Description

[Title of Invention] METHOD FOR PRODUCING ACETYLATED SPHINGOID BASE

[Field of the Invention]

[0001]

The present invention relates to a modified

microorganism of the genus Starmerella producing an acetylated sphingoid base and a method for producing an acetylated sphingoid base by using the microorganism.

[Background of the Invention]

[0002]

Sphingolipid is obtained. by biosynthesis starting from a condensation reaction between L-serine and an acyl CoA such as palmitoyl-CoA. The basic structure of a sphingolipid, i.e., a sphingoid base, is mainly

synthesized as a molecule having a chain length of 18 carbon atoms and known as e.g., sphingosine,

phytosphingosine , dihydrosphingosine (sphinganine) and 6- hydroxy sphingosine. Each of these sphingoid bases is bound to a fatty acid via an amide bond to synthesize a ceramide .

[0003]

Sphingolipid has many physiological functions.

Particularly ceramide and a sphingoid base, which are involved in skin-moisturizing function and skin barrier function, suppress moisture evaporation from the skin and play a role in protecting human bodies from various external stimuli. Phytosphingosine is reported to have a growth inhibitory effect against Staphyrococcus aureus, Streptococcus pyogenes, Micrococcus luteus,

Propionibacterium acnes, Candida albicans and

Trichophyton mentagrophytes (Non Patent Literatures 1 and 2) . In particular, the antibacterial effect of

phytosphingosine on Propionibacterium acnes is known to be higher than that of erythromycin, which is one of macrolide antibiotics (Non Patent Literature 3) .

[0004]

It is known that supply of a ceramide or a sphingoid base by external application shows an effect of improving skin properties. Furthermore, it has been confirmed that when phytosphingosine and tetraacetylphytosphingosine , which is an acetylated phytosphingosine, are applied to the skin, they are permeated into the skin and converted into a ceramide (Patent Literature 1) . Accordingly, external application of ceramide, a sphingoid base or acetylated phytosphingosine is expected to have an improving effect on skin property and a growth inhibitory effect against microorganisms responsible for infection.

[0005]

Recently, a technique for specifically analyzing the ceramide composition of skin has been established and it has been found that there are 12 classes (340 or more species) of ceramide molecular species formed by combinations of a fatty acid and a sphingoid base (Non Patent Literature 4) . For example, as ceramide NP formed by a combination of a saturated fatty acid and a

phytosphingosine, a combination of a fatty acid having a chain length of from 23 to 30 carbon atoms and a

phytosphingosine having a chain length of from 16 to 26 carbon atoms is found and a molecule having a chain length of from 40 to 52 carbon atoms in total of the fatty acid and phytosphingosine is known to be present. As ceramide NS formed by a combination of a saturated fatty acid and a sphingosine, a combination of a fatty acid having a chain length of from 16 to 30 carbon atoms and a sphingosine having a chain length of from 16 to 26 carbon atoms is found and a molecule having a chain length of from 40 to 54 carbon atoms in total of the fatty acid and a sphingosine is known to be present (Non Patent Literature 4) . It is known that healthy skin contains a large amount of long-chain ceramide; whereas the content of ceramide is lowered in rough skin and additionally the amount of short-chain ceramide is increased (Non Patent Literature 5) . From this,

usefulness of a long-chain ceramide or a sphingoid base is expected.

[0006]

However, currently commercially available ceramide, a sphingoid base and acetylated phytosphingosine are extremely expensive, e.g., several tens to several hundreds of thousands of yen per kg. In addition, the length of a carbon chain of them is limited. For example, for ceramide NP and ceramide NS, molecules having 34, 36 or 40 carbon atoms are only available,- and for

phytosphingosine and sphingosine, molecules having 18 carbon atoms are only available.

[0007]

Since it is difficult to separate and purify animal- or plant-derived sphingolipids , a method for producing a sphingolipid by yeast fermentation has been recently developed as a method for producing a ceramide and a sphingoid base. Candidate yeast strains include Pichia ciferrii ; at present Wickerhamomyces ciferrii, Candida utilis and Saccharomyces cerevisiae, and a method for obtaining tetraacetylphytosphingosine using

Wickerhamomyces ciferrii , which secrets

tetraacetylphytosphingosine out of the yeast cells, has been positively developed (Patent Literature 6) . The length of carbon chain of acetylated phytosphingosine produced by this method is mostly 18 carbon atoms.

Acetylated phytosphingosine is deacetylated and used as phytosphingosine, or bound to a fatty acid via an amide bond through a chemical synthesis and used as a ceramide.

[0008]

It has been elucidated by in-vitro analysis of biosynthesis pathway that the rate-limiting steps of tetraacetylphytosphingosine synthesis in Wickerhamomyces ciferrii are a condensation reaction between serine and palmitoyl-CoA and an acetylation reaction of phytosphingosine . In addition, two acetylation enzymes for phytosphingosine, SLI1 and ATF2 , are found (Non

Patent Literature 7) . Among them, SLI1 produces

triacetylphytosphingosine when it is expressed in

Saccharomyces cerevisiae (Non Patent Literature 6) . From this, it is considered that SL ^' Il is involved in

acetylation of any three sites of 3 hydroxy groups and a single amino group of phytosphingosine.

[0009]

In the meantime, a microorganism of the genus

Starmerella, for example, Starwerella bombicola (old scientific name: Candida bombicola) , can produce a significant amount of sugar lipid serving as a

biosurfactant out of the cells and is known as a

microorganism having high lipid availability (Non Patent Literature 8) . However, little is known about whether the microorganism produces a ceramide or a sphingolipid .

[0010]

[Patent Literature 1] U.S. Pat. No.5578641

[Patent Literature 2] JP-A-9-504434

[Non Patent Literature 1] Bibel D. J. et . al . , J. Invest. Dermatol., 98, 269, (1992)

[Non Patent Literature 2] Bibel D. J. et . al . , Clin. Exper. Dermatol., 20, 395, (1995)

[Non Patent Literature 3] Park C. et . al . , Fragrance journal, 10, 84, (1999)

[Non Patent Literature 4] Masukawa Y. et . al . , J. Lipid Res., 49, 1466, (2008) [ on Patent Literature 5] Ishikawa J. et . al . , J. Invest. Dermatol., 130, 2511, (2010)

[Non Patent Literature 6] Veld, F. et . al . , Ap l . Microbiol, Biotechnol . , 97, 8537, (2013)

[Non Patent Literature 7] Barenholz, Y. et . al . , Biochim. Biophys . Acta, 306, 341, (1973)

[Non Patent Literature 8] Udo R. et . Al . ,

Biotechnology Letters, 18 (2), 149, (1996)

[Summary of the Invention]

[0011]

The present invention relates to (1) or (2) .

(1) A method for producing an acetylated sphingoid base comprising: culturing a microorganism of the genus Starmerella to which a xenogeneic gene encoding a polypeptide having an activity to acetylate a sphingoid base is introduced.

