Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion

Journal of Biotechnology, 20 (1991)271-278

271

© 1991 Elsevier Science Publishers B.V. All fights reserved 0168-1656/91/$03.50 ADONIS 016816569100136W BIOTEC 00670

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion M. Didek-Brumec

l, V.

Gaberc-Porekar 2 M. Ala~evi6 3, S. Milifii6 1 and H. So~i~ 2

t LEK, Pharmaceutical and Chemical Works, Ljubljana, Yugoslavia, 2 Boris IOdri6 Institute of Chemistry, Ljubljana, Yugoslavia and 3 Faculty of Food and Biotechnology, Zagreb, Yugoslavia

(Received 11 March 1991;revision accepted 1 May 1991)

Summary Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crossings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established. Claviceps purpurea; Protoplast fusion; Ergot alkaloids; Prototrophic isolates

Introduction Claviceps purpurea is a commercially important filamentous fungus used in the production of a broad spectrum of ergot alkaloids with various therapeutic uses. Most submerged active commercial strains were isolated from parasitical strains by mutation and selection techniques. A novel approach to obtaining high yielding

Correspondence to: M. Didek-Brumec, Boris Kidfi~ Institute of Chemistry, 61115 Ljubljana, P.O. Box"

30, Hajdfihova 19, Yugoslavia.

272 strains was provided by genetic recombination, which becomes an important adjunct to mutagenesis once several lineages of mutants have been established. With the development of the protoplast fusion technique, it is now relatively simple to recombine the properties of a wide variety of industrial microorganisms that are relatively uncharacterized genetically or biochemically. In Claviceps sexual crossing may be observed only in parasitic conditions in which genetic materials from different parental auxotrophic and fungicide resistant mutants can recombine (Tudzynski et al., 1982). The recent demonstration (Brauer and Robbers, 1987) that a parasexual cycle can function in Claviceps gave another possibility for genetic recombination on the basis of mitotic crossing over. Spalla and Marnati (1978, 1982) reported that protoplasts of two strains of C. purpurea producing different alkaloids could be fused, giving rise to new heterokaryons in which auxotrophy and alkaloid production were complemented. An intraspecific fusion of two auxotrophic mutants of Claviceps sp. strain SD58 was also performed by Robbers (1984) resulting in fused strains capable of producing greater amounts of alkaloids than the starting strains. In an attempt to change the spectrum of alkaloids produced, SpaIla and Marnati (1982) also used the protoplast technique for an interspecific fusion of C. purpurea and Claviceps paspali auxotrophic strains. Hybrid colonies showed complementation of nutritional requirements, but produced only ergocristine. Robbers (1984) attempted an interspecific fusion between ergotamine producing strain of C. purpurea and the clavine producing strain SD58, which resulted in hybrid strains producing both types of alkaloids. In this report we present recombination studies performed with morphologicaUy and biochemically different C. purpurea strains producing ergotoxines. Combining inactive strains with active strains and vice versa, we tried to find the possibility of activating alkaloid synthesis in the fused strains and also to establish the relationships and appearance of different alkaloids introduced in the cross. Materials and Methods

Microorganisms Claviceps purpurea strains listed in Table 1 were used in the study. The active strains, CP6 and CP10 were obtained from the high yielding ergocornine-ergokryptine strain L-17 and ergocristine strain L-18, respectively (Didek-Brumec et al., 1987, 1988). Inactive strain CP8 was isolated from the selected parasitic ergocristine strain and was incapable of producing alkaloids in submerged conditions. Strain CP3 was isolated from the L-17 strain and produced only traces of alkaloids. Isolation of mutants Auxotrophs were isolated after UV-irradiation (150 J mm -2) and additionally fungicide-resistant clones were selected.

273 TABLE 1 Characteristics of isolated doubly marked Claoicepspurpurea strains Type of strain

Original strains CP8

Parent strains CP46

met capr

Active ECN-EKT

CP6

CP34 .

I/z benr

Inactive ECN-EKT

CP3

CP48

met or cys c a p r

Active ECT

Cpl0

CP47

arg actr

Inactive ECT

Genotypes

ECT, ergocristine;ECN, ergocornine;EKT, ergokryptine;ECN-EKT, ergocornine-ergokryptine.

Preparation and fusion of protoplasts Mycelium was cultivated in S medium for 48 h and treated with /3-glucuronidase-arylsulphatase enzyme as described previously, (Kus et al., 1987). Fusion experiments in P E G (6000) were performed according to Spalla and Marnati (1978).

Culture media Minimal medium SL (Spalla and Marnati, 1978) was used for isolation and maintenance of prototrophs and testing the strains. The complete medium SP (Spalla and Marnati, 1978) served for resistance control and segregation analyses. Fungicides employed in the complete medium were: Benomyl(ben), du Pont (30 /~g ml-1); Captane(cap), Serva (300/zg ml-1); Actidione(act), Serva (15/zg m l - l ) . Amino acids (Merck) were employed in minimal medium (Sermonti, 1969).

Submerged growth This was carried out in media according to Kobel and Sanglier (1976).

