most s u i t a b l e found f o r o  these s t u d i e s .  An  i n c u b a t i o n temperature o f 23 C was  e c t e d f o r optimum pigment p r o d u c t i o n . be n e c e s s a r y f o r pigment  formation  L i g h t was  and  this  c o u l d not be r e p l a c e d by hydrogen p e r o x i d e hydrochloride.  Blue l i g h t was  sel-  shown t o  requirement  o r hydroxylamine  most e f f e c t i v e i n s t i m u l a t i n g  pigment development. The  maximum r a t e o f h i s p i d i n p r o d u c t i o n  t a b l i s h e d c u l t u r a l c o n d i t i o n s was  es-  observed to l a g the max-  imum growth r a t e by about f i v e days. senescent, the y i e l d o f h i s p i d i n was ly.  under the  As  the c u l t u r e s became  observed t o f a l l  I t i s proposed t h a t t h i s d e c l i n e i s a s s o c i a t e d  rapid-  with  o x i d a t i v e a c t i v i t y r e f l e c t e d i n browning o f the c u l t u r e s . P. h i s p i d u s was t u r e s when  observed t o s p o r u l a t e  vigorously-growing  were employed as inoculum. t a i n e d from the d e v e l o p i n g t e r s i x months.  i n agar  h y p h a l t i p s i n agar  Viable basidiospores sporocarp and  blocks  were  ob-  these germinated a f -  I t i s suggested t h a t a c r i t i c a l l y  t u r e l e v e l i n the s u b s t r a t e t r i g g e r e d the  cul-  low  germination.  mois-  101 P_-coumaric, c a f f e i c , p_-hydroxyphenylacetic, b e n z o i c and p r o t o c a t e c h u i c a c i d s were observed n a t u r a l l y i n c u l t u r e s o f the fungus.  p_-hydroxy-  t o occur  By f e e d i n g r a d i o a c t i v e l y -  l a b e l l e d compounds t o these c u l t u r e s , i t was p o s s i b l e t o el u c i d a t e the d e t a i l s o f the cinnamate pathway proposed i n Figure 26.  The d e g r a d a t i o n o f p h e n y l a l a n i n e was a l s o p r o -  posed from the evidence o b t a i n e d  (Fig. 2 4 . ) .  In o t h e r f e e d i n g experiments v a r i o u s r a d i o a c t i v e molecules  the i n c o r p o r a t i o n o f  i n t o h i s p i d i n was examined.  These r e s u l t s p r o v i d e good evidence  f o r the h y p o t h e s i s t h a t  f u n g a l s t y r y l p y r o n e s a r e b i o s y n t h e s i z e d through  the conden-  s a t i o n o f two u n i t s o f a c e t i c a c i d w i t h cinnamic a c i d s ( p o s s i b l y as e s t e r s o f Coenzyme A ) . yangonin presence  In a d d i t i o n , b i s - n o r -  was d e t e c t e d i n c u l t u r e s o f t h e fungus. of this  compound^ t o g e t h e r  with  The  obtaining a  crude enzyme p r e p a r a t i o n capable o f h y d r o x y l a t i n g i t t o h i s p i d i n , suggests  t h a t more than one pathway t o h i s p i d i n i s  o p e r a t i v e i n Polyporus h i s p i d u s ( F i g . 3 0 . ) . Other  crude enzyme p r e p a r a t i o n s have been o b t a i n e d  from c u l t u r e s o f P. h i s p i d u s .  Although  no attempts  were made  t o p u r i f y and c h a r a c t e r i z e the enzymes, demonstrating  their  presence p r o v i d e s the b e s t evidence  con-  version.  for a biochemical  Both PAL and TAL a c t i v i t y were d e t e c t e d i n f u n g a l  e x t r a c t s w i t h maximum PAL a c t i v i t y r e c o r d e d d u r i n g the  102 COOH  COOH  COOH  I I  I I I  OH OH  o  OH  O il J-SCoA  C-SCoA II  V  OH H  HO  OH  HO.  T V I I  V I I I  'OH OH FIG.  OH  29. A l t e r n a t e r o u t e s proposed f o r the b i o s y n t h e s i s o f h i s p i d i n i n Polyporus h i s p i d u s .  