Zbl. Bakt. II. Abt., Bd. 133, S. 551-556 (1978)
[National Research Centre, Dokki, Cairo, Egypt, and Institute of Microbiology, Czechoslovak Academy of Sciences, Prague.]
Variability of Requirements to Fermentation Media purpurea Strains
III
Claviceps
O. HAMED EL-SAYED and Z. REHACEK With 3 Figures
Summary The alkaloid-synthesizing activity of the saprophytic Claviceps purpurea strains was Yariable. This fact was partly due to different requirements of the cultures to nutriticnal conditions. Not only single strains, but also formation of intra- and extracellular alkaloids differed in this respect. In case of extracellular alkaloids, the influence of medium was more expressive. A positive correlation between accumulation of cell polysaccharides and the level of intracellular alkaloids was evident. Constant or decreasing cell proteins were favourable for alkaloid formation.
Zusammenfassung In saprophytischen Claviceps purpurea-Stammen wurde eine variable Aktivitat der Alkaloidbildung festgestellt. An dieser Variabilitat widerspiegeln sich die Unterschiede in den Al~spriichen der einzelnen Stamme an das Medium. Auch die Biosynthese der intra- und extrazellularen Alkaloide war von der Zusammensetzung des Nahrmediums abhangig. 1m Faile der extrazellularen Alkaloide war der Einflu13 des Mediums besonders deutlich. - Es wurde eine positive Korrelation zwischen der Speicherung von Zellpolysacchariden und intrazellularen Alkaloiden beobachtet. - Ein gleichbleibender oder sinkender Zellproteinspiegel war fUr die Alkaloidbildul'g giinstig.
The first convincing demonstration of a production of ergot alkaloids in vitro (ABE 1951) was followed by a series of papers on the saprophytic nutritional requirements. However, it is still impossible to make any generalization of the factors, influencing alkaloid formation, because of great differences among the strains. In addition, nutritional conditions that favour maximum primary shunt metabolism in a given case may not be expected to favour maximum alkaloid synthef'is (REHACEK 1971). In this investigation, submerged cultures of three strains of Claviceps purpurea (Fr.) Tu!. on different fermentation media were examined. The purpose of this presentation was to gain information that may lead to better understanding of the physiology of alkaloid biosynthesis.
Materials and Methods Organisms and culture conditions The following saprophytic strains of Claviceps purpurea (Fr.) Tul. were used in this work: Pia 4, Plo 2, and ATCC 14934. The strain Pia 4 (HRONES and KYBAL 1962) was prepared from the ground plectenchymatous tissues of the sclerotia (lhclcovA and REH..\.CEK 1968). 36"
552
O. H. EL-SAYED and Z. REHACEK
Nutrient media of the following types were used in fermentation experiments: A B C D E F
-
mannite, ammonium succinate (Bu'LooK and BARR 1968); mannite, glucose, ammonium succinate (VINING 1970); mannite, sucrose, ammonium succinate, yeast extract (KAPLAN et al. 1969); sorbitol, ammonium succinate (O'BAYASHI and KANIE 1966); sucrose, ammonium citrate (MAIER et al. 1971); glucose, ammonium phosphate (KOBEL et al. 1964).
The fermentation experiments were carried out in 300 ml Erlenmeyer flasks, containind 100 ml of medium. The flasks were incubated at 24° ± 1 °C on a rotary shaker, operating at 240 rpm with a 5.5 cm stroke. Each flask was shaken and inoculated with washed and blended (15 sec) vegetative cell inoculum, equivalent to 10 mg of dry weight. The inoculum was obtained from 5-day-ok1 shaken cultures, grown in 4.5 % of malt extract broth (Difco) in distilled water. The experimental design consisted of growing the cultures in a rich medium that did not favour alkaloid production and of subsequent transfer to a nutritionally poor medium in which alkaloids are known to be formed.
Analytical methods Protein was determined by the method of LOWRY et al. (1951), total alkaloids according to ROEDER et al. (1967). The alkaloid results were expressed as ergometrine equivalents. For determination of total polysaccharides by the phenol sulphuric acid method (Du BOIS et al. 1956), the mycelia were washed with distilled water, then with ethyl alcohol (80%), and homogenized for 5 minutes (high speed rate). The mycelial pellet was resuspended in 10 ml of distilled water and homogenized again for 5 minutes. The final suspension was taken for determination of total polysaccharides.