(2) A Starmerella microorganism to which a

xenogeneic gene encoding a polypeptide having an activity to acetylate a sphingoid base is introduced.

[Brief Description of Drawings]

[0012]

[Figure 1] Figure 1 is a graph showing the amount of acetylated phytosphingosines expressed where (A) shows production of triacetylphytosphingosine (TriAPS) and (B) shows production of tetraacetylphytosphingosine (TAPS) . [Detailed Description of the Invention]

[0013]

The present invention relates to providing a means for producing an acetylated sphingoid base by using a modified Starmerella microorganism, particularly a modified Starmerella bambicola.

[0014]

The present inventors conducted studies on a

Starmerella microorganism known as a microorganism having high lipid availability. As a result, they found that the microorganism cannot produce a ceramide or a

sphingolipid in an amount sufficient to be available. They further investigated. As a result, they

unexpectedly found that an acetylated sphingoid base can be produced in an amount sufficient to be available by introducing a xenogeneic gene encoding a polypeptide having an activity to acetylate a sphingoid base into a microorganism of the genus Starmerella and culturing the modified microorganism, and that the length of carbon chain of the product is mostly 19 or 20 carbon atoms, which differs from 18 carbon atoms in case of using

Wickerhamomyces ciferrii.

[0015]

According to the present invention, it is possible to produce an acetylated sphingoid base, preferably an acetylated phytosphingosine having a chain length of 19 or 20 carbon atoms, useful as an intermediate for synthesizing a ceramide or a ceramide precursor substance in an amount sufficient to be available by using a

Starmerella microorganism, which does not basically produce a ceramide or a sphingolipid in an amount

sufficient to be available.

[0016]

In the specification, homology between amino acid sequences refers to the rate (%) of the number of sites at which the identical amino acid residues exists between two amino acid sequences when aligned, relative to the total number of amino acid residues. More specifically, the homology is calculated in accordance with the Lipman- Pearson method (Science, 227, 1435, (1985)) and

computationally obtained based on the homology analysis

(Search homology) program of genetic information

treatment software, Genetyx- in (Ver. 5.1.1; Software Development) by setting the Unit size to compare (ktup) at 2.

[0017]

Furthermore, the term "gene" includes not only double-stranded DNA but also single stranded DNA

molecules, such as a sense chain and an anti-sense chain, constituting the double-stranded DNA, and is not limited by its length. Furthermore, as the polynucleotide, RNA and DNA can be mentioned as an example. DNA includes cDNA, genomic DNA and synthesis DNA.

In the specification, a gene encoding a polypeptide having an activity to acetylate a sphingoid base is referred to also as an acetyltransferase gene and a gene encoding SLI1 is referred to also as SLI1 gene.

In the specification, "xenogeneic" refers to being derived from a microorganism or an organism classified into the genera except the genus Starmerella.

In the present invention, the "sphingoid base" refers to a long-chain amino alcohol having a chain length of from 18 to 20 carbon atoms and having the following group:

[0019]

[0020]

Examples of a sphingoid base having a chain length of 18 carbon atoms include (2S, 3S, 4R) -2-aminooctadecane- 1,3,4-triol (phytosphingosine) , (2S , 3R, 4E) -2 -amino-4 - octadecene- 1 , 3 -diol (sphingosine) and (2S,3R)-2- aminooctadecane- 1 , 3 -diol (sphinganine) ; examples of a sphingoid base having a chain length of 19 carbon atoms include (2S , 3S , 4R) -2 -aminononadecane- 1 , 3 , 4 -triol (C19 phytosphingosine) , (2S, 3R, 4E) -2 -amino-4 -nonadecene- 1 , 3- diol (C19 sphingosine) and (2S, 3R) -2 -aminononadecane- 1, 3- diol (CI9 sphinganine) ,- and examples of a sphingoid base having a chain length of 20 carbon atoms include

(2S, 3S, 4R) -2-aminoicosane-l, 3 , 4 -triol (C20 phytosphingosine) , (2S, 3R, 4E) -2 -amino-4 - icosene- 1 , 3-diol (C20 sphingosine) and (2S , 3R) -2 -aminoicosane- 1 , 3 -diol (C20 sphinganine) . Among them, phytosphingosine having a chain length of from 19 to 20 carbon atoms is preferable and phytosphingosine having a chain length of 20 carbon atoms is more preferable.

[0021]

In the present invention, the gene to be introduced into a microorganism of the genus Starmerella is not limited as long as the gene encodes a polypeptide having an activity to acetylate a sphingoid base, and is present in a microorganism or an organism classified in the genera except the genus Starmerella . Preferable examples include genes found in Wickerhamomyces ciferrii,

Saccharomyces cerevisiae or Pichia pastoris, encoding acetyltransferase designated as SLI1, or a polypeptide deduced from the polypeptide, more specifically, a polypeptide consisting of the amino acid sequence

selected from the following (a) to (i) and having an acetyltransferase activity:

(a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO : 2 ,

(b) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO : 2 , (c) a polypeptide consisting of the amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 2;

(d) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO : 4 ,

(e) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO : 4 ,

(f) a polypeptide consisting of the amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO:4;

(g) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6,

(h) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO : 6 , and

(i) a polypeptide consisting of the amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO : 6.

[0022]

Herein, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is SLI1 derived from Wickerhamomyces ciferrii; the polypeptide consisting of the amino acid sequence represented by SEQ ID NO : 4 is SLI1 derived from Saccharomyces cerevisiae; the

polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is SLI1 derived from Pichia pastoris, and all of them have an acetyltransferase activity, favorably an activity to acetylate a sphingoid base .

[0023]

Furthermore, "one to several" in the polypeptides of (b) , (e) and (h) , means from 1 to 80, preferably from 1 to 40, more preferably from 1 to 20, even more preferably from 1 to 10.

[0024]

The amino acid sequence (c) having a homology of 80% or more with the amino acid sequence represented by SEQ ID N0:2, refers to an amino acid sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, with the amino acid sequence of SEQ ID NO : 2 , when the corresponding sequence thereof is properly aligned with the amino acid sequence represented by SEQ ID NO : 2. The amino acid sequence (f) having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 4 refers to an amino acid sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, with the amino acid sequence of SEQ ID NO: 4, when the corresponding sequence thereof is properly aligned with the amino acid sequence represented by SEQ ID NO: 4. The amino acid sequence (i) having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 6 refers to an amino acid sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, with the amino acid sequence of SEQ ID NO: 6, when the

corresponding sequence thereof is properly aligned with the amino acid sequence represented by SEQ ID NO : 6.

The genes encoding polypeptides (a) to (i) may have any selected codons as long as the amino acid sequence of the peptide corresponds to the amino acid sequences (a) to (i) . For example, codons suitable for a microorganism of the genus Starmerella are preferably selected.

