Patent Publication Number: US-2007122871-A1

Title: Microbial strains producing sphingosine

Description:
The present invention relates to microbial strains that are capable of producing the sphingoid base sphingosine.  
      The term “sphingolipids” refers to a group of lipids that are derived from sphingoid bases like sphingosine. Sphingolipids occur frequently in cellular membranes of animals, plants and microorganisms.  
      Ceramides are a specific group of sphingolipids containing sphingosine, phytosphingosine or dihydrosphingosine (sphinganine) as a base (sphingoid base) in amide linkage with a fatty acid. Ceramides are the main lipid component of the stratum corneum, the upper layer of the skin. Topical application of compositions comprising sphingolipids, such as ceramides, improves the barrier function and moisture-retaining properties of the skin (Curratolo, 1987, Pharm. Res. 4, 271-277; Kerscher et al., 1991, Eur. J. Dermatol. 1, 39-43). Furthermore, sphingoid bases as such are known to mediate several physiological effects as inhibiting the activity of protein kinase C and are therefore included in cosmetic or dermatological compositions for their anti-inflammatory and antimicrobial activity.  
      As sphingosine is the major sphingoid base component of sphingolipids in human, it is of considerable commercial interest to produce sphingosine and sphingosine-containing sphingolipids for food, pharmaceutical and cosmetic applications.  
      Currently, several routes for the chemical synthesis of sphingosine (and sphinganine) have been developed. However, due to the presence of two stereocenters in these molecules, chemical synthesis results in a racemic mixture with only 25% representing the naturally occurring stereochemical configuration. Moreover, extensive protection chemistry has to be applied due to the presence of three functional groups within these molecules. Consequently, sphingosine and sphinganine produced via chemical synthesis are extremely expensive, which does not allow for its incorporation into food and cosmetic formulations. This is also true for pure sphingosine and sphinganine isolated from natural sources, such as brain or chicken eggs. Heterogeneous sphingolipids preparations, which have been extracted from animal sources, are also available. Though cheaper than the pure compounds, they suffer from compositional heterogeneity and are potentially unsafe as they might contain pathogenic agents.  
      Microorganisms as the yeast  Pichia ciferrii  (Wickerham and Stodola, 1960, J. Bacteriol. 80, 484-491) were shown to produce sphingoid bases and derivatives thereof, but mainly C18-phytosphingosine and acetylated derivatives thereof. These can be extracted and chemically converted into other sphingoid bases and/or ceramides, thereby obtaining pure cosmetic ingredients (see e.g. WO 93/20038).  
      However, an economically feasible way of producing the naturally occurring stereoisomers of sphingosine and sphinganine as pure compounds is not available and is extremely desirable.  
      The present invention surprisingly shows the identification of microorganisms that are able to convert phytosphingosine into sphingosine. The ability to perform this conversion is due to the presence in the microorganism of a suitable enzyme activity, i.e. a dehydratase (see  FIG. 1 ). The identification of such a dehydratase was not a simple task, as such enzymes often catalyse both forward and reverse reactions and in water (or in acidic conditions) usually hydratation is favored; therefore the equilibrium of the enzymatic reaction lies towards the alcohol (phytosphingosine). Only if the alkene (sphingosine) is extremely stabilized by, for example, phenyl or other conjugating groups or the kinetics of the enzyme favor one direction of the reaction, the equilibrium may lie less extremely towards one side. Chemically, elimination of water only takes place under drastic conditions like concentrated H 2 SO 4 , gas phase heating over AL 2 O 3  or H 3 PO 4 .  
      Surprisingly, the present invention provides microbial strains and enzymes capable of dehydratation of phytosphingosine.  
      In a first aspect, the present invention provides a process to screen a series of candidate microorganisms for their capacity to convert exogenous phytosphingosine into sphingosine and to identify a microorganism that is able to convert at least part of the phytosphingosine into sphingosine.  
      Preferably, the process comprises:  
      providing a series of candidate microorganisms,  
      incubating said series of candidate microorganisms in the presence of a suitable amount of phytosphingosine,  
      analyzing the composition of the sphingoid base obtained from each incubated microorganism, and  
      identifying a microorganism that is able to convert at least part of the phytosphingosine into sphingosine.  
      The microorganism that is subjected to the process of the invention can be any microorganism, i.e. a bacterium, a yeast or a fungus. Preferably, eukaryotic microorganisms or Actinomycete bacteria are used for the screening. The candidate microorganisms are cultured until a desired cell density is reached, whereupon for each microorganism the cells are harvested, according to procedures known in the art.  
      If the relevant microbial enzyme(s) accumulate(s) intracellularly, the microbial cells are preferably permeabilised prior to incubation with phytosphingosine, to ensure that the relevant microbial enzyme(s) are in contact with the exogenously added, hydrophobic substrate phytosphingosine. Permeabilisation may be done by any suitable treatment that results in cell wall/membrane permeability. For instance, a suitable amount of microbial cells may be treated with an organic solvent not miscible with water, such as toluene, under vigorous stirring. Treatment typically is done at a pH around neutral, e.g. a pH of 6-8, and a temperature that is similar to the temperature used for culturing the microorganism. Permeabilisation may also be done by using an agent capable of introducing pores in the cell membrane. An example of such an agent is nystatin (Avéret N, Fitton V, Bunoust O, Rigoulet M, Guérin B. 1998. Yeast mitochondrial metabolism: from in vitro to in situ quantitative study. Mol Cell Biochem 184: 67-79). Permeabilisation may also be done by vigorous vortexing without the addition of any specific chemicals. The permeabilising treatment is done until the permeability of the cells reaches a sufficient level. Care should be taken to prevent too far cell permeabilisation, resulting in cell lysis. The extent of permeability may be tested by testing the activity of a suitable intracellular indicator enzyme, for example isocitrate dehydrogenase or by testing whether suitable dyes, e.g. fluorescent dyes, can enter the cells.  