Results and Discussion Three investigated strains of C. purpurea produced a mixture of extra- and intracellular alkaloids. This alkaloid mixture comprised partly clavines and simple lysergic acid derivatives (extracellular), partly peptide alkaloids (intracellular). The amount of intracellular alkaloids was lower than that of extracellular ones (Table 1). The alkaloid-synthesizing activity of the strains was variable. This fact was due to different susceptibility of producing cells to the composition of fermentation medium. Not only single strains (Fig. 1a, b, c; 2) but also formation of intra- and extracellular alkaloids (Table 1) differed in their claims to nutritional conditions. The influence of medium was more expressive in case of extracellular alkaloids than in case of intracellular ones. Strain Plo-2, with the highest alkaloid-synthesizing activity (Table 1), was observed to be most medium-dependent, especially on its sugar component. In this strain mannite (medium A) was a very suitable carbon source during synthesis of extracellular alkaloids. The positive role of mannite was reduced when also glucose or sucrose was present (medium B, C). In contrast to strain Plo-2, strain Pla·4 exerted intensive alkaloid synthesis both in medium containing mannite together with glucose (B) and in medium with sucrose (E). Strain ATCC 14934 was very active on medium with glucose (F) and on medium with sucrose (E). Differences in susceptibility to medium were also noted in extra· and intracellular alkaloid synthesis (Table 1). In strain Plo-2, medium with mannite (A) increased the formation of extracellular alkaloids, while medium with sorbitol (D) and that with glucose (F) were suitable for formation of intracellular alkaloids. On the other hand, in strain ATCC 14934 extracellular alkaloid formation was intensified in medium with glucose (F), while medium with mannite and glucose (B) gave rise to synthesis of intracellular alkaloids.
Variability of Requirements to Fermentation Media
553
Table 1. Some characteristics of mycelial) of Glaviceps purpurea strains in different fermentation media Strain
~Iedium
Dry weight Proteins mg' 10 ml- l pg . mg D.W.-l
Polysaccharides Alkaloids (pg . 10 mg D.W. -1) pg . mg D.W.-l Intracellular Extracellular Total
Pla-4
A
12 2 5 3 4 2
41 28 34 7 55 20
24 25 20 24 22 20
3 2 2 2 2 2
80 400 210 240 380 300
83 402 212 242 382 302
1 7 6 1 2 1
9 17 21 13 6 6
15 16 21 36 6 :37
5 5 3 60 8 60
900 130 170 600 430 600
905 135 173 660 438 660
A
II
B
44 16 22
82 25 17 7 28 19
22 30 20 33 55 22
5 50 5 3 20 10
80 120 80 220 400 540
85 170 85 223 420 550
B
C D E F Plo-2
A B
C D E F ATCC 14934
C D E F
22 12
I) 9-day-old shaken cultures with maximum alkaloid yield.
The physiological conditions necessary for the culture growth differed from those that determined the start of alkaloid synthesis. Increasing alkaloid formation was negatively correlated with the growth-rate capacity. Media inducing intensive culture growth were usually unsuited to alkaloid formation (Table 1). During alkaloid-producing phase different cell components were formed with different intensities. Increasing cell-protein level corresponded to relative decrease of the alkaloid-synthesizing activity. Constant or decreased cell proteins were favourable for alkaloid formation and indicated a non-proliferating culture (Fig. 3). In the light of this fact, and according to previous data (Bu'LoCK and BARR 1968, KAPLAN et al. 1969, thHACEK et al. 1971) which demonstrated the occurrence of no protein synthesis at a constantly decreasing rate and the almost invariable level of cell proteins as well as the low protease activities, we suggest a positive association between the rate of protein turnover and alkaloid-synthesizing activity. The level of cell polysaccharides was less variable than that of cell proteins. It is noteworthy that a positive correlation between accumulation of both cell polysaccharides and intracellular alkaloids was evident. The results of this presentation demonstrate partly a variability of O. purpurea in its claims to fermentation medium, partly some consequences of this property in physiology of the culture and alkaloid formation. Our data are in harmony with the hypothesis (TABER and VINING 1963, TABER 1964, Bu'LoCK 1965) that nutritional conditions which favour maximum primary shunt metabolism in a given case are not expected to favour maximum secondary shunt metabolism.
554
o. H.
EL-SAYED
and Z.
REHACEK
150
140
100
~
-;
PLO-2
-7'100
E
E
Q Do
Q .;.