[0025]

The acetyltransferase activity, specifically, includes an activity to catalyze an acetylation reaction with a sphingoid base, preferably an activity to catalyze an acetylation reaction with a hydroxy group and an amino group of phytosphingosine .

[0026]

The gene of the present invention can be easily obtained by a customary PCR method using primers prepared with reference to the nucleotide sequences represented by SEQ ID NO:l, 3 or 5 and genomic DNA of a microorganism having each of the genes as a template.

More specifically, the gene of the present invention can be obtained, for example by chemically synthesizing oligonucleotide A, which consists of a sequence

containing N-terminal initiation codon of SLI1 gene represented by SEQ ID NO : 1 , and oligonucleotide B, which consists of a sequence complementary to a sequence containing a termination codon of the gene; and performing PCR using one set of these oligonucleotides A and B and genomic DNA of Wickerhamomyces ciferrii as a template. To efficiently clone the gene fragment thus obtained to a plasmid vector, etc., a sequence for restriction enzyme digestion can be added to the 5 ' - terminal of an oligonucleotide primer. As a primer herein, nucleotides chemically synthesized based on the information on the nucleotide sequence of SLI1 gene can be generally used; however, SLI1 gene already obtained or a fragment thereof can be satisfactorily used. Examples of the nucleotides include a partial nucleotide sequence corresponding to SEQ ID NO:l and consisting of from 10 to 50 continuous bases, preferably from 15 to 35 continuous bases.

PCR conditions are, for example, 98°C for 2 minutes, (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 1 minute) x 30 cycles.

[0027]

Furthermore, the gene of the present invention can be obtained by artificial synthesis by using a DNA synthesizer in accordance with the nucleotide sequence or amino acid sequence. In synthesizing DNA, another codon encoding the same amino acid residue may be selected in place of the codon originally used (codon conversion) . An aspect of DNA obtained by the codon conversion

includes DNA in which a codon present in DNA encoding SLI1 of Wickerhamomyces ciferrii but rare in a

microorganism of the genus Starmerella (codon used less frequently in the microorganism) is converted into a codon encoding the same amino acid and highly frequently used in a translation mechanism of a microorganism in the genus Starmerella . More specifically, DNA consisting of the nucleotide sequence represented by SEQ ID NO : 7 is mentioned. Similarly, as a DNA in which a codon present in DNA encoding SLI1 of Saccharomyces cerevisiae is converted to a codon encoding the same amino acid and highly frequently used in a Starmerella microorganism, DNA consisting of the nucleotide sequence represented by SEQ ID NO: 8 is mentioned. Furthermore, as a DNA in which a codon present in DNA encoding SLI1 of Pichia pastris is converted to a codon encoding the same amino acid and highly frequently used in a Starmerella microorganism, DNA consisting of the nucleotide sequence represented by SEQ ID NO: 9 is mentioned.

[0028]

In the present invention, introduction of an

acetyltransferase gene into a microorganism of the genus Starmerella includes a method of introducing the

acetyltransferase gene such that the gene can be

expressed in the microorganism of the genus Starmerella .

[0029]

The method for introducing the gene such that the gene can be expressed is not particularly limited. A nucleic acid fragment, which contains the

acetyltransferase gene and is properly bound to a. DNA fragment containing a transcription initiation regulatory region or a transcription initiation regulatory region and a ribosome binding site, upstream thereof, may be introduced .

Such a fragment can be genetically stably maintained in a host microorganism by (1) being directly introduced as a nucleic acid fragment, or introduced as a nucleic acid fragment in a plasmid vector, etc.; or by (2) being introduced as a nucleic acid fragment with partial genome sequences of the host organism at both ends for

homologous recombination. The number of copies of the gene to be introduced is not particularly limited. In other words, a single copy and multiple copies of the gene may be introduced.

Examples of the method (1) of introducing a nucleic acid fragment into a host microorganism include an electroporation method and a lithium acetate method.

Furthermore, if the fragment is introduced in accordance with (2) , homologous recombination takes place at the site corresponding to the .sequence of the host chromosome, added to the nucleic acid fragment and the nucleic acid fragment introduced is integrated into the chromosome of the microorganism.

[0030]

Note that the transcription initiation regulatory region or transcription initiation regulatory region and the ribosome binding site to bind to a site upstream the acetyltransferase gene are not particularly limited as long as they function in a host microorganism. As an example, the original transcription initiation regulatory- region or transcription initiation regulatory region and ribosome binding site of the acetyltransferase gene, or another known transcription initiation regulatory region or transcription initiation regulatory region and

ribosome binding site is mentioned. Alternatively, e.g., promoters of a glyceraldehyde- 3 -phosphate dehydrogenase gene, a cytochrome P450 monooxygenase and a UDP- glucosyltransferase gene can be used.

[0031]

The target microorganism of the genus Starmerella to which the gene is to be introduced is not limited as long as the microorganism has a metabolic system producing a sphingoid base, at least sphingosine or phytosphingosine . Examples thereof include Starmerella bombicola, Candida apicola and Candida floricola . Among them, Starmerella bombicola is preferable. More specifically, e.g.,

Starmerella bombicola KSM36 strain (JP-A-61-31084) or NBRC10243 strain is mentioned. Starmerella bombicola is known to produce sophorolipid (SL) (Non Patent Literature 6) but incapable of producing an acetylated sphingoid base .

[0032]

In the present invention, the acetylated sphingoid base refers to a compound obtained by substituting at least one of hydrogen atoms of an acetylatable group (a hydroxy group, an amino group, etc.) that a sphingoid base has with an acetyl group. Examples thereof include acetylated compounds of (2S , 3S , 4R) -2 -aminooctadecane- 1,3,4-triol (phytosphingosine) , (2S , 3R, 4E) -2-amino-4- octadecene-1 , 3 -diol (sphingosine) , (2S,3R)-2- aminooctadecane-1, 3-diol (sphinganine) , (2S , 3S , 4R) - 2 - aminononadecane- 1 , 3 , -triol (C19 phytosphingosine),

(2S, 3R, 4E) -2-amino-4-nonadecene-l, 3-diol (C19

sphingosine), (2S , 3R) -2 -aminononadecane-1 , 3 -diol (C19 sphinganine), (2S , 3S , 4R) -2 -aminoicosane-1 , 3 , 4 -triol (C20 phytosphingosine) , (2S, 3R, 4E) -2-amino-4-icosene-l, 3-diol

(C20 sphingosine) and (2S , 3R) -2 -aminoicosane- 1 , 3 -diol

(C20 sphinganine) . Among them, an acetylated sphingoid base obtained by substituting at least one of the

hydroxyl group and amino group of a sphingoid base having a chain length of 19 or 20 carbon atoms with an acetyl group, is preferable. Among them, an acetylated

phytosphingosine obtained by acetylating at least one of the hydroxyl group and amino group of a phytosphingosine having a chain length of 19 or 20 carbon atoms, is more preferable. Furthermore, an acetylated phytosphingosine obtained by acetylating at least one of the hydroxyl group and amino group of a phytosphingosine having a chain length of 20 carbon atoms, is even more preferable.