      The permeabilised cells are then incubated with a suitable amount of phytosphingosine, whereby phytosphingosine preferably is solubilised in an aqueous solution. To solubilise phytosphingosine in aqueous solution, a suitable amount of an alcohol, e.g. ethanol, and/or a surfactant, e.g. Tween-80, is present in the phytosphingosine solution. Ethanol and Tween-80 may be used at concentrations up to 20%. Incubation may be done at a temperature of 10° to 40° C., typically at a temperature that is similar to the culture temperature of the microorganism. The incubation may be for 10 minutes to 24 hours, typically 2 hours, at a pH around neutral. The phytosphingosine concentration in the incubation mixture may be from 0.1 to 100 mM, preferably from 1 to 20 mM. After incubation, the obtained reaction product is dried, preferably by lyophilisation, optionally preceded by storage of the liquid reaction product at −70° C.  
      To identify a microorganism that is able to convert at least part of the exogenously added phytosphingosine into sphingosine, the reaction product of the incubation of (permeabilised) cells with phytosphingosine is analysed for its sphingoid base composition. To this end, the sphingoid base is extracted from the microbial cell residue present in the reaction product and treated to derivatise the sphingoid base.  
      Prior to extraction of the sphingoid base, the reaction product may be pulverised or milled, as the sample used for sphingoid base extraction and derivatisation should preferably consist of finely divided solids.  
      Extraction of the sphingoid base is done by vigorous mixing of the reaction product with a suitable organic solvent , e.g. chloroform, methanol and/or ethanol. Any crystals that are present may be removed by e.g. ultrasonic treatment. In a preferred embodiment, derivatisation is done on the sphingoid base as present in the reaction mixture without firstly performing a separation of extracted sphingoid base. More preferably, the derivatisation agent is already present during extraction.  
      Derivatisation may be done by treatment of the sphingoid base with, for instance, acetic anhydride, trifluoro acetic anydride or o-phthalaldehyde, in a suitable solvent. The treatment may be for 30 minutes at 35° C. Depending on the matrix (e.g. microorganism) and specific conditions, optimization of the treatment may be needed. This could result in incubation times ranging from 1 to 240 minutes and temperatures ranging from 20 to 50° C.  
      The sphingosine content of the extracted sphingoid base is determined by e.g. HPLC or NMR analysis. Extracted sphingoid base comprises sphingoid bases natively present in the microbial cells as well as, where applicable, sphingosine as produced from exogenously added phytosphingosine.  
      According to the invention, microorganisms are identified that are able to convert at least part of exogenously added phytosphingosine into sphingosine to such an extent that the sphingosine content of extracted sphingoid base is at least 5%, preferably at least 10%, more preferably at least 15%, most preferably at least 20% (w/w), using phytosphingosine in a concentration of 10 mM. Examples of identified microorganisms are microorganisms from the genus (species)  Pseudomonas  ( aurantiaca ),  Grifola  ( frondosa ),  Streptomyces, Inonotus  ( dryophilus ),  Hericium  ( coralloides ),  Phytophthora  ( capsici ),  Tricellula  ( aurantiaca ),  Pythium  ( adhaerens ).  
      In  FIG. 2  an overview of identified microorganisms is provided.  
      In a second aspect, the present invention provides a process for the preparation of sphingosine by cultivating a microorganism obtainable by the process of the first aspect, incubating the microorganism in the presence of phytosphingosine to convert at least part of the phytosphingosine into sphingosine and recovering the sphingosine.  
      Incubation of the microorganism in the presence of phytosphingosine may conveniently be done using conditions similar to those mentioned for the identification of microorganisms that are capable of converting phytosphingosine to sphingosine.  
      Preferably, the phytosphingosine used is obtainable from  Pichia cifferrii.    
      The sphingosine obtained after incubation of the micro-organisms with phytospingosine can directly be recovered from the total broth, or more preferably after acylation according to methods known in the art for sphingolipid recovery. The acylation can range from mono- to tri-acylated forms of sphingosine. Preferably, the acylation is acetylation.  
      Classical mutagenesis methods may be used to isolate variants of the phytosphingosine to sphingosine converting microorganisms, e.g. variants that can resist higher concentrations of phytosphingosine and/or perform the conversion at a higher rate.  
      In a third aspect, the present invention provides for the use of the microorganism obtainable by the process of the first aspect to isolate the enzyme activity that converts phytosphingosine into sphingosine. This enzyme activity preferably is a dehydratase activity. In addition, the present invention provides the use of the microorganism obtainable by the process of the first aspect to obtain a microorganism that produces sphingosine, by isolating the gene(s) encoding the particular enzyme (dehydratase) activity, transforming the gene(s) into a suitable phytosphingosine-producing microorganism, preferably  Pichia ciferrii , and isolating a transformed microorganism that produces sphingosine. Such a transformed microorganism can be advantageously used to directly produce sphingosine.  
    