I cinnamic a c i d , I I p_-coumaric a c i d , I I I c a f f e i c a c i d , I V cinnamoylCoenzyme A, V p_-coumaroylCoenzyme A, V I c a f feoylCoenzyme A, V I I s t y r y l pyrone, V I I I b i s - n o r y a n g o n i n , DC h i s p i d i n .  103 l o g a r i t h m i c phase o f growth.  Enzymes capable  l a t i n g cinnamic a c i d , b e n z o i c  a c i d and b i s - n o r y a n g o n i n  were p r e s e n t  i n c e l l - f r e e preparations.  o f hydroxy-  Also, l a t e r  evi-  dence has shown t h a t a crude enzyme p r e p a r a t i o n from t h i s fungus w i l l h y d r o x y l a t e  p_-coumaric a c i d t o c a f f e i c a c i d (Nam-  b u d i r i , p e r s . comm.). Although the maximum y i e l d o f h i s p i d i n from c u l t u r e s o f P. h i s p i d u s was o n l y 10% o f t h a t from c u l t u r e s o f P. schweinitzii  ( H a t f i e l d 1970), j?. h i s p i d u s has proved t o be  an e x c i t i n g organism f o r b i o c h e m i c a l  study.  Cultures of this  fungus perform numerous enzymatic c o n v e r s i o n s compounds r e p o r t e d from o n l y a few o t h e r and  o f aromatic  Basidiomycetes,  i t has been shown t h a t a c t i v e e x t r a c t s o f s e v e r a l o f these  enzymes can be o b t a i n e d  readily.  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S t u d i e s on 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 n Neurospora c r a s s a . A r c h . Biochem. Biophys. 50:71-80.  113 APPENDIX A. C h a r a c t e r i s t i c s o f Wrattan f i l t e r s employed i n experiments d e t e r m i n i n g the e f f e c t s o f the wavelength o f the l i g h t  source  on growth and pigment development. Minimum 10% T F i l t e r No.  C o l o u r o f l i g h t passed  Bandpass  (nm)  H-45  Blue  430 - 540  B-58  Green  500 - 620  G-15  Red & Y e l l o w  520 - 700+  A-25  Red  590 - 700+  114 APPENDIX B. Spray reagents  employed i n the d e t e c t i o n o f v a r i o u s compounds  s e p a r a t e d by paper and t h i n - l a y e r chromatographic 1. D i a z o t i z e d p _ - n i t r o a n i l i n e reagent 5 ml o f 0.3% p _ - n i t r o a n i l i n e ,  techniques  (Ibrahim and Towers 1960) : 1 ml o f 5% sodium n i t -  r i t e and 15 ml o f 20% sodium a c e t a t e were combined, i n t h a t order,  j u s t p r i o r t o use.  A f t e r spraying with t h i s  the chromatogram was allowed  t o d r y b r i e f l y and then oversprayed  w i t h a 5% aqueous s o l u t i o n o f NaOH. appear as v a r i o u s l y c o l o u r e d  solution  P h e n o l i c compounds  spots.  2. F e r r i c c h l o r i d e reagent: 2% f e r r i c c h l o r i d e i n 95% e t h a n o l ; d e t e c t s dihydroxy  ortho-  p h e n o l i c compounds as g r e e n i s h o r brownish s p o t s .  3. M o d i f i e d E h r l i c h s reagent: 1  2% £-dimethylaminobenzaldehyde i n e t h a n o l - H C l (3:1) d e t e c t s s t y r y l p y r o n e s as shades o f b l u e , green o r y e l l o w . 4. p_-dimethylaminocinnamaldehyde 0.5%  reagent:  p_-dimethylaminocinnamaldehyde i n 0.5N HC1 d e t e c t s  s t y r y l p y r o n e s v a r i o u s l y as p u r p l e s , greens o r b l u i s h 5. Bromocresol green 0.04%  greens.  reagent:  i n 96% e t h a n o l w i t h the a d d i t i o n o f 0.1 N NaOH  u n t i l the s o l u t i o n j u s t t u r n s b l u e . as y e l l o w spots on a b l u e background.  Detects  a c i d i c compounds  A l l traces of a c i d i c  s o l v e n t s must be removed from chromatograms b e f o r e  spraying.  

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