[0033]

A microorganism thus prepared has an ability to produce an acetylated sphingoid base, and preferably an acetylated phytosphingosine having a chain length of 19 or 20 carbon atoms. The acetylated sphingoid base is accumulated in a culture medium when the microorganism is cultured.

[0034]

An acetylated sphingoid base can be produced by culturing a microorganism according to the present

invention as mentioned above in a medium,, accumulating an acetylated sphingoid base in a culture solution and recovering the acetylated sphingoid base from the culture solution .

As described later in Examples, an acetylated

sphingoid base having a chain length of 19 or 20 carbon atoms (for example, acetylated C19 phytosphingosine and acetylated C20 phytosphingosine) can be produced by using a microorganism of the present invention. Furthermore, the amount or ratio of an acetylated sphingoid base having a chain length of 19 carbon atoms produced can be increased by culturing the microorganism . in a culture medium supplemented with a pentadecanoic acid alkyl ester, a heptadecanoic acid alkyl ester or a nonadecanoic acid alkyl ester. Furthermore, the ratio of an acetylated sphingoid base having a chain length of 20 carbon atoms produced can be increased by culturing the microorganism in a culture medium supplemented with an octadecanoic acid alkyl ester.

Herein, as an alkyl ester, an alkyl ester having from 1 to 4 carbon atoms is mentioned and preferably a methyl ester or ethyl ester is mentioned. The amount of the fatty acid alkyl ester added is preferably 1 mass% or more and preferably 30 mass% or less, more preferably 10% or less, even more preferably 3% or less. In other words, the amount added is

preferably from 1 to 30 mass%, more preferably from 1 to 10 mass%, even more preferably 1 to 3 raassl.

[0035]

As the medium to be used for culture, a general medium containing a carbon source, a nitrogen source, inorganic salts, if necessary, organic micronutrients such as amino acids and vitamins can be used. Both a synthesis medium and a natural medium can be used. Any type of carbon source and nitrogen source may be used in a medium as long as a yeast strain to be cultured can utilize it.

[0036]

As the carbon source, sugars such as glucose,

glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and syrup can be used. Other than these, organic acids such as acetic acid and citric acid and alcohol such as ethanol can be used singly or in combination with another carbon source. As the nitrogen source, e.g., ammonia and an ammonium salt such as

ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, or a nitrate can be used. As the organic micronutrient , e.g., amino acids, vitamins, fatty acids, nucleic acids; and peptone,

casamino acid, a yeast extract and a soybean protein decomposition product containing these can be used. If an auxotrophic mutant requiring amino acids for growth is used, it is preferable to add the nutrients to be

required. As the inorganic salts, e.g., a phosphate, a magnesium salt, a calcium salt, an iron salt and a manganese salt can be used.

[0037]

Culture is preferably performed while controlling the culture temperature at from 20 to 35°C. If culture is performed under such conditions, preferably for about 24 hours to 120 hours, an acetylated sphingoid base can be accumulated in a culture solution.

[0038]

After completion of culture, an acetylated sphingoid base is recovered from the culture solution. The

recovering method is not particularly limited and

recovering may be made in accordance with a known

recovery method. An acetylated sphingoid base can be recovered, for example, by removing yeast cells from the culture solution, followed by applying a concentration crystallization method or column chromatograph .

[0039]

Regarding the aforementioned embodiments, the following aspects are disclosed in the present invention.

<1> A method for producing an acetylated sphingoid base comprising culturing a microorganism of the genus Starmerella to which a xenogeneic gene encoding a polypeptide having an activity to acetylate a sphingoid base is introduced.

<2> The method for producing an acetylated sphingoid base according to <1>, in which the polypeptide having an activity to acetylate a sphingoid base consists of an amino acid sequence selected from the following (a) to (i) :

(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2,

(b) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO : 2 ,

(c) a polypeptide consisting of an amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO : 2 ,-

(d) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 4,

(e) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO : 4 ,

(f) a polypeptide consisting of an amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 4;

(g) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO : 6 , (h) a polypeptide consisting of an amino acid

sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 6, and

(i) a polypeptide consisting of an amino acid

sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 6.

<3> The method for producing an acetylated sphingoid base according to <2>, in which the one to several amino acids in (b) , (e) and (h) means 1 to 80, preferably 1 to 40, more preferably 1 to 20, even more preferably 1 to 10 amino acids .

<4> The method for producing an acetylated sphingoid base according to <2>, in which the polypeptide (c) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO : 2 ; the polypeptide (f) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO: 4; and the polypeptide (i) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO: 6.

<5> The method for producing an acetylated sphingoid base according to any one of <1> to <4>, in which the microorganism of the genus Starmerella is Starmerella bo bicola . <6> The method for producing an acetylated sphingoid base according to <5>, in which the Starmerella bombicola is Starmerella bombicola KSM36 strain or Starmerella bombicola NBRC10243 strain.

<7> The method for producing an acetylated sphingoid base according to any one of <1> to <6>, in which the acetylated sphingoid base is an acetylated

phytosphingosine .

<8> The method for producing an acetylated sphingoid base according to any one of <1> to <7>, in which the acetylated sphingoid base is an acetylated sphingoid base having a chain length of 19 or 20 carbon atoms.

<9> The method for producing an acetylated sphingoid base according to <8>, in which the acetylated sphingoid base having a chain length of 19 or 20 carbon atoms is an acetylated phytosphingosine having a chain length of 19 or 20 carbon atoms.

<10> The method for producing an acetylated

sphingoid base according to any one of <1> to <9>, in which at least one fatty acid alkyl ester selected from a pentadecanoic acid alkyl ester, a heptadecanoic acid alkyl ester, an octadecanoic acid alkyl ester and a nonadecanoic acid alkyl ester is added to a culture medium .

<11> The method for producing an acetylated

sphingoid base according to <10>, in which the fatty acid alkyl ester is an ester of an alkyl having 1 to 4 carbon atoms and a fatty acid. <12> The method for producing an acetylated

sphingoid base according to <10> or <11>, in which an amount of the fatty acid alkyl ester added to the medium is preferably from 1 to 30 mass%, more preferably from 1 to 10 mass%, even more preferably from 1 to 3 mass%.

<13> A microorganism in the genus Starmerella to which a xenogeneic gene encoding a polypeptide having an activity to acetylate a sphingoid base is introduced.