    
     LEGENDS TO THE FIGURES  
       FIG. 1 . Phytosphingosine (I) conversion into Sphingosine (II) via a dehydratase enzyme.  
       FIG. 2 . Overview dehydratase positive organisms.  
       FIG. 3 . Sphingoid base analysis using Acetic Anhydride (AA).  
       FIG. 4 . Dehydratase activity by  Pseudomonas aurantiaca  (Nakhimovskaya 1948, VKM B-1524). TFAA-based HPLC analysis of Sphingosine produced from Phytosphingosine. A. direct sample analysis; B. sphingosine spiked to the sample to confirm formation of sphingosine. 
    
    
     EXAMPLE 1  
     Analysis of Sphingoid Bases Via Derivatization with Acetic Anhydride  
      As a derivatization reagent 600 μl acetic anhydride was mixed with 40 mg 4-(dimethylamino) pyridin and 200 μl triethylamine. Chloroform was added till 10 ml and the solution was mixed on a magnetic stirrer. Two mg of either phytosphingosine or sphingosine were mixed with 250 μl derivatization reagent and sonicated until the crystals are dissolved. The solutions were placed in a 35° C. water bath for 30 min, acetonitrile was added up to 5 ml and mixed with a vortex. Ten μl was analysed by HPLC, using the following conditions.  
                               HPLC conditions                                        Mobile phase   0.5 ml trifluoro acetic acid + in           1000 ml acetonitrile       Column   GL Science, Inertsil ODS 80-A, 4.6 × 250 mm       Flow   1 ml/min       UV-detector Wavelength   200 nm                  
 