Cell volumes These were calculated by the equation: ,

v = ~-~

(Sipiczky and Ferenczy, 1977), a and b representing the long and the short axes of" a cell.

274

Nuclei These were Giemsa stained (Didek-Brumec et al., 1987).

Genetic stability This was followed on the basis of the phenotypes observed after transferring colonies from the master plates of complete media on which segregational events occurred to a series of diagnostic media, each lacking one of the substances required by the haploid parents and containing a drug to which one of the haploid parents is resistant and the other sensitive.

Alkaloids Total alkaloids were determined as described earlier (So~i~ et al., 1985). For quantitative analyses of individual alkaloids a fluoro-densitometric method was used (Pro~ek et al., 1977).

Results and Discussion

For genetic recombination studies by protoplast fusion we introduced auxotrophic and resistance markers to various fungicides into different Claviceps purpurea strains. In this way numerous stable, doubly marked mutants of morphologically and biochemically different C. purpurea strains were isolated and characterized (Table 1). Resistance markers to similar fungicides (benomyl, cercobin, bavistin), as well as auxotrophic markers, were also employed by Tudzynski et al. (1982), while some other authors (Spalla and Marnati 1978, 1982; Robbers, 1984; Brauer and Robbers, 1987) performed recombination studies with auxotrophic Claviceps strains. By crossing the various doubly marked mutants we obtained several prototrophic isolates, which were quite stable on minimal media. In comparison with the original and parental strains, the isolates differed in their morphological and cytological characteristics. On minimal media violet pigmented prototrophic colonies were observed (Fig. 1), mostly larger and completely different from the parental colonies. Similar observations were made by Spalla and Marnati (1978) but colonies of their fused strains, although different, exhibited the characteristics of one or other parent or of an intermediate character. The same authors reported (1982) that strains resulting from interspecific fusion formed colonies with sectors when growing on minimal and complete media. We have observed such sectoring of prototrophic colonies on complete media, the individual sectors differing in morphology, pigmentation and nutritional requirements. These results indicate that complete media could be employed in segregational analyses of offspring derived from prototrophic isolates without addition of agents promoting haploidization as used by Brauer and Robbers (1987).

275

J

/I CP34

2P46

~'~-'~-

"

[ CP46 x CP34 )

\ 0 vP.-k-~._::T]';:, . "~ ~r,

Fig. 1. Morphological characteristics of Claviceps purpttrea prototrophic isolates after crossing of: (a) inactive ergocristine (CP46) with active ergocornine-ergokryptine strain (CP34) and (b) inactive ergocornine-ergokr3,ptine strain (CP48) with active ergocristine strain (CP47).

During growth we also observed a typical and significant differentiation of fused strains, which was even more pronounced than the morphological differentiation of highly-productive C. purpurea strains (Didek-Brumec et al., 1987). Sclerotia-like morphology with polynuclear cells in the vegetative mycelium and especially in the productive mycelium was typical for both types of prototrophic isolates, these phenomena being consistent with the findings of Spalla and Marnati (1978). Prototrophic clones were further examined for their cell size (Table 2). As expected, the size of the cells of both types of isolates was significantly greater TABLE 2 Cell size of prototrophic Ctauiceps purpurea isolates Strain Original strains CP8 CP6 CP3 CP10 Prototrophic isolates CP46 × CP34 CP48 × CP47

Length of cells (/zm)

Width of cells (jzm)

Volume of cells (/~m 3)

9.08 5.58 8.92 13.86

3.07 3.33 4.22 3.30

51.62 37.54 91.93 88.16

10.94 11.02

6.97 9.0

386.55 377.77

Results represent the mean value of 50 cell measurements of an average sample.

276 TABLE 3 Alkaloids synthesized by prototrophic isolates of Claviceps purpurea Strain

Specific * alkaloid content (mg g - l )

ECT

ECN

EKT

(%)

CP8 original

0

-

-

-

CP46 parent

0

-

-

-

CP6 original

8.38

0

8.62

CP34 parent

0

-

-

(CP46 x CP34) Prototrophic isolate

1.45

8.47

21.28

19.07

CP3 original

0.4

0

17.4

22.42

CP48 parent

0

-

-

-

0

0

-

-

CP10 original

11.25

57.02

23.6 -

CP47 parent

0

× CP47) Prototrophic isolate

2.3

-

(CP48

16.54

24.07

24.25

ECT, ergocristine; ECN, ergocornine; EKT, ergokryptine. * Each result represents the average of 10 analyses.