<14> The microorganism in the genus Starmerella according to <13>, in which the polypeptide having an activity to acetylate a sphingoid base consists of an amino acid sequence selected from the following (a) to (i) :

(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO : 2 ,

(b) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 2,

(c) a polypeptide consisting of an amino acid sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO : 2 ;

(d) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO : 4 ,

(e) a polypeptide consisting of an amino acid sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 4, (f) a polypeptide consisting of an amino acid

sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 4;

(g) a polypeptide consisting of an amino acid

sequence represented by SEQ ID NO : 6 ,

(h) a polypeptide consisting of an amino acid

sequence which has a deletion, substitution or addition of one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 6,

(i) a polypeptide consisting of an amino acid

sequence having a homology of 80% or more with the amino acid sequence represented by SEQ ID NO: 6.

<15> The microorganism in the genus Starmerella according to <14>, in which the one to several amino acids in (b) , (e) and (h) means 1 to 80, preferably 1 to 40, more preferably 1 to 20, even more preferably 1 to 10.

<16> The microorganism in the genus Starmerella according to <14>, in which the polypeptide (c) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO : 2 ; the polypeptide (f) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO:4; and the polypeptide (i) is an amino acid sequence having a homology of preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NO: 6. <17> The microorganism in the genus Starmerella according to any one of <13> to <16>, in which the microorganism in the genus Starmerella is Starmerella bombicola .

<18> The Starmerella microorganism according to <17>, in which the Starmerella bombicola is Starmerella

bombicola KSM36 strain or Starmerella bombicola NBRC10243 strain.

[0040]

The content of the present invention will be

described more specifically by way of the following

Examples .

[Examples]

[0041]

Example 1: Preparation of a strain having cSLIl gene derived from Wickerhamomyces ciferrii introduced therein

(1) Construction of gene fragment to be introduced Acetyltransferase gene (WcSLIl) (SEQ ID NO: 7) derived from Wickerhamomyces ciferrii was artificially synthesized in accordance with codon usage of Starmerella bombicola . Then, PCR was performed using the

acetyltransferase gene (WcSLIl) as a template and primers of SEQ ID NOs:10 and 12 or primers of SEQ ID NOs:ll and 12 to obtain a WcSLIl gene fragment. The conditions of PCR are, for example, 98°C for 2 minutes, (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 1 minute) x 30 cycles. For expression of WcSLIl, a promoter of

Glyceraldehyde-3 -phosphate dehydrogenase (5'-GAPDH) and a promoter of UDP-glucosyltransferase (5'-UGT) gene were used. Individual promoter sequences were obtained by PCR using primers of SEQ ID NOs : 13 and 14 and primers of SEQ ID NOs -.15 and 16 and using genomic DNA of Star arella bombicola KSM36 strain as a template. In addition, a terminator of Cytochrome C(3'-CYC) was used. The

sequence of 3 ' -CYC was obtained by PCR using primers of SEQ ID NOs : 17 and 18 and genomic DNA of Starmerella bombicola KSM36 strain as a template. These were ligated by use of SOE-PCR to obtain [5'-GAPDH or 5'- UGT] [WcSLIl] [3' -CYC] gene fragment. The gene fragment and plasmid pHsp70A/RbcS2 -Chlamy (Chlamydomonas Resource Center) were treated with restriction enzymes Sacl and Ncol and ligated by use of in-Fusion cloning kit

(Clontech) to obtain plasmid 1. A transformant was screened by use of a hygromycin resistant gene (SEQ ID NO: 19) . The hygromycin resistant gene was obtained by PCR using primers of SEQ ID NOs: 20 and 21 and plasmid loxP-PG -gb2 -hygro- loxP (Gene Bridges) having a

hygromycin resistant gene as a template, and then, a promoter and a terminator of URA3 gene were separately amplified by PCR using genomic DNA of Starmerella

bombicola KSM36 strain as a template and primers of SEQ ID NOs: 22 and 23 or 24 and 25. The hygromycin resistant gene was ligated with the products thus amplified by means of SOE-PCR to obtain a gene fragment [5'- UR7A3] [hygromycin resistant gene] [3'-URA3] . Furthermore, plasmid pUC-Arg7 - lox-B ARG7 was subjected to PCR with primers of SEQ ID NOs:26 and 27 to amplify the region except ARG7, which was ligated with amplified product by SOE-PCR by use of in-Fusion cloning kit (Clontech) to obtain plasmid 2. Ligation was performed using loxP sequences of plasmid 1 and plasmid 2 by a ere recombinase reaction to obtain a sequence of [5 ' -GAPDH or 5 ' - UGT] [WcSLIl] [3 ' -CYC] - [5 ' -URA3] [hygromycin resistant gene] [3'-URA3] as plasmid 3. Plasmid 3 was subjected to PCR using primers of SEQ ID NOs : 13 and 25 or 15 and 25 to obtain a WcSLIl- introduced gene fragment.

Furthermore, a gene fragment to be introduced for deleting cyp52Ml, was prepared as follows. The upstream region of cyp52Ml gene was amplified by PCR using primers of SEQ ID NOs: 28 and 29; the downstream region thereof by PCR using primers of SEQ ID Nos:30 and 31; and URA3 gene by using primers of SEQ ID Nos:32 and 33, using genomic DNA of Starmerella bombicola KSM36 strain as a template. The three fragments thus obtained were ligated by SOE-PCR. The resultant fragment was used as a cyp52Ml deficient fragment .

The primers used in Example 1 are summarized in Table 1.

[0042] [Table 1]

[0043]

(2) Acquisition of uracil auxotrophic strain Star erella bombicola SM36 strain (FERM BP-799) was inoculated into SD-U agar medium containing 0.68% Yeast Nitrogen Base w/o amino acids, 2% glucose, 0.03% uracil and 1.5% Agar and then cultured at 30°C for one month. The obtained yeast cells were taken by a platinum loop and suspended in 1 mL of 0.8% saline solution. 100 μΐ of the suspension was spread on SD-UF agar medium containing 0.68% Yeast Nitrogen Base w/o amino acids, 2% glucose, 0.03% uracil, 5 - fluoroorotic acid and 1.5% Agar and cultured at 30°C for 2 weeks. The grown colonies were cultured again in SD-UF agar medium. Thereafter,

cultured products were each confirmed for. uracil

auxotrophy and 5-fluoroorotic acid resistance, and then, an uracil auxotrophic strain was obtained.

Starmerella bombicola KSM36 strain and the obtained uracil auxotrophic strain were each taken by a platinum loop and inoculated into 5 mL of 50 g/L YPD Broth

(manufactured by Japan BD) contained in a 100 -mL volume test tube and cultured at 30°C and 250 rpm for 48 hours. The culture solution (1 mL) was centrifuged at 5000 rpm at 4°C for 5 minutes to collect yeast cells. From the yeast cells, genomic DNA was extracted by using

Dr.GenTLE™ (TAKARA Bio) in accordance with the method described in the attached instruction. Using primers (SEQ ID. NOs:32, 33) listed in Table 1 and KOD-plus. ver 2 (TOYOBO) , URA3 gene encoding orotidine decarboxylase involved in uracil biosynthesis was amplified. Sequence of the URA3 gene was analyzed using the PCR product as a template and compared to the sequence of Starmerella bombicola NBRC10243 strain (GenBank accession No.