~
~
til
til
[National Research Centre, Dokki, Cairo, Egypt, and Institute of Microbiology, Czechoslovak Academy of Sciences, Prague.]
Variability of Requirements to Fermentation Media purpurea Strains
III
Claviceps
O. HAMED EL-SAYED and Z. REHACEK With 3 Figures
Summary The alkaloid-synthesizing activity of the saprophytic Claviceps purpurea strains was Yariable. This fact was partly due to different requirements of the cultures to nutriticnal conditions. Not only single strains, but also formation of intra- and extracellular alkaloids differed in this respect. In case of extracellular alkaloids, the influence of medium was more expressive. A positive correlation between accumulation of cell polysaccharides and the level of intracellular alkaloids was evident. Constant or decreasing cell proteins were favourable for alkaloid formation.
Zusammenfassung In saprophytischen Claviceps purpurea-Stammen wurde eine variable Aktivitat der Alkaloidbildung festgestellt. An dieser Variabilitat widerspiegeln sich die Unterschiede in den Al~spriichen der einzelnen Stamme an das Medium. Auch die Biosynthese der intra- und extrazellularen Alkaloide war von der Zusammensetzung des Nahrmediums abhangig. 1m Faile der extrazellularen Alkaloide war der Einflu13 des Mediums besonders deutlich. - Es wurde eine positive Korrelation zwischen der Speicherung von Zellpolysacchariden und intrazellularen Alkaloiden beobachtet. - Ein gleichbleibender oder sinkender Zellproteinspiegel war fUr die Alkaloidbildul'g giinstig.
The first convincing demonstration of a production of ergot alkaloids in vitro (ABE 1951) was followed by a series of papers on the saprophytic nutritional requirements. However, it is still impossible to make any generalization of the factors, influencing alkaloid formation, because of great differences among the strains. In addition, nutritional conditions that favour maximum primary shunt metabolism in a given case may not be expected to favour maximum alkaloid synthef'is (REHACEK 1971). In this investigation, submerged cultures of three strains of Claviceps purpurea (Fr.) Tu!. on different fermentation media were examined. The purpose of this presentation was to gain information that may lead to better understanding of the physiology of alkaloid biosynthesis.
Materials and Methods Organisms and culture conditions The following saprophytic strains of Claviceps purpurea (Fr.) Tul. were used in this work: Pia 4, Plo 2, and ATCC 14934. The strain Pia 4 (HRONES and KYBAL 1962) was prepared from the ground plectenchymatous tissues of the sclerotia (lhclcovA and REH..\.CEK 1968). 36"
552
O. H. EL-SAYED and Z. REHACEK
Nutrient media of the following types were used in fermentation experiments: A B C D E F
-
mannite, ammonium succinate (Bu'LooK and BARR 1968); mannite, glucose, ammonium succinate (VINING 1970); mannite, sucrose, ammonium succinate, yeast extract (KAPLAN et al. 1969); sorbitol, ammonium succinate (O'BAYASHI and KANIE 1966); sucrose, ammonium citrate (MAIER et al. 1971); glucose, ammonium phosphate (KOBEL et al. 1964).
The fermentation experiments were carried out in 300 ml Erlenmeyer flasks, containind 100 ml of medium. The flasks were incubated at 24° ± 1 °C on a rotary shaker, operating at 240 rpm with a 5.5 cm stroke. Each flask was shaken and inoculated with washed and blended (15 sec) vegetative cell inoculum, equivalent to 10 mg of dry weight. The inoculum was obtained from 5-day-ok1 shaken cultures, grown in 4.5 % of malt extract broth (Difco) in distilled water. The experimental design consisted of growing the cultures in a rich medium that did not favour alkaloid production and of subsequent transfer to a nutritionally poor medium in which alkaloids are known to be formed.
Analytical methods Protein was determined by the method of LOWRY et al. (1951), total alkaloids according to ROEDER et al. (1967). The alkaloid results were expressed as ergometrine equivalents. For determination of total polysaccharides by the phenol sulphuric acid method (Du BOIS et al. 1956), the mycelia were washed with distilled water, then with ethyl alcohol (80%), and homogenized for 5 minutes (high speed rate). The mycelial pellet was resuspended in 10 ml of distilled water and homogenized again for 5 minutes. The final suspension was taken for determination of total polysaccharides.
