WO1999057279A1 - Plasmid for gene expression in pichia ciferrii and transformation method using the same - Google Patents

Abstract

Expression cassettes for transforming Pichia ciferrii and their use are disclosed. The present invention relates to expression cassettes containing Pichia ciferrii ribosomal DNA fragment, CYHr gene resistant to cycloheximide, and a desired gene. Expression cassette further comprising Pichia ciferrii GAPDH promoter gene which allows an increase in the expression of the desired gene also provided. Moreover, the present invention provides a process for producing tetraacetyl phytosphingosine using transformed Pichia ciferrii cells with the expression cassettes in a higher yield.

Description

1

PLASMID FOR GENE EXPRESSION IN PICHIA CIFERRII AND TRANSFORMATION METHOD USING THE SAME

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

The present invention relates to expression cassettes for transforming Pichia ciferru. More particularly, it relates to expression cassettes containing Pichia ciferru ribosomal DNA fragment, CYH^r gene resistant to cycloheximide, and a desired gene, and to uses thereof.

2. DESCRIPTION OF THE PRIOR ARTS

Pichia ciferru has been used to biologically desulfurize coals (Stevens et al., USP 4,851,350), to produce D-alpha-amino acids (Takeichi et al, USP 5,068,187), to produce (S)-l-phenyl-l,3-propandiol (Ajinomoto, JP 6-90789A) or to produce secondary alcohols by stereospecific ketone reduction (Merck, EP-300287). Further, it produces and secretes tetraacetyl phytosphingosine (TAPS) which is a precursor of ceramides (Barenholz et al, Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341, 1973).

Phytosphingosines including TAPS, like ceramides, show an activity of surface skin-protection and of preventing excessive water-loss and dry out of the skin, facilitating their uses in cosmetics. They can be obtained from various microorganisms and easily converted to ceramides by N-acylation.

TAPS productions by wild type Pichia ciferru ATCC 14091 and F-60-10 (NRRL 1301) are not satisfactory for commercial uses. To improve the production of TAPS in the strains of Pichia ciferru, attempts to provide mutants which are capable of producing a higher level of TAPS have been made (Wickerham & Burton, 2

J. Bacteriol, 80, 484, 1960; USP 5,618,706). The present inventors also developed novel useful mutant (KFCC- 10937) which allows a larger amount of TAPS production in a shorter time (KR 98-49305 A).

Pichia ciferrii had been classified into genus Hansenula and is recently reclassified into genus Pichia by 5S-RNA analysis (Yamada et al, Biosci, Biotechnol Biochem., 58, 1245, 1994). By this reason, the genetic study of the Pichia yeasts is not sufficient and transformation method of Pichia ciferru has not been established.

The present invention provides plasmid prACL2 comprising Pichia ciferru serine palmitoyl transferase gene and a transformed Pichia ciferru cell which allows an improved production of TAPS.

The inventors found that the known transformation method for Candida utilis (Kondo et al., J. Bacteriol., 177, 7171, 1995) can be modified and applied to the Pichia ciferru. They cloned Pichia ciferrii ribosomal protein L41 -coding gene to determine its nucleotide sequence and manipulated to give a resistance to cycloheximide, an antibiotic from yeasts, so as to be used as a selection marker. Thus recombinant gene may be linked to a plasmid which carry a desired gene to give an expression cassette which is useful to transform Pichia ciferrii in which the desired gene is expressed. Moreover, the inventors succeeded in cloning of Pichia ciferrii GAPDH promoter gene and found that its insertion into the expression cassette allows an unexpected improvement of the expression level. In fact, they increased the production amount of TAPS by transforming the strain of Pichia ciferrii with the expression cassette caπying LCB2 gene as a desired gene and culturing the resulting transformed cells. LCB2 gene codes for palmitoyl transferase which is involved in the TAPS synthesis in the living body. 3

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to determine and use genetic information of Pichia ciferrii ribosomal protein L41 gene.

Another object of the present invention is to provide an expression cassette for transforming Pichia ciferrii, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferrii L41 gene as a selection marker, and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.

Another object of the present invention is to provide a method for transforming Pichia ciferru with a plasmid containing the expression cassette.

The present invention determines and uses genetic information of Pichia ciferrii glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene and GAPDH promoter gene.

