JP2002519070A - Improved microbial strains producing sphingoid bases - Google Patents

Abstract

  (57) [Summary] The present invention reveals a Pichia strain with substantially enhanced sphingoid base production capacity as compared to Pichia ciferrii Y-1031 F-60-10. The present invention further discloses a method of obtaining a microorganism strain with enhanced sphingoid base production, comprising an initial screening step based on the antimicrobial activity of sphingoid bases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field of the Invention The present invention, microbial strains sphingoid base productivity of itself is improved, and to their preparation.

BACKGROUND OF THE INVENTION [0002] The term "sphingolipid" refers to a group of lipids derived from sphingoid bases such as sphingosine. Sphingolipids often appear on the cell membranes of animals, plants and microorganisms. Although the exact function of human sphingolipids is not yet known, it is clear that this group of compounds is involved in nervous system electrical signaling and cell membrane stabilization. Glycosphingolipids have functions in the immune system; in particular, certain glycosphingolipids have been shown to function as receptors for bacterial toxins, and possibly also as bacteria and viruses.

[0003] Ceramides are a specific group of sphingolipids that include sphingosine, dihydrosphingosine or phytosphingosine as the base of an amide bond with a fatty acid. Ceramide is the major lipid component of the stratum corneum, the upper layer of the skin. The stratum corneum has an important barrier function, with external compounds generally being kept outside of the barrier and with limited water loss. Topical application of a composition containing sphingolipids such as ceramide improves, for example, skin barrier function and moisture retention properties (C
uratolo, Pharm. Res., 4: 271-277 (1987); Kerscher et al., Eur. J. Dermatol., 1:
39-43 (1991)).

[0004] Sphingoid bases themselves have been shown to mediate some physiological effects by inhibiting the activity of protein kinase C, a key enzyme in signal transduction pathways. In addition, sphingoid bases are included in cosmetic compositions or dermatological compositions for their anti-inflammatory and antimicrobial activities. Currently, heterogeneous sphingolipid preparations for cosmetics are mainly extracted from animal sources. Clearly, this is a relatively expensive method on an industrial scale. Furthermore,
It has been found that these substances may be unsafe due to the potential for bovine spongiform encephalopathy (BSE) in bovine tissue, for example. Thus, the cosmetics industry has shown increasing interest in new sources of pure, well-defined sphingolipids available from sources other than animal tissue.

[0005] Microorganisms such as the yeast Pichia ciferrii have been found to produce sphingolipids themselves, as well as sphingosine, phytosphingosine and / or their derivatives (Wickerham and Stodola, J. Bacteriol., 80: 484). -491 (1960)). This discovery provides a source of sphingolipids per se, and a source of starting materials to produce other commercially valuable compounds, and is feasible for the use of animal sources of these compounds A good substitute.

For example, sphingosine, dihydrosphingosine and acetylated derivatives of phytosphingosine produced by Pichia ciferrii are deacetylated and the sphingosine, dihydrosphingosine or phytosphingosine so obtained is ceramide, It is chemically converted into pseudo-ceramides and / or related compounds such as glycoceramides, which can then be applied in cosmetic and dermatological products (WO 93/20083).

[0007] Unfortunately, none of the yeast strains studied to date (Pichia ciferri
i Even NRRL Y-1031 F-60-10), produce sphingolipid bases such as sphingosine, phytosphingosine or their derivatives in sufficient quantities to provide an efficient, economical and attractive supply of such compounds Nothing is the source.

[0008] WO 95/12683 discloses a Pichia ciferrii strain that has enhanced productivity of TAPS compared to the parent strain NRRL Y-1031 F-60-10. However, the productivity of these "improved" strains is at most 1.6 times. Despite the great desire to obtain strains with substantially higher productivity, it is uncertain whether further improvements in productivity will be achieved due to the toxicity of sphingoid bases to microbial cells. (Pinto et al., J. Bacteriol., 174: 2565-2574 (1992); Bibel et al., J.
Invest. Dermatol., 93: 269-273 (1992)).