DQ916828) . As the result, it was confirmed that

Starmerella bombicola KSM36 strain has the same sequence as that of the URA gene of Starmerella bombicola NBRC10243 strain; the uracil auxotrophic strains all have a mutation (cysteine is changed to tyrosine) at the 54- position. The obtained uracil auxotrophic strain was used as Starmerella bombicola KSM36-ura3 strain.

[0044]

(3) Acquisition method for uracil auxotrophic strain Since the mutation position of the uracil

auxotrophic strain was determined in the section (2) , it became possible to easily prepare an uracil auxotrophic strain, for example, by using the following gene

recombinant means .

URA3 gene was amplified using genomic DNA of

Starmerella bombicola KSM36 strain as a template and primers of SEQ ID NOs:32 and 33. The amplified gene fragment was introduced into an appropriate vector to introduce a point mutation for changing cysteine at the 54 -position to tyrosine. The vector to which a point mutation was introduced was amplified by using the primers of SEQ ID NOs:32 and 33 to obtain a transformed fragment containing the mutation introduced in URA3 gene. Starmerella bombicola KSM36 was taken by a platinum loop, inoculated into YPD Broth (5 mL) contained in a 100 mL- volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution was inoculated so as to obtain a concentration of 1% in YPD Broth (50 mL) contained in a Sakaguchi flask and cultured at 30°C and 120 rpm until OD600 reaches 1 to 2. The yeast cells proliferated were collected by centrif gation at 3000 rpm and 4°C for 5 minutes and washed twice with ice-cooled sterilized water (20 mL) . The yeast cells were suspended in 1 mL of an ice-cooled 1M sorbitol solution and

centrifuged at 5000 rpm and 4°C for 5 minutes. After the supernatant was discarded, 400 μΐι of a 1M sorbitol solution was added. The mixture was allowed to stand on ice and suspended by pipetting. The yeast suspension solution (50 μΐι) was taken and dispensed, and DNA (1 μg) for transformation was added. The yeast suspension solution was transferred to an ice-cooled chamber having a gap of 0.2 cm (BIO-RAD) and thereafter, a pulse (25 μΡ, 350 Ω, 2.5 kV) was applied by use of GENE PULSER II

(BIO-RAD) . An ice-cooled 1M sorbitol-containing YPD Broth was added and the mixture was transferred to a 1.5 mL-volume tube, shaken at 30°C for 2 hours and then centrifuged at 5000 rpm and 4°C for 5 minutes to recover the yeast cells. The yeast cells were resuspended in 200 μ1_, of a 1M sorbitol solution. An aliquot (100 μΐ^ was taken, spread on a selective medium and cultured at 30°C for about one week. As the selective medium, SD-UF agar medium containing 0.68% Yeast Nitrogen Base w/o amino acids, 2% glucose, 0.03% uracil, 5 - fluoroorotic acid and 1.5% Agar was used. The colonies grown were cultured again in the SD-UF agar medium. Thereafter, the cultured products were each confirmed for uracil auxotrophy and 5- fluoroorotic acid resistance. In this manner, a uracil auxotrophic strain could be obtained.

[0045] (4) Acquisition of cyp52Ml gene deficient strain Starmerella bombicola KSM36-ura3 strain obtained above was taken by a platinum loop, inoculated into YPD Broth (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution was inoculated so as to obtain a concentration of 1% in YPD Broth (50 mL) contained in a Sakaguchi flask and cultured at 30°C and 120 rpm until OD600 reached 1 to 2. The yeast cells proliferated were collected by centrifugation at 3000 rpm and 4°C for 1 minute and washed twice with ice-cooled sterilized water (20 mL) . The yeast cells were suspended in 1 mL of an ice-cooled 1M sorbitol solution and centrifuged at 5000 rpm and 4°C for 5 minutes. After the supernatant was discarded, 400 μL of a 1M sorbitol solution was added. The mixture was allowed to stand on ice and suspended by pipetting. The yeast suspension solution (50 μL) was taken and dispensed, and 1 μg of DNA (cyp52Ml deficient gene fragment) for transformation was added. The yeast suspension solution was transferred to an ice-cooled chamber having a gap of 0.2 cm (BIO-RAD) and thereafter, a pulse (25 μΡ, 350 Ω, 2.5 kV) was applied by use of GENE PULSER II (BIO-RAD) . An ice-cooled 1M sorbitol - containing YPD Broth was added and the mixture was transferred to a 1.5 mL-volume tube, shaken at 30°C for 2 hours and then centrifuged at 5000 rpm and 4°C for one minute to recover yeast cells. The yeast cells were resuspended in 200 μL of a 1M sorbitol solution. An aliquot (100 μΐ-ι ) was taken, spread on a selective medium and cultured at 30°C for about one week. As the

selective medium, SD-ura agar medium containing 0.68% Yeast Nitrogen Base w/o Amino Acids, 2% glucose, 0.077% CSM-ura (Funakoshi) and 1.5% Agar was used. The colonies grown were subjected to colony PGR using primers of SEQ ID NOs:28 and 31 by KOD-FX-Neo (TOYOBO) . After

confirming that the length of the sequence amplified was changed, cyp52Ml gene deficient strain was obtained.

[0046]

(5) Introduction of WcSLIl gene into Starmerella bombicola

Starmerella bombicola cyp52Ml gene deficient strain and Starmerella bombicola SM36 strain were each taken by a platinum loop, inoculated into YPD Broth (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The culture solution obtained was inoculated so as to obtain a concentration of 1% in YPD Broth (50 mL) contained in a Sakaguchi flask and cultured at 30°C and 120 rpm until OD600nm reached 1 to 2. The yeast cells proliferated were collected by centrifugation at 3000 rpm and 4°C for 5 minutes and washed twice with ice-cooled sterilized water (20 mL) . The yeast cells were suspended in 1 mL of an ice-cooled 1M sorbitol solution and centrifuged at 5000 rpm and 4°C for one minute. After the supernatant was discarded, 400 μL of a 1M sorbitol solution was added. The mixture was allowed to stand on ice and suspended by pipetting. The yeast suspension solution (50 μΐι) was taken and dispensed, and 1 μg of DNA ( cSLIl introduced gene fragment) for transformation was added. The yeast suspension solution was transferred to an ice-cooled chamber having a gap of 0.2 cm (BIO-RAD) and thereafter, a pulse (25 μΡ, 350 Ω, 2.5 kV) was applied by use of GENE PULSER II (BIO-RAD) . An ice-cooled 1M sorbitol-containing YPD Broth was added and the mixture was transferred to a 1.5 mL-volume tube, shaken at 30°C for 2 hours, and then centrifuged at 5000 rpm and 4°C for one minute. The yeast cells were