      An UV-detector was used for the analysis of the acetic anhydride derivatives. phytosphingosine (PS) and sphingosine (So) were separated with retention times of 7.5 and 7.1 minutes, respectively (see  FIG. 3 ). The detection limit was determined at: 20 μM PS.  
     EXAMPLE 2  
     Analysis of Sphingoid Bases Via Derivatization with Trifluoro Acetic Anhydride  
      As a derivatization reagent 600 μl trifluoro acetic anhydride was mixed with 40 mg 4-(dimethylamino) pyridin and 200 μl triethylamine. Chloroform was added till 10 ml and the solution was mixed on a magnetic stirrer. Two mg of either phytosphingosine or sphingosine were mixed with 250 μl derivatization reagent and sonicated until the crystals are dissolved. The solutions were placed in a 35° C. water bath for 30 min, acetonitrile was added up to 5 ml and mixed with a vortex. Ten μl was analysed by HPLC using conditions of Example 1.  
      An UV-detector was used for the analysis of the acetic anhydride derivatives. PS and So were well separated with retention times of 10.1 and 8.9 minutes, respectively . The detection limit was determined at: 10 μM PS.  
     EXAMPLE 3  
     Analysis of Sphingoid Bases Via Derivatization with O-phthalaldehyde  
      As a derivatization reagent 50 mg OPA (o-phthalaldehyde) was dissolved in 1 ml ethanol and 50 μl 2-mercaptoethanol. Subsequently 99 ml 3% boric acid in water, pH 10,5 (with KOH) was added. Two mg of either phytosphingosine or sphingosine were mixed with 2.5 ml MeOH and sonicated until the crystals are dissolved. Then 2.5 ml OPA derivatization reagent was added, after mixing the samples were incubated for 15 minutes at room temperature. Before analysis with HPLC, the samples were cleared via centrifugation. The HPLC conditions were as follows.  
                               HPLC conditions                                                Mobile phase   90% (v/v) methanol in 0.5 mM potassium               phosphate buffer pH 7.0           Column   Radial Pak C18 column Waters (8 × 100 mm)           Flow   2 ml/min           Fluorescence           detector           Excitation   340 nm           wavelength           Emission   455 nm           wavelength                      
 
      For detection a scanning fluorescence detector was used. OPA derivatives of PS and So were well separated with retention times of 5.1 and 8.0 minutes, respectively. The detection limit was determined at: 2 μM PS  
     EXAMPLE 4  
      To find organisms capable of producing sphingosine the sphingoid base substrates, which are very hydrophobic compounds, need to be in contact with the necessary enzymes in their right environment. Therefore, cells of different species (see Table 1) were grown to mid-exponential phase, washed and incubated with 0.5% toluene for half an hour while gently shaking (10 to 100 rpm) during the whole incubation period . Cells were washed again and tested for permeability by performing an internal enzyme assay according to methods commonly known to the person skilled in the art. A suitable enzyme is isocitrate dehydrogenase. A portion of permeabilized cells is incubated with the necessary substrates and co-factors, incubated at the appropriate temperature and cofactor/product formation is analysed afterwards.  
     EXAMPLE 5  
      To evaluate the recovery of sphingoid bases from cell matrices, permeabilised cells were incubated with 300 μM of phytosphingosine for 2 hours. Afterwards, samples were spun down to remove excess phytosphingosine. Cells were subsequently resuspended in 50 mM TRIS-HCl pH 7.4, frozen and freeze-dried overnight. After lyophilization the sphingoid bases were extracted by addition of pure methanol and periodically vortexing for half an hour. The supernatant obtained after centrifugation was derivatized according to the o-phthalaldehyde method (see Example 3) and the average recovery was at least 71% with high reproducibility (see Table 1).  
               TABLE 1                          Results from phytosphingosine recovery                                 Recovery               phytosphingosine           Microorganism   (%)                         Acinetobacter calcoaceticus     82 4             Escherichia coli     75 8             Mycobacterium neoaurum     90 1             Ochrobactrum anthropi     71 1             Pseudomonas putida     82 4             Rhodococcus erythropolis     87 7             Saccharomyces cerevisiae     75 1                      
 
     EXAMPLE 6  
     Protocol for Incubation of Biological Samples with Phytosphingosine  
     
         
          1. Pre-grow the organisms in 10 ml medium in a 50 ml shake flask. 
        Bacteria for ˜24-36 hours; fungi for ˜72-96 hours.    
     