DESCRIPTION OF THE INVENTION The present invention discloses a Pichia strain that exhibits a substantially enhanced ability to produce sphingoid bases or derivatives thereof. In the context of the present invention, the substantially enhanced ability to produce sphingoid bases or derivatives thereof, when measured under the same conditions, of Pichia ciferrii Y-1031 F-60-10 deposited as CBS 408.94 It is associated with a productivity that is at least twice as high as the production capacity (WO 95
/ 12683). Preferably, the substantially enhanced ability to produce sphingoid bases or derivatives thereof is Pichia ciferrii Y-10 deposited as CBS 408.94.
It is associated with a productivity that is at least 4-fold, more preferably at least 6-fold, even more preferably at least 8-fold, and most preferably at least 10-fold higher than that of 31 F-60-10. The present invention further discloses a novel method for isolating a microorganism strain that exhibits enhanced ability to produce sphingoid bases or derivatives thereof.

[0010] The novel method of the present invention determines the ability of a series of strains to produce sphingoid bases and the antimicrobial activity of sphingoid bases or derivatives thereof as produced by each individual strain present in the series of strains. A first screening step, as determined by This first screening step advantageously screens a substantial amount of the sphingoid base-producing ability of the individual strain and of the strain subpopulation grown on the strain that exhibits enhanced productivity compared to the reference strain. Enable selection.

In particular, the present invention relates to the selection or isolation of microbial cells or strains derived from those that exhibit enhanced sphingoid base or derivative thereof production ability relative to a reference strain. The reference strain is preferably Pichia ciferrii Y-1031 F-60-10 or a derivative thereof.

[0012] The method of the invention essentially comprises the following steps:-Sphingoids of a series of strains by determining the antibacterial activity of sphingoid bases produced by each individual strain present in the series of strains Screening for the ability to produce id bases; selecting a subpopulation of strains for at least one further passage of cells; and further transferring each individual strain within the selected subpopulation to the next medium. Transplanting a certain amount of cells,
And subculturing the cells by culturing the amount of the cells in the next medium under conditions that produce sphingoid bases; compared to the ability to produce a reference strain when cultured under the same conditions; This is a step of selecting a strain having enhanced sphingoid base-producing ability.

As used herein, the term “sphingoid base” includes sphingoid base derivatives specifically set forth below, but the term “derivative” is not always specifically mentioned. Should be understood.

It should be further understood that the above-described method of the present invention represents one cycle of screening and selection. However, repeated cycles of screening and selection can also be applied to obtain microbial strains that exhibit enhanced sphingoid base production.

As a first step in the initial screening of a series of strains described above, cells from each individual strain present in the series are mixed with cells from different individual strains in an appropriate culture medium. Is inoculated, taking care not to cause illness. In the next step,
The cells are cultured under conditions that produce sphingoid base compounds. If sufficient growth has occurred, the ability of each individual culture to produce sphingoid bases is determined by determining the antimicrobial activity of the culture supernatant containing sphingoid bases.

Culturing the cells to obtain culture supernatants for determining their antimicrobial activity can be conveniently performed in any suitable system. For example, culturing can be performed in shake flasks or on microtiter plates.

[0017] The antibacterial activity of the culture supernatant containing sphingoid bases is advantageously
It is determined by measuring the degree of growth inhibition of the indicator strain produced by incubation of the appropriate indicator strain in the presence of the culture supernatant. A suitable indicator strain can be any microbial strain susceptible to the antibacterial activity of sphingoid bases or derivatives thereof. Preferably, the indicator strain is a bacterial strain, more preferably a gram positive strain.