t h a n t h a t of the original strains, a n d m a y serve as a d d i t i o n a l e v i d e n c e that the p r o t o t r o p h i c clones isolated a r e real fusion p r o d u c t s . In following t h e alkaloid p r o d u c t i o n o f the strains o b t a i n e d a f t e r fusion of inactive E C T with the active E C N - E K T strain, it was f o u n d that the isolates w e r e c a p a b l e o f p r o d u c i n g all t h r e e o f the e r g o t o x i n e a l k a l o i d s ( T a b l e 3). Similar results w e r e o b t a i n e d a f t e r fusion o f inactive E C N - E K T strains with t h e active E C T strain w h e r e synthesis o f E C N - E K T was activated. T h e m e c h a n i s m s o f the activation o f a l k a l o i d synthesis a r e u n k n o w n , b u t we s u p p o s e t h a t an inactive strain m u s t c o n t a i n the c o m p l e t e g e n e t i c i n f o r m a t i o n for the synthesis o f various e r g o t a l k a l o i d species, which can b e e x p r e s s e d only a f t e r fusion by m e a n s of some, yet unknown, i n d u c e r m o l e c u l e , which originally f u n c t i o n e d only in t h e active strain. A n o t h e r possible e x p l a n a t i o n could b e a kind o f d a m a g e o f t h e r e g u l a t o r y g e n e in t h e

277 inactive strain, the activity of which can b e u n d e r t a k e n , after c o m p l e m e n t a t i o n , by the a p p r o p r i a t e regulatory g e n e of the active strain. I n o u r e x p e r i m e n t s we observed that the c o m p l e m e n t a t i o n of alkaloid synthesis p r o c e e d e d in q u a n t i t a t i v e ratios in b o t h types of crosses. However, in c o m p a r i s o n with the original strains the biosynthetic activity of the fused strains was m u c h lower, b u t as regards the p a r e n t a l doubly m a r k e d m u t a n t strains used for fusions, which were completely inactive in s u b m e r g e d culture, the alkaloid yields o b t a i n e d were high e n o u g h to d e m o n s t r a t e the activation of distinct alkaloid biosynthesis in inactive p a r t n e r strains. As previously r e p o r t e d ( F e r e n c z y et al., 1987), genetic m a r k e r s exhibit a negative i n f l u e n c e o n productivity, b u t their i n t r o d u c t i o n is necessary to prove that r e c o m b i n a t i o n really proceeds. T h e biosynthetic activity as well as the alkaloid spectra of the p r o t o t r o p h i c isolates r e m a i n e d d u r i n g several g e n e r a t i o n s . T h e fused strains were perfectly stable o n m i n i m a l m e d i u m , b u t o n c o m p l e t e m e d i u m segregation of p r o t o t r o p h i c

TABLE 4 Segregational analyses of different Claviceps purpurea prototrophic isolates A. Prototrophic isolate (ben r liz+ met +) Parent strains: benr liz- met + xben s liz+ metPhenotype

Types of segregants (%) Stages I

ben + ben ben ben + + (+ ben +

+ liz liz liz + + liz + met met

met + + met + met met +) or liz or liz

12 40 0 0 0 0 0 48 0 0

II

III

IV

4 27 0 1 0 0 0 68 0 0

0 32 0 0 0 0 4 64 0 0

0 24 0 0 10 0 0 66 0 0

0 18 0 0 0 0 0 76 6 0

0 64 0 0 4 0 8 24 0 0

B. Prototrophic isolate (act r arg + met + ) Parent strains: act r arg- met + × act s arg + metTypes of segregants (%) Phenotype Stages I act + act act act + + (+ act +

+ arg arg arg + + arg + arg arg

met + + met + met met +) or met or met

II

III

76 0 0 0 12 0 0

20 0 0 0 20 4 0

12

48

0 0

0 8

40 8 0 0 4 4 0 0 0 36

IV 32 4 0 0 8 4 0 44

4 4

0 16 0 16 0 0 0

52 0 0 0 24 4 0

0 0 0 0 73 0 0

44 0 0 0 48 0 0

24 0 0 0 36 0 O

64

20

27

4

0 4

0 0

0 0

0 4

32 8 0

278 c h a r a c t e r i s t i c s a p p e a r e d . T h e t y p e s o f s e g r e g a n t s w e r e d e t e r m i n e d on t h e basis o f t h e i r p h e n o t y p e s , i n c l u d i n g two n u t r i t i o n a l m a r k e r s a n d o n e r e s i s t a n c e m a r k e r for e a c h t y p e o f crossing b e i n g a n a l y s e d ( T a b l e 4). A n a l y s e s o f s e g r e g a n t p h e n o t y p e s s h o w e d c h a r a c t e r i s t i c s o f a p a r a s e x u a l cycle, which a r e e v i d e n t f r o m t h e r e a r r a n g e m e n t s o f p a r e n t a l s t r a i n characteristics. F u r t h e r , it is e v i d e n t f r o m t h e s e g r e g a n t p h e n o t y p e s t h a t p r o t o t r o p h i c isolates a r e h e t e r o k a r y o n s o r diploids. T h e results o b t a i n e d suggest t h a t h y b r i d i z a t i o n by p r o t o p l a s t fusion c a n b e a d v a n t a g e o u s n o t only for c o m p l e m e n t a t i o n b u t also for activation o f distinct e r g o t alkaloid production.

Acknowledgement T h e financial s u p p o r t o f t h e M i n i s t r y for R e s e a r c h a n d T e c h n o l o g y o f Slovenia is g r a t e f u l l y a c k n o w l e d g e d .

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