          2. Dilute 100× fold by transferring 200 μl of the pre-grown culture in to 20 ml fresh medium in 100 ml shake flasks (for fungi the amount to be transferred may be adapted if they grow in pellets or to slow) and grow to exponential phase (if Optical Density measurements are applied then up to an OD 600 nm  of ˜4-5). 
        For bacteria ˜6-8 hours; for fungi ˜24-72 hours (depending on the growth rate of the fungi).    
     
          3. Harvest cells by centrifugation.  
          4. Resuspend the pellet in 2.5 ml of Tris/HCl (50 mM; pH 7.4) in 12 ml test tubes (glass or plastic).  
          5. Add 2.5 ml of 1% toluene in Tris/HCl (50 mM; pH 7.4). 1% toluene does not dissolve in the buffer; therefore the mixture must be stirred vigorously.  
          6. Incubate for 10 minutes on a magnetic stirrer with 1200 rpm at the same temperature as the applied growth temperature in steps 1 and 2.  
          7. Harvest cells by centrifugation.  
          8. Resuspend the pellet in 10 ml Tris/HCl (50 mM; pH 7.4).  
          9. Harvest cells by centrifugation.  
          10. Resuspend the pellet in 2 ml Tris/HCl (50 mM; pH 7.4).  
       
    
      The permeabilized cells can be used immediately or may be stored at −70° C. 
      11. Add 71 μl of Phytosphingosine stock solution to the 1 ml of permeabilized cells. 
        Stock solution Phytosphingosine: 
            440 mg phytosphingosine (free base)     8 ml 20% (v/v) Tween-80 in milliQ (=de-ionized water)     2 ml 96% ethanol.     Adjust pH with HCl until Phytosphingosine is dissolved (pH ˜5.8)     Final concentration of stock solution is 140 mM    
           
        12. Incubate for 2 hours with shaking (100 rpm) and apply same temperature as used for growth in step 1 and 2.      13 . Store the samples at −70° C. until you have enough samples for lyophilization. 
        Lyophilise samples in the 12 ml plastic test tube overnight.         14. Without any additions pulverize the residue using a vortex shaker.     15. Add 2.5 ml Trifluoroacetic anhydride derivatization reagent. 
        Trifluoroacetic anhydride derivatization reagent (for 100 reactions): 
            2,0 g 4-(dimethylamino) pyridin     30 ml Trifluoroacetic anhydride     10 ml triethylamine     Fill up to 500 ml with chloroform and mix the solution with a magnetic stirrer.     The solution is stable for one month, stored at 4° C.    
           
        16. Vortex vigorously to obtain an homogenous mixture.     17. Apply ultrasonic treatment to remove any crystals.     18. Incubate the mixture for 30 minutes in a water bath at 35° C.     19. Centrifuge the mixture.     20. Transfer the supernatant to a new tube.     21. Store supernatant at less then 8° C. and analyse within 48 hours due to the stability of the derivatized material.     22. Prepare derivatized standards of phytosphingosine and sphingosine fresh each time.     23. Phytosphingosine standard: 
        2 mg phytosphingosine     add 250 μl Trifluoroacetic anhydride derivatization reagent     apply ultrasonic treatment to remove any crystals     Trifluoroacetic anhydride derivatization reagent (for 40 standard solutions): 
            40 mg 4-(dimethylamino) pyridin     600 μl Trifluoroacetic anhydride     200 μl triethylamine     Fill up to 10 ml with chloroform and mix the solution with a magnetic stirrer.     The derivatization solution is stable for one month, stored at 4° C.    
           