Briefly, an appropriate dilution of the indicator strain is incubated with culture supernatant from an individual strain that is screened for sphingoid base production. Suitable dilutions can be obtained by diluting a stationary phase culture of the indicator strain, for example, an overnight culture. Proper dilution of the overnight culture is
Typically about 100 to about 500 times. The degree of growth inhibition as produced by the culture supernatant containing sphingoid bases is determined by measuring the optical density of the incubated sample. Incubation conditions depend on the indicator strain used and the ability to produce sphingoid bases. For example,
Incubation times can vary from 4-8 hours to overnight incubation, depending on the degree of growth inhibition obtained. The incubation temperature depends on the optimal growth conditions for the indicator strain. Generally, the incubation temperature is 37 ° C. By extending the incubation, the temperature can be reduced to about 25 ° C. In a preferred embodiment of the present invention, the indicator strain is Staphylococcus aur
eus or a bacterial strain of the species Streptococcus pyogenes.

In a preferred embodiment of the invention, the initial screening step can be performed using a microtiter plate system for the incubation and analysis steps. An advantage of using a microtiter plate system is that large numbers of individual strains can be screened under substantially automated conditions.

Based on the results of the antimicrobial assay, a subpopulation of the strain is selected for at least one further passage of the cells. Further passage of the aforementioned cells may be performed under conditions that transplant cells from each individual strain present in the selected strain subpopulation into the next medium, and subsequently produce sphingoid base compounds of each individual strain. Including cultivation underneath. The culture media or their appropriate dilutions are then analyzed for sphingoid base production using an appropriate assay.

For example, cells from each individual strain present in a selected subpopulation are cultured in shake flask cultures or in test tube cultures. The ability of each culture to produce sphingoid bases is determined by analysis of the culture medium or appropriate dilutions thereof, preferably by quantitative analysis. The strain is then identified as satisfying a certain increase in productivity compared to the reference strain.

The above-mentioned series of strains to be subjected to the screening and selection methods of the present invention can be obtained by several methods, but these methods are not important for the present invention. For example, the set of strains described above can include a set of strains unrelated to the strain to be analyzed for sphingoid base-producing ability, such as strains of different species, different genera, different families, and the like. The strains to which the screening and selection methods of the present invention can be applied include yeasts, which can naturally produce sphingoid bases and / or derivatives thereof,
Bacterial and fungal strains are included. Preferably, the strain selected by the method of the present invention is a strain that exhibits a high endogenous production level of the desired product.

[0023] The yeast strain used in the present invention includes Saccharomyces, Kluveromyces, Deb
aromyces, Pichia, Hansenula, Lipomyces, Sporobolomyces, Crypto
genus coccus, genus Torulopsis, genus Endomycopsis, genus Candida, genus Trichosporon, Hanse
It can be selected from the species of the genus niaspora and Rhodotorula. Preferred yeast strains belong to the genera Pichia, Hansenula, Endomycopsis, Candida, Saccharomyces and Hanseniaspora, and especially Pichia ciferrii, Candida utilis and Sacc.
haromyces cerevisiae. Most preferred are yeast strains belonging to the genus Pichia, and Pichia ciferrii species are most preferred (particularly Pichia ciferrii NRRL Y
-1 031 F-60-10).

Preferred fungi are the genus Aspergillus and the genus Penicillium, more preferably
Aspergillus sydowi and Penicillium notatum species. Preferred bacteria are of the genus Sphingobacterium (especially S. versatilis, S. multivorum
And S. mizutae), genus Acetobacter (especially, A. xylinum), genus Bacteroides (especially,
B. melaninogenicus, B. fragilis, B. ruminicola, and B. thetaiotaomicron)
Genus, Bdellovibrio (especially Bdellovibrio bacteriovus), Xanthomonas (especially Xa
nthamonas campestris), and Flavobacterium (particularly, Flavobacterium devorans)
It is.

In a preferred embodiment of the invention, the above-mentioned strain series is obtained by mutagenesis of a suitable parent strain. According to this embodiment of the invention, the parent population of cells is subjected to a mutagenesis treatment in order to introduce a genetic mutation into the population. The type of mutagenesis treatment applied is not critical to the invention. Mutagenesis treatments typically include so-called classical treatments, but can also include DNA-mediated transformation.