The present invention provides an expression cassette for transforming Pichia ciferrii, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferru L41 gene as a selection marker, Pichia ciferrii GAPDH promoter gene and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.

The present invention further provides an expression cassette for transforming Pichia ciferru, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferrii L41 gene as a selection marker, Pichia ciferrii GAPDH promoter gene, a desired gene and

Pichia ciferru ribosomal DNA. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.

The present invention determines and uses genetic information of Pichia ciferrii serine palmitoyl transferase which is involved in TAPS synthesis.

The present invention provides plasmid prACL2 comprising an expression cassette having Pichia ciferrii serine palmitoyl transferase gene and a transformed Pichia ciferru cell with an improved production of TAPS.

The present invention further provides plasmid prACGL2 comprising an expression cassette having Pichia ciferrii serine palmitoyl transferase gene and a Pichia ciferrii transformant with an improved production of TAPS.

The present invention further provides plasmid prHECGL2 comprising an expression cassette having Pichia ciferru serine palmitoyl transferase gene and a Pichia ciferrii transformant with an improved production of TAPS.

The present invention still further provides a method for producing TAPS by culturing the transformed Pichia ciferru cells.

The objects mentioned above, other features and applications of the present invention would be much more apparent by those of ordinary skills in the art from the following explanation in detail.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a restriction map of various plasmids having Pichia ciferrii ribosomal DNA incorporated with L41 genes of Pichia ciferrii at different sites for each plasmid, in which hatched boxes indicate ribosomal DNA fragments; restriction enzymes in bold letters indicate the enzymes used for linearization of the respective plasmids; arrows indicate the transcription direction of the genes; and plasmid prACL2 carries LCB2 gene coding for serine palmitoyl transferase from Pichia ciferru.

Figure 2 is a restriction map of Pichia ciferrii LCB2 gene, and the orientation and the length of the arrows indicate the direction and degree of nucleotide sequencing, respectively.

Figure 3 represents the construction and restriction map of plasmid prACL2.

Figure 4 is a restriction map of Pichia ciferru GAPDH gene, and the orientation and the length of the arrows indicate the direction and degree of nucleotide sequencing, respectively.

Figure 5 represents the construction and restriction map of plasmid prACGL2.

Figure 6 represents the construction and restriction map of plasmid prHECGL2.

Figure 7 is results of Southern blot analysis carried out to measure the copy number of LCB2 gene transformed into host cell.

DETAILED EXPLANATION OF THE INVENTION

Kondo et al describes a transformation method for Candida utilis in which an antibiotic resistant gene from yeast is used as a marker gene instead of the conventional bacterial one (Kondo et al., J. Bacteriol., 177, 7171, 1995). The present inventors made attempts to apply such an idea to Pichia ciferrii. For this purpose, they cloned Pichia ciferrii ribosomal protein L41 gene and deteπnined its nucleotide sequence. Further, they identified 56th arnino acid which is responsible for the sensitivity to cycloheximide is proline and replaced it by glutamine to give cycloheximide-resistance to L41 protein. In the present invention, L41 gene is used as a selection marker.

1. Isolation of Pichia ciferrii ribosomal protein L41 gene

By following and modifying the method by Kondo et al (Kondo et al, J. Bacteriol., 177, 7171, 1995), two primers CYH1 and CYH4 were synthesized. CYH1 : 5'-CGC GTA GTT AAY GTN CCN AAR AC-3' CYH4 : 5'-GCC TGG CCY TTY TGY TTY TTN TC-3' The two primers are also represented as SEQ. ID. NO. 7 and SEQ. ID. NO. 8, respectively in SEQUENCE LISTING.

PCR was performed using the two primers to isolate Pichia ciferrii ribosomal protein L41 gene of about 300bp, which was labeled to be used as a probe. Thus obtained probe was used to carry out Southern blot analysis for Pichia ciferru genome DNA. A genome DNA digested with EcoRI gives a desired band at 1.9 kb. EcoRI-digested DNA fragments of 1.9 kb was collected and used to construct pCYHl.9 which is used for nucleotide sequencing. The results are shown in Figure

1. DNA sequence of Pichia ciferrii ATCC 14091 ribosomal protein L41 gene was submitted on March 7, 1998 to GenBank under accession number AF 053457. Pichia ciferru ATCC 14091 ribosomal protein L41 gene has 737 base pairs including 419 bp intron. The putative amino acid sequence deduced from the nucleotide sequence shows a homology of 90% or more to those of other yeasts. It was also identified that the cycloheximide-sensitive amino acid is amino acid 56, proline.