Results and Discussion Three investigated strains of C. purpurea produced a mixture of extra- and intracellular alkaloids. This alkaloid mixture comprised partly clavines and simple lysergic acid derivatives (extracellular), partly peptide alkaloids (intracellular). The amount of intracellular alkaloids was lower than that of extracellular ones (Table 1). The alkaloid-synthesizing activity of the strains was variable. This fact was due to different susceptibility of producing cells to the composition of fermentation medium. Not only single strains (Fig. 1a, b, c; 2) but also formation of intra- and extracellular alkaloids (Table 1) differed in their claims to nutritional conditions. The influence of medium was more expressive in case of extracellular alkaloids than in case of intracellular ones. Strain Plo-2, with the highest alkaloid-synthesizing activity (Table 1), was observed to be most medium-dependent, especially on its sugar component. In this strain mannite (medium A) was a very suitable carbon source during synthesis of extracellular alkaloids. The positive role of mannite was reduced when also glucose or sucrose was present (medium B, C). In contrast to strain Plo-2, strain Pla·4 exerted intensive alkaloid synthesis both in medium containing mannite together with glucose (B) and in medium with sucrose (E). Strain ATCC 14934 was very active on medium with glucose (F) and on medium with sucrose (E). Differences in susceptibility to medium were also noted in extra· and intracellular alkaloid synthesis (Table 1). In strain Plo-2, medium with mannite (A) increased the formation of extracellular alkaloids, while medium with sorbitol (D) and that with glucose (F) were suitable for formation of intracellular alkaloids. On the other hand, in strain ATCC 14934 extracellular alkaloid formation was intensified in medium with glucose (F), while medium with mannite and glucose (B) gave rise to synthesis of intracellular alkaloids.
Variability of Requirements to Fermentation Media
553
Table 1. Some characteristics of mycelial) of Glaviceps purpurea strains in different fermentation media Strain
~Iedium
Dry weight Proteins mg' 10 ml- l pg . mg D.W.-l
Polysaccharides Alkaloids (pg . 10 mg D.W. -1) pg . mg D.W.-l Intracellular Extracellular Total
Pla-4
A
12 2 5 3 4 2
41 28 34 7 55 20
24 25 20 24 22 20
3 2 2 2 2 2
80 400 210 240 380 300
83 402 212 242 382 302
1 7 6 1 2 1
9 17 21 13 6 6
15 16 21 36 6 :37
5 5 3 60 8 60
900 130 170 600 430 600
905 135 173 660 438 660
A
II
B
44 16 22
82 25 17 7 28 19
22 30 20 33 55 22
5 50 5 3 20 10
80 120 80 220 400 540
85 170 85 223 420 550
B
C D E F Plo-2
A B
C D E F ATCC 14934
C D E F
22 12
I) 9-day-old shaken cultures with maximum alkaloid yield.
The physiological conditions necessary for the culture growth differed from those that determined the start of alkaloid synthesis. Increasing alkaloid formation was negatively correlated with the growth-rate capacity. Media inducing intensive culture growth were usually unsuited to alkaloid formation (Table 1). During alkaloid-producing phase different cell components were formed with different intensities. Increasing cell-protein level corresponded to relative decrease of the alkaloid-synthesizing activity. Constant or decreased cell proteins were favourable for alkaloid formation and indicated a non-proliferating culture (Fig. 3). In the light of this fact, and according to previous data (Bu'LoCK and BARR 1968, KAPLAN et al. 1969, thHACEK et al. 1971) which demonstrated the occurrence of no protein synthesis at a constantly decreasing rate and the almost invariable level of cell proteins as well as the low protease activities, we suggest a positive association between the rate of protein turnover and alkaloid-synthesizing activity. The level of cell polysaccharides was less variable than that of cell proteins. It is noteworthy that a positive correlation between accumulation of both cell polysaccharides and intracellular alkaloids was evident. The results of this presentation demonstrate partly a variability of O. purpurea in its claims to fermentation medium, partly some consequences of this property in physiology of the culture and alkaloid formation. Our data are in harmony with the hypothesis (TABER and VINING 1963, TABER 1964, Bu'LoCK 1965) that nutritional conditions which favour maximum primary shunt metabolism in a given case are not expected to favour maximum secondary shunt metabolism.
554
o. H.
EL-SAYED
and Z.
REHACEK
150
140
100
~
-;
PLO-2
-7'100
E
E
Q Do
Q .;.
~
~
til
til