        24. Sphingosine standard: 
        1 mg sphingosine     add 250 μl Trifluoroacetic anhydride derivatization reagent     apply ultrasonic treatment to remove any crystals    
        25. Incubate both mixtures for 30 minutes in a water bath at 35° C.     26. add 1750 μl acetonitrile.     27. mix by vortexing.     28. centrifuge.     29. use 10 μl of the supernatant for HPLC analysis.     30. vortex and centrifuge samples from step 20.     31. use 10 μl of the supernatant for HPLC analysis.    

      32. HPLC conditions:  
                                                      Mobile phase   0.5 ml Trifluoroacetic acid in 1000 ml               acetonitrile           Column   GL Science, Inertsil ODS 80-A, 4.6 × 250 mm           Flow   1 ml/min           UV-detection   at 200 nm           RetentionTime   Phytosphingosine: 10.1 minutes           RetentionTime   Sphingosine: 8.9 minutes                      
      33. Each sample takes 15 minutes to be analysed. The sample is run trough (phytosphingosine and sphingosine come after 10.1 and 8.9 minutes) and the column is washed for the same time; there is no need for additional washing.    

     EXAMPLE 7  
     Sphingosine Production by  Pseudomonas    
       Pseudomonas aurantiaca  (VKM B-1524) was precultivated on PDA agar plates (per liter: Potato extract, 230 ml; glucose, 20.0 g; agar, 20.0 gr; pH 6.0) for 4 days at 25 C. This was used to inoculate shake flasks with P-M medium (per liter: meat extract, 5.0 g; pepton, 10.0 g; yeast extract, 3.0 g; NaCl, 3.0 g; glucose, 10.0 g; KH 2 PO 4 , 3.0 g; MgSO 4 ×7H 2 O5.0 g; pH 7.2) for 2 days at 25 C. The cells were processed as described in example 6 and 10-28% of the extracted sphingoid bases was sphingosine (see  FIG. 4 ), while no sphingosine could be detected in the control sample. To identify the formed products sphingosine was spiked to the sample and analysed again. Clearly the peak at 4.85/4.86 minutes is increased, confirming the formation of sphingosine (compare  FIGS. 4A and 4B ).  
     EXAMPLE 8  
     Sphingosine Production by  Grifola    
       Grifola frondosa  (VKM F-3125) was precultivated on liquid malt with glass beads (per liter: Malt extract, 300 ml; pH 6.0) for 10 days at 25 C. This was used to inoculate (using 5% of the preculture) shake flasks with agaric medium (per liter: pepton, 2.0 g; yeast extract, 2.0 g; glucose, 20.0 g; K 2 HPO 4 , 1.0 g; KH 2 PO 4 , 0.5 g; MgSO 4 ×7H 2 O, 0.2 g; pH 6.3-6.4) for 10 days at 25 C. The cells were processed as described in example 6 and 13% of the extracted sphingoid bases was sphingosine , while no sphingosine could be detected in the control sample.  
     EXAMPLE 9  
     Sphingosine Production by  Streptomyces    
       Streptomyces  sp. (Ac-109) was precultivated on P-Y medium (per liter: pepton, 5.0 g; yeast extract, 3.0 g; glucose, 5.0 g; KH 2 PO 4 ,0.2 g; pH 7.0-7.2) for 3 days at 26 C. This was used to inoculate a second shake flask with P-Y medium using 5% of the preculture as an inoculum, for 3 days at 26 C. The cells were processed as described in example 6 and 19% of the extracted sphingoid bases was sphingosine, while no sphingosine could be detected in the control sample.  
     EXAMPLE 10  
     Sphingosine Production by  Inonotus    