Classical mutagenesis treatments include N-methyl, N′-nitro, N-nitrosoguanidine (NT
G) or treatment with a chemical mutagenic reagent, such as an alkylating agent such as ethylmethane sulfonate (EMS), or physical treatment, such as UV irradiation, or a combination of both treatments. This treatment may be performed in a manner that results in a cell population that has been mutagenized so that the substantial cell mass undergoes a single mutation, and / or the desired product is isolated from its parent strain when cultured under the same conditions. It is preferably performed in a manner that provides a mutant strain that can be produced at a relatively enhanced level.

[0027] DNA-mediated transformation is the transformation of random fragments of the same or different nucleic acids, and / or, for example, DNA fragments containing one or more genes involved in the biosynthesis of sphingoid bases or derivatives thereof. And transformation with a specifically selected DNA fragment.

For example, the genes LCB1 and LCB2 (encoding the subunit of serine palmitoyltransferase) for the first biosynthesis step are cloned.
(Nagiec et al., Proc. Natl. Acad. Sci. USA, 91: 7899-7902 (1994)). Increased expression of these genes, for example, due to increased gene input, can be expected to result in higher carbon capture from the central metabolic pathway (amphoteric pathway) to the sphingoid base biosynthetic sequence. Similarly, SUR2 (encoding a hydroxylase that introduces the 4-OH group of phytosphingosine; Haak et al., J. Biol. Chem., 272: 2970).
4-29710 (1997)) and LCB3 (involved in sphingoid base transport; Oie et al., J. Biol.
Overexpression of Biol. Chem., 272: 16110-16117 (1997)) would lead to higher sphingoid base production capacity. In addition, ELO2 and ELO3 (involved in the biosynthesis of very long chain fatty acids; Oh et al., J. Biol. Chem., 272: 17376-17384 (1997)) and AUR1 (involved in binding ceramide to the inositol moiety). Nagioe et al., J. Biol. Chem., 272: 98.
A number of genes, such as 09-9817 (1997)), have been shown to be involved in the synthesis of ceramide from sphingoid bases. It is difficult to predict the effects of modulating the expression of these genes, but the down-tuning of this expression results in a larger fraction of sphingoid bases being acetylated and excreted in a detectable manner. There is a clear possibility of being connected.

In a preferred embodiment of the invention, the parent strain suitable for undergoing the mutagenesis treatment is a strain of the genus Pichia, more preferably a strain of the species Pichia ciferrii, most preferably P
ichia ciferrii Y-1031 F-60-10 or a derivative thereof.

Thus, the present invention provides a method for isolating a microorganism strain capable of producing sphingoid bases or derivatives thereof at enhanced levels compared to a reference strain.

In particular, sphingosine and / or its keto, glycosylated or acetylated derivatives, such as 3-ketosphingosine, triacetylsphingosine, diacetylsphingosine and N-acetylsphingosine; dihydrosphingosine and / or its keto, glycosylated or Acetylated derivatives, such as
3-ketodihydrosphingosine, triacetyldihydrosphingosine, diacetyldihydrosphingosine and N-acetyldihydrosphingosine; and / or phytosphingosine and / or their keto, glycosylated or acetylated derivatives such as tetraacetyl phytosphingosine, triacetyl Microbial strains are provided that are capable of producing acetyl phytosphingosine, diacetyl phytosphingosine and N-acetyl phytosphingosine at enhanced levels. Preferred strains are capable of producing phytosphingosine and its partially or fully acetylated derivatives at enhanced levels, such as triacetyl phytosphingosine (TriAPS) and tetraacetyl phytosphingosine (TAPS). It is a strain.