2. Impartation of cycloheximide resistance to L41 gene : Construction of plasmid pCYH1.9^r for use in the selection of transformed cell.

Site-directed mutagenesis was carried out to replace proline (aa 56) in L41 gene with glutamine in order to impart cycloheximide resistance to L41 gene. Thus obtained gene-manpulated gene is designated as 'plasmid CYH1.9

Hereinafter, the L41 gene is indicated by an abbreviation of YT and the cycloheximide-resistant L41 gene is indicated by ^ΛCYH throughout the description.

3. Isolation of Pichia ciferrii ribosomal DNA : Construction of plasmid prDX9.0 for improving integration efficiency of desired gene into chromosome

In order to improve the efficiency of integration of the desired gene into the chromosomal DNA, ribosomal DNA was employed. Within the cell, several hundred copies of ribosomal DNA occur. PCR primers were synthesized by using the partial nucleotide sequence of Pichia ciferru ribosomal RNA (Yamada et al, Biosci. Biotechnol Biochem., 58, 1245, 1994). PCR of Pichia ciferrii ATCC 14091 genome DNA was carried out by using the primers to isolate ribosomal DNA fragment of 6.0 kb. The fragment was used as a probe for Southern blot analysis to isolate Pichia ciferrii ATCC 14091 ribosomal DNA fragment of 9 kb, which is inserted into plasmid pBluescript KS+ to produce plasmid prDX9.0.

Partial sequence of the ribosomal DNA fragment (9 kb) was determined, and the location and orientation of 5S, 26S, 5.8S and 18S ribosomal protein genes are shown in Figure 1.

4. Construction of recombinant plasmids

Ribosomal DNA was digested with various restriction enzymes and ligated to CYH^r to give various plasmids as shown in Figure 1 which will be analyzed for transformation efficiency in regard with (1) the transcription directions of respective genes; (2) the arrangement of ribosomal DNA and CYH^r gene; and (3) the kind of insertion site, ribosomal RNA structural gene or non-transcribed region.

The characteristics of the plasmid in Figure 1 are summarized in Table 1.

Table 1

Plasmid Characteristics prXHNC Ribosomal DNA (3.5 kb) obtained by Xbal digestion is treated with Hpal/Ncol to remove 1.6 kb ribosomal DNA and then ligated to COT. prEHC Ribosomal DNA (3.8 kb) obtained by EcoRI digestion is treated with Hpal to remove 2 kb ribosomal DNA and then ligated to COT. prCEX CYH^r is ligated to EcoRI site of 1.1 kb ribosomal DNA

obtained by EcoRI/Xbal digestion prCRX CYH^r is ligated to EcoRV site of 1.3 kb ribosomal DNA obtained by EcoRV/Xbal digestion prXCH CYH^r is ligated to Hpal site of 3.5 kb ribosomal DNA obtained by Xbal digestion prXCE CYH^r is ligated to EcoRl site of 3.5 kb ribosomal DNA obtained by Xbal digestion prXHC1.9 COT is ligated to EcoRI site of 1.6 kb ribosomal DNA obtained by Xbal/Hindlll digestion prAC1.9 COT is ligated to EcoRV site of 0.6 kb ribosomal DNA obtained by HinάTII/EcoRV digestion prEC1.9 COT is ligated to EcoRI site of 1.4 kb ribosomal DNA obtained by Hindlll EcoRI digestion prHEC1.9F COT is ligated to EcoRV site of 1.4 kb ribosomal DNA obtained by Hindlll/EcoRI digestion prHEC1.9R Same as prHEC1.9F except that the insertion orientation of

CYH^r is reversed

In the nomenclature of the plasmid, for example prAC, the letter ^Λp^λ indicates a plasmid, Y indicates that the plasmid has a ribosomal DNA, and ^' means for "CYHV The letters 'A' in prAC1.9 and Ε^" in prEC1.9 indicate restriction enzymes used for liberalization by digesting the ribosomal DNA. In case of other plasmids, X^Λ means Xbal, Ε^x means EcoRI, "R^λ means EcoRV, and Η means Hindlll, respectively. Therefore, the plasmid containing these letters in their name comprise ribosomal DNA frgaments digested with these respective restriction enzymes. Moreover, numerical number after C indicates the size (base pairs) of the L41 gene, while the letter or ^λR^λ means the orientaion of ribosomal DNA fragment and COT.