       Inonotus dryophilus  (VKM F-434) was precultivated on liquid malt with glass beads (per liter: Malt extract, 300 ml; pH 6.0) for 6 days at 25 C. This was used to inoculate (using 5% of the preculture) shake flasks with agaric medium (per liter: pepton, 2.0 g; yeast extract, 2.0 g; glucose, 20.0 g; K 2 HPO 4 ,1.0 g; KH 2 PO 4 ,0.5 g; MgSO 4 ×7H 2 O, 0.2 g; pH 6.3-6.4) for 3 days at 25 C. The cells were processed as described in example 6 and 7% of the extracted sphingoid bases was sphingosine.  
     EXAMPLE 11  
     Sphingosine Production by  Hericium    
       Hericium coralloides  (VKM F-3128) was precultivated on liquid malt with glass beads (per liter: Malt extract, 300 ml; pH 6.0) for 10 days at 25 C. This was used to inoculate (using 5% of the preculture) shake flasks with agaric medium (per liter: pepton, 2.0 g; yeast extract, 2.0 g; glucose, 20.0 g; K 2 HPO 4 , 1.0 g; KH 2 PO 4 , 0.5 g; MgSO 4 ×7H 2 O, 0.2 g; pH 6.3-6.4) for 3 days at 25 C. The cells were processed as described in example 6 and 18% of the extracted sphingoid bases was sphingosine, while no sphingosine could be detected in the control sample. EXAMPLE 12  
      Growth of  Phytophthora    
       Phytophthora capsici  (VKM F-2062) was precultivated on V8 agar plates (per liter: V8 vegetable juice, 125 ml; CaCO 3  2.5 g; agar 20 g). One square cm blocks were cut and used to inoculate 15 ml liquid medium in plastic petri dishes without shaking. Several media were used: Ph (per liter: yeast extract, 5.0 g; glucose, 5.0 g; L-Asparagine, 1.0 g; KH 2 PO 4 , 0.5 g; MgSO 4 ×7H 2 O, 1.0 g; thiamine, 0.001; pH 6.0), V8 and HV8 (same as V8, but after mixing of V8 vegetable juice and CaCO 3  the debris was removed). All media were supplemented with pimaricin (100 mg/l), rifampicin (10 mg/l), vancomycin (10 mg/ml) and ampicillin (100 mg/l). After 64 hours growth was clearly visible.  
     EXAMPLE 13  
      Sphingosine Production by  Phytophthora    
       Phytophthora capsici  (VKM F-2062) was precultivated on PDA agar plates (per liter: Potato extract, 230 ml; glucose, 20.0 g; agar, 20.0 gr; pH 6.0) for 14 days at 25 C. This was used to inoculate liquid Ph medium (per liter: yeast extract, 5.0 g; glucose, 5.0 g; L-Asparagine, 1.0 g; KH 2 PO 4 , 0.5 g; MgSO 4 ×7H 2 O, 1.0 g; thiamine, 0.001; pH 6.0) for 3 days at 25 C. The cells were processed as described in Example 6 and 29% of the extracted sphingoid bases was sphingosine, while no sphingosine could be detected in the control sample.  
     EXAMPLE 14  
     O-Phthalaldehyde Analysis of  Pseudomonas  and  Tricellula  Sphingoid Bases  
       Tricellula aurantiaca  (VKM F-2456) and  Pseudomonas aurantiaca  (VKM B-1524) were cultivated as described in example 8. After freeze drying the samples were derivatised using o-phthalaldehyde, as described in example 3. Both species produced sphingosine after incubation with phytosphingosine.  
     EXAMPLE 15  
     Sphingosine Production and NMR Analysis by  Pythium    
       Pythium adhaerens  (VKM F-1921) was precultivated on malt agar petri dishes (per liter: Malt extract, 300 ml; agar, 20.0 g; pH 6.0) for 20 days at 25 C. Blocks with fungus were cut from these agar plates and used to inoculate (using 20 blocks of the preculture) liquid agaric medium (per liter: pepton, 2.0 g; yeast extract, 2.0 g; glucose, 20.0 g; K 2 HPO 4 , 1.0 g; KH 2 PO 4 , 0.5 g; MgSO 4 ×7H 2 O, 0.2 g; pH 6.3-6.4) for 3 days at 25 C. The cells were processed as described in example 6, but analysed with Nuclear Magnetic Resonance (NMR) and sphingosine was observed.