A description of the growth conditions as detailed in the examples is provided as a reference for determining the level of production of the desired product by the strains of the present invention. However, other culture conditions known to those skilled in the art and performed to produce the desired product may be used without departing from the spirit of the invention. For example, a strain that exhibits some enhancement of sphingoid base production when compared to a reference strain when analyzed in a shake flask culture is typically substantially similar when measured on a larger scale. Demonstrate the enhancement.

[0033] Quantitative analysis can be performed by various methods known in the art. For example, a quantitative analysis based on TAPS production levels may be performed on culture supernatant of shake flask supernatant, the culture supernatant is extracted with a suitable solvent such as CDCl _3, NMR
Analyzed by TAPS is preferably used as a standard for NMR analysis.

Sphingosine, phytosphingosine and dihydrosphingosine; sphingosine, phytosphingosine and triacetyl, diacetyl and N-acetyl derivatives of dihydrosphingosine; sphingosine, phytosphingosine and glycosylated derivatives of dihydrosphingosine; 3-ketodihydrosphingosine and Other desired products, such as / or 3-ketosphingosine, can be measured quantitatively in a similar manner, and TAPS standards are used as a reference.

In an embodiment of the present invention, an enhanced amount of Tr compared to the reference strain Y-1031 F-60-10
A Pichia strain, preferably a Pichia ciferrii strain, that produces iAPS is isolated. In a preferred embodiment of the present invention, the Pichia strain is one that produces TriAPS as the predominant sphingoid base, ie, that has low or no TAPS levels formed.

A further aspect of the present invention provides for the production of a sphingoid base or a derivative thereof, including fermentation of a strain obtainable by the method of the invention under conditions that produce a sphingoid base or a derivative thereof. Related to how you do. The sphingoid base or derivative thereof thus produced can be recovered from the fermentation broth, preferably from the fermentation supernatant, using conventional extraction and recovery techniques.

[0037] Once the desired product is obtained, it can be used directly or further processed depending on the desired application. For example, acetylated derivatives of sphingosine, dihydrosphingosine and / or phytosphingosine can be deacetylated enzymatically or chemically. Deacetylated sphingoid base derivatives, such as sphingosine, dihydrosphingosine and / or phytosphingosine, can be used directly or afterwards with ceramides (see WO 83/20038), glycoceramides and pseudoglycans. It can be used as a starting material in the synthesis of sphingolipid products of commercial value, such as ceramide. The direct use of the sphingoid base compounds obtained according to the invention includes their use in cosmetic and dermatological compositions.

The following examples are provided to enable those skilled in the art to better understand the complete disclosure and description of the present invention, and are not intended to limit what the inventors consider as the present invention. .

Example 1 Testing of Indicator Strains for TAPS Production Bioassay The following indicator strains were tested on agar plates for their sensitivity to TAPS standards: Staphylococcus aureus ATCC 14458 Streptococcus pyogenes ATCC 12344 Micrococcus luteus ATCC 10204 Serratia marcescens ATCC 14041 These four bacterial strains were grown in 500 ml shake flasks in 100 ml brain heart infusion (BHI) broth at 37 ° C. until stationary phase was reached. Subsequently, BHI agar (45 stationary phase cultures 0.5 ml, melted containing 1 mM CaCl ₂
C) with 100 ml. This mixture was poured into a Petri dish and allowed to solidify. A sterile metal ring (6 mm id) was then placed on the surface of the agar and 50 μl of a 50 mg / ml TAPS stock solution in 50% ethanol was placed in the ring. Thereafter, the agar plate was incubated at 37 ° C. and the growth inhibition zone extending from the metal ring was evaluated. Distinct growth inhibition was observed in S. aureus and S. pyogenes, and to a lesser extent for M. luteus. In contrast, growth of S. marcescens was not inhibited by TAPS solution under these conditions.