5. Transformation and selection of transformed cells

The method described by Klass and Peter (Klass & Peter, Curr. Genet., 25, 305, 1994) is followed. That is to say, cells of Pichia ciferrii ATCC 14091 grown in YEPD medium to OD_600nm of 1.5 were collected by centrifugation and mixed with the plasmid. Electroporation was performed at 500V, 50μF and 800Ω so as to transform the cells with the plasmid and subjected to growth in YEPD solid medium to which cycloheximide was added to select transformed cells containing cycloheximide-resistant L41 gene(CYH^r).

The transformation efficiency is shown in Table 2 (Example 17). The data in Table 2 reveals that prHEC1.9F in which the non-transcribed region between 5S and 26S ribosomal RNA structural genes is used as an insertion site has the highest transformation efficiency. And, prHECl.pR in which the transcription direction of 5S RNA gene is opposite to that of CYΗ shows significantly decreased transformation efficiency.

6. Analysis of transformed cells by Southern blot analysis Genomic DNA was isolated from the transformed cells and the insertion patterns of CYH^r were analyzed by Southern blot analysis. As a result thereof, it was found that 4-5 copies of CYH^r were present in the chromosome of the transformed cells. This result indicates that the transformation of Pichia ciferrii with the expression cassette of the present invention optimizes the integration of the desired gene into the chromosome of transformed Pichia ciferrii cells.

7. Construction of prACL2 for TAPS production; transformation; and the production of TAPS by cultivation of transformed cells

An expression plasmid was constructed from an expression cassette comprising LCB2 gene for producing serine palmitoyl transferase and transformed into the mutant Pichia ciferrii KFCC- 10937 to evaluate the TAPS production. The desired gene, LCB2 gene codes for serine palmitoyl transferase, which is involved in TAPS synthesis, and the mutant KFCC- 10937 was developed by the inventors (KR 10

98-49305A).

Serine palmitoyl transferase (3-ketosphinganine synthase, EC 2.3.1.50) is involved in the first step, which is the rate limiting step of the overall reaction, of the sphingolipids synthesis and forms 3-ketosphinganine having 18 carbon atoms by condensing serine and palmitoyl-CoA (Barenholz et al, Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341, 1973). 3-ketosphmganine serves as one of long-chain compounds in animals and as a precursor for phytosphingosine in plants and fungi.

In the present invention, the TAPS production can be significantly improved by integrating multiful copies of LCB2 gene into the chromosome of Pichia ciferrii. The transformed Pichia ciferru cell carrying multiful copies of LCB2 gene on its chromosome produces a large amount of TAPS in a shortened time.

7-1. Isolation of LCB2 gene coding for serine palmitoyl transferase

For the purpose of cloning the gene coding for serine palmitoyl transferase from Pichia ciferrii ATCC 14091 genomic DNA, probes were prepared with reference to the subunits of the known serine palmitoyl transferase gene.

Serine palmitoyl transferase coding gene from Saccharomyces cerevisiae consists of two subunits, LCB1 and LCB2. LCB is abbreviation for long chain base/

(Nagiec et al, Proc. Natl. Aca. Sci. USA, 91, 7899, 1994). DNA sequence of LCB2 genes from other organisms such as human, mouse, Klebsiella lactis, and Schizosaccharomyces pombe (Nagiec et al., Gene, 177, 237, 1996; Hanada et al, J.

Biol Chem., 272, 32108, 1997; Weiss & Stoffel, Eur. J. Biochem., 249, 239, 1997) are reported.

First, by referring the nucleotide sequence of LCB 1 gene from Saccharomyces cerevisiae (Nagiec et al, Proc. Natl. Aca. Sci. USA, 91, 7899, 1994), PstI fragment (1 kb) of LCB 1 gene was used as probe for Southern blot analysis of genomic DNA of Pichia ciferrii ATCC 14091. In this analysis, no desired band was detected. On the other hand, the Southern blot analysis using Sail fragment (0.9 kb) of LCB2 gene 11 as probe gave DNA band of 12 kb LCB2 gene. This band was collected and inserted into plasmid pBluescript KS+ to give a library. By repeating the Southern blot analysis, Scal/Afllll fragments (3.0 kb) were obtained and plasmid pL2SA was constructed (Figure 2). Nucleotide sequence of LCB2 gene was determined and is represented as SEQ. ID. NO. 3 together with its putative amino acid sequence (SEQ. ID. NO. 4) in SEQUENCE LISTING. The sequence was submitted on March 7, 1998 to GenBank under accession number AF053456.