recovered and resuspended in 200 μΐ, of a 1M sorbitol solution. An aliquot (100 μΐ.) was taken, spread on a selective medium and cultured at 30°C for about one week. As the selective medium, SD-ura + hygromycin agar medium containing 0.68% Yeast Nitrogen Base w/o Amino Acids, 2% glucose, 0.077% CSM-ura (Funakoshi) , 200 μg/mL· hygromycin and 1.5% Agar was used. The colonies grown were

subjected to colony PCR using primers of SEQ ID NOs : 13 and 18 or 15 and 18 by KOD-FX-Neo (TOYOBO) . After

confirming that the gene was inserted in the genome,

Acyp52Ml/pGAPDH-WcSLIl strain (GAPDH promoter was used) and Acyp52Ml/pUGT-WcSLIl strain (UGT promoter was used) having the gene introduced in Starmerella bombicola cyp52Ml deficient strain; and pGAPDH-WcSLIl strain and pUGT- cSLIl strain having the gene introduced in

Starmerella bombicola KSM36 strain, were obtained.

Furthermore, as a control, a strain having the hygromycin resistant gene alone introduced therein was obtained. [0047]

Example 2: Analysis of acetylated phytosphingosine productibility in strain having WcSLIl gene introduced therein (1)

(1) Analysis of lipid composition of the strain having the gene introduced therein

A control strain (hygromycin resistant gene) ,

Acyp52Ml/pGAPDH-WcSLIl strain and Acyp52Ml/pUGT-WcSLIl strain were each spread on YPD agar medium. The colonies grown were taken by a platinum loop, inoculated into SD- Ura medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution (500 ΐ was inoculated into SD-Ura medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 to 72 hours. Then, lipid composition was analyzed.

[0048]

(2) Quantification of acetylated phytosphingosine After the culture solution (1 mL) was recovered, 4 mL of a solution mixture containing chloroform and methanol in a ratio of 2:1 was added, vortexed and then allowed to stand still for 15 rain. The mixture was centrifuged at 3000 rpm for 15 minutes and the lower layer (chloroform layer) was collected. The solution collected was dried by blowing nitrogen gas, suspended in 1 mL of methanol, appropriately diluted and filtered through a filter. After filtration, the sample was measured by LC-MS/MS. The conditions of LC-MS/MS are as follows:

LC conditions: Capcell core C18 2.7 μπκρ 2.1 x 50 mm (Shiseido Co., Ltd.), Oven Temp. 40°C, Sol. A: 0.1% HC0 ₂ H in water, Sol. B: MECN, (A60%, B40%) 0.5 min → [(A60%, B40%) → (A0%, B100%) 5.5 min] → B100¾ 2 min, [(A0%, B100%) → (A60%, B40%) O.Olmin] → (A60%, B40%) 2 min, Flow rate 0.6 ml/min. , Inject 5 μΐ , MS/MS apparatus:

API3200QTrap (AB SCIEX) .

[0049]

[Table 2]

Ions used for quantification of acetylated phytosphingosines

[0050]

Lipid compositions after culture for 24 and 48 hours were shown in Figure 1. In the control strain, neither triacetylphytosphingosine (TriAPS) (length of carbon chain: 18) nor tetraacetylphytosphingosine (TAPS) (length of carbon chain: 18) were produced; whereas, in

Starmerella bombicola having WcSLIl gene introduced therein, production of TriAPS and TAPS were confirmed. The amounts of acetylated phytosphingosin.es produced at 72nd hour after initiation of culture and the ratios of the products are shown in Table 3 and Table 4.

[0051]

[Table 3]

Amounts of triacetylated phytosphingosines having a chain length of from 18 to 20 carbon atoms produced and the ratios of the products

[0052]

[Table 4]

Amounts of tetraacetylated phytosphingosines having a chain length of from 18 to 20 carbon atoms produced and the ratios of the products

[0053] Example 3 : Analysis of acetylated phytosphingosines productibility in strain having WcSLIl gene introduced therein (2)

Acyp52Ml/pGAPDH-WcSLIl strain was spread on YPD agar medium. The colonies grown were taken by a platinum loop, inoculated into SD-Ura medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution (100 μΐ was inoculated into SD-Ura medium (5 mL) with a C15 to C19 fatty acid ethyl ester (50 mM) contained in a 100 mL- volume test tube and cultured at 30°C and 250 rpm for 72 hours. Then, lipid composition was analyzed in the same manner as in Example 2.

[0054]

The amounts of C18 to C20 acetylated

phytosphingosines produced at 72nd hour after initiation of culture and the ratios of the products are shown in Table 5 and Table 6

[0055]

[Table 5]

Amounts of triacetylated phytosphingosines having a chain length of 18 to 20 carbon atoms produced and the ratios of the products Amount produced Ratio (%)

(mg/L) C18 C19 C20

No additives 23.3 22.5 7.5 70.0

Ethyl pentadecanoate 18.4 0.6 94.4 5.0

Ethyl hexadecanoate 19.9 52.7 3.6 43.6

Ethyl heptadecanoate 27.7 0.4 97.0 2.7

Ethyl stearate 22.6 4.3 2.3 93.4

Ethyl nonadecanoate 28.7 6.2 68.6 25.2

[00-56]

[Table 6]

Amounts of tetraacetylated phytosphingosines having a chain length of from 18 to 20 carbon atoms produced and the ratios of the products

[0057]

Reference Example: Analysis of acetylated

phytosphingosine productibility in strain having WcSLIl gene introduced therein (3)

Acyp52Ml/pGAPDH-WcSLIl strain, pGAPDH-WcSLIl strain, Acyp52Ml/pUGT-WcSLIl strain and pUGT-WcSLIl strain were each spread on YPD agar medium. The colonies grown were taken by a platinum loop, inoculated into YPD medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution (100 μL) was inoculated into modified YPD medium

(10% glucose, 10% ethyl hexadecanoate , 2% Peptone, 1% Yeast Extract, 25 mM CaCl ₂ -2H ₂ 0, 50 m L-serine) (5 mL) contained in a 100 mL-volume test tube, and cultured at 30°C and 250 rpm for 7 days. Then, lipid composition was analyzed in the same manner as in Example 2. The results of lipid analysis are shown in Table 7. Furthermore, fatty acid ethyl ester or fatty acid was extracted from the culture solution (1 mL) by using 2 mL of hexane .

After the hexane layer was collected, ethyl acetate (2 mL) was added to the remaining water layer to extract sophorolipid and the ethyl acetate layer was collected. The ethyl acetate layer was dried and analyzed. As the result, it was confirmed that Acyp52Ml/pGAPDH-WcSLIl strain and Acyp52Ml/pUGT-WcSLIl strain produced no sophorolipid; whereas, pGAPDH-WcSLIl strain and pUGT- WcSLIl strain produced sophorolipid.