Example 2 Bioassay for TAPS Production in Microtiter Plates : The primary selection indicator strain, Staphylococcus aureus ATCC 14458, was stored under flash freezing conditions. One frozen test tube was used to inoculate a 100 ml shake flask containing 25 ml BHI medium. The culture was incubated overnight at 37 ° C. (16-17
time). The overnight culture was then placed in fresh BHI medium containing 1 mM CaCl ₂ for 250
Diluted ~ 500-fold.

[0041] This production strain was separated from the agar surface by 33 g / l dextrose, 1 g / l yeast extract, NH_Four Cl 4.83 g / l, KH_TwoPO_Four 1.0 g / l, MgSO_Four7-hydrate 0.88 g / l, NaCl 0.06 g / l, CaCl _Two ・ Dihydrate 0.2 g / l, KH-phthalate 20 g / l, concentrated trace element solution 0.3 ml / l, concentrated bita
100 with 25 ml of medium containing 1.5 ml / l of the Min solution and 0.16 ml / l of the Structol defoamer
Inoculate ml shake flasks. After 72 hours of growth (25 ° C) on a rotary shaker,
Samples were taken from the samples and the samples were centrifuged at 4000 rpm for 10 minutes.

10 μl of the supernatant fraction was transferred to the wells of a 96-well flat bottom microtiter plate. This operation was repeated seven times, and one row of the microtiter plate was filled with one sample of eight replicates. Each microtiter plate contained eight replicates of the TAPS standard solution, as well as a 2 * 8 media blank.

Thereafter, 150-200 μl of the diluted suspension of the indicator strain was added to these wells. Microtiter plates were incubated for 5-6 hours at 37 ° C. in a Flow Laboratories “Titertek” shaking incubator (speed 4). After incubation, the optical density of the wells was measured at 620 nm using an Anthos HtIII microtiter plate reader and the average of eight replicates for one strain was taken as a measure of its TAPS production. If growth inhibition was severe, incubation was extended to 26 ° C. overnight.

Example 3 Use of Bioassays in Secondary Selection In order to check whether the strains selected by the primary selection bioassay were in fact high producers, they were subjected to secondary selection in triplicate. did. Each selected strain was prepared from the rapidly frozen glycerol suspension using the medium 100 described in Example 2.
A 500 ml baffled shake flask containing ml was inoculated. After growth on a rotary shaker at 25 ° C. for 40 hours, the seed culture was used to inoculate three 500 ml baffled shake flasks containing 100 ml of the same medium at a level of 5% by volume.
These production cultures were then incubated for a further 48 hours on a shaker and samples were taken. The samples were processed as described in Example 2.

The supernatant fraction was subjected to the bioassay described in Example 2, but this time the TAPS concentration was also measured directly using NMR spectroscopy. Strains were selected based on NMR results and used to evaluate whether this bioassay could discriminate between relatively high production levels. If the sample supernatant was diluted in proportion to the TAPS concentration,
This has always proven to be the case. Using dilutions up to 5-fold correspond to the higher TAPS-concentration bioassays observed in the cultures of the more advanced strains.

As an example of the assay capacity of a bioassay to discriminate between strains of different production levels, the results for strains with many known production levels are shown in the table below:

[0047]

Example 4 Direct comparison of COS 1A and COS 10 strains Two strains were compared: COS 1A, a working cell bank of the haploid wild-type strain Pichia ciferri F-60-10. And COS 10, a fermenting cell bank for COS 106, one of these strains was obtained by chemical mutagenesis and selected by the primary selection described in Example 2. COS 10 was announced on July 23, 1998 as `` Cen
traal Bureau voor Schimmelcultures "and deposited as CBS 101070. The comparison was performed as in Example 3. The results are shown in the table below:

[0049]

It is clear that there is a good correlation between growth inhibition in this bioassay and TAPS production as measured by NMR. As a result, strains producing two times more TAPS than wild type under the same conditions were selected.

Example 5 Comparison of COS 17 and COS 144 In the next strain improvement cycle (mutagenesis and selection), the best strain COS 17 thus obtained was used as a control. In this experiment, the results for the selected strain COS144 were compared to controls using the method described in Example 3, except that the sample supernatant was diluted 4-fold.

[0052]

It is clear that strain COS 144 shows increased TAPS production, about 25% more than COS 17, which also indicates that the production level of COS 144 is 302% compared to COS 1A. ing. Thus, a fermenting cell bank was prepared from this strain and designated COS 19A, which showed that it had the same TAPS production as COS144.

Example 6 Bioassay Capability for Selection of TriAPS Producing Strains In addition, this bioassay was used to select TriAPS producers because the growth inhibition induced by this compound was less than that of TAPS. This is because it is known that the number of digits is the same as the size. As shown in the following table, a strain mainly producing TAPS, a strain producing both TAPS and TriAPS, and a strain producing only TriAPS were selected by using the operation described in Example 2. I understood.

[0055]

The ability to produce total acetylated phytosmolcin was apparently the highest in COS154, indicating that this strain had a 515% production level compared to the wild-type COS1A. .

[Procedure amendment]

[Submission date] July 24, 2001 (July 24, 2001)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

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Claims (13)

[Claims] 1. Deposited as CBS 408.94 when cultured under the same conditions
Pichia ciferrii Y-1031 A strain of the genus Pichia that exhibits a sphingoid base or a derivative thereof at least twice as high as the ability to produce F-60-10. 2. Deposited as CBS 408.94 when cultured under the same conditions.
2. The production capacity of Pichia ciferrii Y-1031 F-60-10 is at least 4 times, preferably at least 6 times, more preferably at least 8 times, most preferably at least 10 times higher. Strains. 3. The strain according to claim 1, which belongs to the species Pichia ciferrii. 4. The strain according to claim 3, which is a derivative of Pichia ciferrii sp. Y-1031 F-60-10. 5. A method for isolating a microbial cell or a strain derived therefrom with enhanced ability to produce sphingoid bases or their derivatives, comprising the steps of:
Screening the sphingoid base-producing ability of the set of strains by determining the antibacterial activity of sphingoid bases as produced by each individual strain present in the set of strains; at least one additional step Selecting a strain subpopulation for cell passage; transplanting an amount of cells from each individual strain in said selected subpopulation into the next medium;
And further subculturing the cells by culturing this amount of cells in the next medium under conditions that produce sphingoid bases or derivatives thereof; and • Production of a reference strain when cultured under the same conditions. Selecting a strain having an enhanced ability to produce sphingoid bases or derivatives thereof as compared to the ability to produce sphingoid bases. 6. The method according to claim 5, wherein said series of strains is obtained by subjecting a parent strain to a mutagenesis treatment. 7. The method according to claim 5, wherein the reference strain is Pichia ciferrii Y-1031 F-60-10. 8. The method according to claim 8, wherein the strains are of the genus Pichia, preferably Pichia ciferrii.
8. The method according to any one of claims 5 to 7, wherein the method belongs to a species, more preferably Pichia ciferrii sp. Y-1031 F-60-10 or a derivative thereof. 9. Fermentation of the strain according to any one of claims 1 to 4 under conditions that produce said sphingoid base or a derivative thereof, and optionally said sphingoid base or from a fermentation broth. A method for producing a sphingoid base or a derivative thereof, comprising recovering the derivative. 10. Fermentation of a strain obtained by the method according to any one of claims 5 to 8 under conditions for producing the sphingoid base or a derivative thereof,
And optionally a recovery of the sphingoid base or derivative thereof from the fermentation broth. 11. The method according to claim 9, wherein the sphingoid base or a derivative thereof is acetylated phytosphingosine. 12. The method of claim 11, further comprising deacetylating the acetylated phytosphingosine to obtain phytosphingosine. 13. A method for preparing a ceramide, comprising N-acylation of a sphingoid base or a derivative thereof, as produced by a method according to any one of claims 9 to 12.