[0058]

[Table 7]

Amounts of triacetylated phytosphingosines having a chain length of 18 to 20 carbon atoms produced and the ratios of the products Amount Ratio (%)

produced

(mg/L) C18 C19 C20

Acyp52Ml/pGAPDH-WcSLIl strain 34.4 71.7 2.3 26.1

Acyp52Ml/pUGT-WcSLIl strain 39.1 73.5 2.1 24.4 pGAPDH-WcSLIl strain 71.1 44.6 3.2 52.2 pUGT-WcSLIl strain 68.1 51.1 3.1 45.8

[0059]

Example 4 Preparation of strain having various xenogeneic SLI1 introduced

(1) Preparation of a fragment for introduction

An upstream site of CYP52M1 gene was amplified by PCR using primers of SEQ ID NOs:34 and 35 and genomic DNA of Starmerella bombicola KSM36 strain as a template and ligated with plasmid 1, which was obtained by

amplification using primers of SEQ ID NOs:36 and 37, by use of in-Fusion cloning kit (Clontech) to insert the upstream site of CYP52 1 gene ahead of the GAPDH promoter. This was designated as plasmid 1-A. Subsequently, a region containing a promoter and a terminator of URA3 gene was amplified by PCR using primers of SEQ ID N0s:22 and 25 and genomic DNA of Starmerella bombicola KSM36 strain as a template. Furthermore, the region except ARG7 of plasmid pUC-Arg7- lox-B ARG7 was amplified using primers of SEQ ID NOs:26 and 27 and ligated with the amplification product of URA3 , by use of in-Fusion

cloning kit (Clontech) . The obtained plasmid was

designated as plasmid 2 -A. Furthermore, a downstream site of CYP52 1 gene was amplified by PCR using primers of SEQ ID NOs -.38 and 39 and genomic DNA of Starmerella bombicola KSM36 strain as a template and ligated with plasmid 2 -A which was obtained by amplification using primers of SEQ ID NOs:40 and 41, by use of in-Fusion cloning kit (Clontech) to insert the downstream site of CYP52 1 gene backward into the URA3 terminator. This was designated as plasmid 2-B. Plasmid 1-A and Plasmid 2-B were ligated by Cre recombinase reaction to obtain a plasmid 4. PCR was performed using primers of SEQ ID NOs:28 and 31 and plasmid 4 as a template to obtain cyp52Ml : : GAPDH-WcSLIl fragment.

[0060]

(2) Preparation of fragment for introducing

xenogeneic SLIl

SLIl genes derived from Saccharomyces cerevisiae and Pichia pastoris were artificially synthesized in

accordance with codon usage of Starmerella bombicola to obtain sequences represented by SEQ ID NOs : 8 and 9, respectively. Using these as templates and primers of SEQ ID NOs: 42, 43 and 44, 45, PCR was performed to obtain ScSLIl and PpSLIl fragments. Subsequently, using primers of SEQ ID NOs : 46 and 47 and plasmid 4 as a template, PCR was performed to amplify the region except cSLIl. The ScSLIl fragment and PpSLIl fragment were ligated with a plasmid by use of in-Fusion cloning kit (Clontech) to obtain plasmids 5 and 6, respectively. Using primers of SEQ ID NOs:28 and 31 and plasmids 5 or 6 as a template, PCR was performed to obtain cyp52Ml : :pGAPDH-ScSLIl

fragment and cyp52Ml _{: :} pGAPDH-PpSLIl fragment.

[0061]

(3) Preparation of various SLI1- introduced strains Starmerella bombicola KSM36-ura3 strain as mentioned above was taken by a platinum loop and inoculated into YPD Broth (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The culture solution obtained was inoculated so as to obtain a concentration of 1% in YPD Broth (50 mL) contained in a

Sakaguchi flask, and cultured at 30°C and 120 rpm until OD600 reached 1 to 2. The yeast cells proliferated were collected by centrifugation at 3000 rpm and 4°C for 5 minutes and washed twice with ice-cooled sterilized water (20 mL) . The yeast cells were suspended in 1 mL of an ice-cooled 1M sorbitol solution and centrifuged at 5000 rpm and 4°C for one minute. After the supernatant was discarded, 400 μL of a 1M sorbitol solution was added.

The resultant mixture was placed on ice and suspended by pipetting. The yeast suspension solution (50 μL) was taken and dispensed, and 1 μg of DNA for transformation was added. The yeast suspension solution was transferred to an ice-cooled chamber having a gap of 0.2 cm (BIO-RAD) , and thereafter, a pulse (25 μΡ, 350 Ω, 2.5 kV) was

applied by use of GENE PULSER II (BIO-RAD) . An ice- cooled 1 sorbitol -containing YPD Broth was added and the mixture was transferred to a 1.5 mL-volume tube, shaken at 30°C for 2 hours and then centrifuged at 5000 rpm and 4°C for one minute. The yeast cells were recovered and resuspended in 200 μΣ, of a 1 sorbitol solution. An aliquot (100 μΐ>) was taken, spread on a selective medium and cultured at 30°C for about one week. As the

selective medium, SD-ura agar medium containing 0.68% Yeast Nitrogen Base w/o Amino Acids, 2% glucose, 0.077% CSM-ura (Funakoshi) and 1.5% Agar was used. The colonies grown were subjected to colony PCR using primers of SEQ ID NOs:28 and 31 by KOD-FX-Neo (TOYOBO) . After

confirming that the length of the sequence amplified changed, cyp52Ml : :pGAPDH-WcSLIl strain, cyp52Ml : :pGAPDH- ScSLIl strain and cyp52Ml : : pGAPDH-PpSLIl strain were obtained. The primers used in Example 4 are summarized in Table 8.

[0062]

[Table 8]

[0063]

Example 5 Evaluation of acetylated phytosphingosine productibility of various SLI1 expressing strains

Three strains obtained in Example 4 and cyp52Ml deficient strain were each spread on YPD agar medium. The colonies grown were taken by a platinum loop and inoculated into SD-Ura medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 24 hours. The obtained culture solution (100 μΐ.) was inoculated into SD-Ura medium (5 mL) contained in a 100 mL-volume test tube and cultured at 30°C and 250 rpm for 120 hours. Then, lipid composition was analyzed in the same manner as in Example 2. The amount of C18 to C20 acetylated phytosphingosine produced at 120th hour after initiation of culture and the ratio of the products are shown in Table 9 and Table 10.

[0064]

[Table 9]

Amounts of monoacetylated phytosphingosines having a chain length of 18 to 20 carbon atoms produced and the ratios of the products

[0065]

[Table 10]

Amounts of triacetylated phytosphingosines having a chain length of 18 to 20 carbon atoms produced and the ratios of the products