Pichia ciferrii ATCC 14091 LCB2 gene comprises 1688 base pairs and has no intron. The putative amino acid sequence shows a high homology to that from Saccharomyces cerevisiae. Transmembrane helix region is present spanning the 55th- 79th amino acids. The region containing lysine, which forms Schiff base together with pyridoxal phosphate, has identical amino acids sequence to those of Saccharomyces cerevisiae.

1-2. Construction of plasmid prACL2 Plasmid pL2SA was treated with Hindlll, Klenow and BamHI, in this order, to give a 3.0 kb fragment of LCB2 gene. This fragment was inserted to plasmid prHEC1.9F, which is treated with Eco47III and BamHI, to give plasmid prACL2. Plasmid prACL2 has the Pichia ciferru ribosomal DNA fragment, CYH^r(L41) and LCB2 gene, linked to each other, in this order. Plasmid prACL2 was deposited with Korea Collection of Type Cultures in Taejon on May 4, 1998 according to the

Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and given an accession number of KCTC-

0468BP.

7-3. Transformation Plasmid prACL2 was transformed into the mutant Pichia ciferrii KFCC- 10937 by following the procedure in the step 5 described above and transformed cells 12 having a high copy number of gene were selected.

7-4. TAPS production by transformed cell cultivation

The transformed cells obtained in the step 7-3 above were cultivated in YGM optimum medium (glycerol 100 g/liter, yeast extract 2 g/liter, KN0₃ 3g/liter, (NH₄)₂S0₄0.5 g/liter, MgS0₄-7H₂0 0.3 g/liter, NaCl 0.5 g/liter, CSL 3 g/liter and LS- 300 1 g/liter) for 4(four) days to produce TAPS.

The transformed cells according to the present invention exhibited TAPS production at least 1.3 times greater than the parent strain KFCC- 10937.

8. Isolation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter gene from Pichia ciferrii

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis and converts glyceraldehyde-3 -phosphate into 1,3-bis- phosphogly cerate. It is a constitutively expressed enzyme. Since GAPDH promoter is hardly affected by carbon source, it attracts the researchers in gene manipulation filed (Kniskern et al, Gene, 46, 135, 1986; Travis et al, J. Biol Chem., 260, 4384, 1985; Hallewell et al, Biotechnol, 5, 363, 1987; Rosenberg et al, Method Enzymol., 185, 341, 1990; Waterham et α/., Gene, 16, 37, 1997).

Based on these studies, the present inventors cloned Pichia ciferrii GAPDH promoter gene and evaluated whether the insertion of GAPDH promoter increases the expression of the desired genes.

8-1. Isolation of GAPDH gene

GAPDH gene from Saccharomyces cerevisiae 2805 was used to clone

GAPDH gene from the genomic DNA of Pichia ciferrii ATCC 14091. By using thus cloned GAPDH gene, plasmid pGH2.2 was constructed (Figure 4). The nucleotide sequence of GAPDH gene of Pichia ciferrii ATCC 14091 was determined and is 13 represented as SEQ. ID. NO. 5 together with its putative amino acid sequence (SEQ. ID. NO. 6) in SEQUENCE LISTING. This sequence was submitted on March 7, 1998 to GenBank under accession number of AF053300.

Pichia ciferrii ATCC 14091 GAPDH gene comprises 1004 base pairs and has no intron. The nucleotide sequence and the putative amino acid sequence show 69.3% and 76.2% homology to that from Saccharomyces cerevisiae, respectively. This suggests that Pichia ciferrii ATCC 14091 GAPDH gene is new.

8-2. Isolation of GAPDH promoter gene

PCR using plasmid pGH2.2 and primers (primer Nos. 3 and 4; SEQ. ID. NO. 9 and SEQ. ID. NO. 10, respectively in SEQUENCE LISTING) was performed to isolate Pichia ciferrii GAPDH promoter gene (600 bp). This gene was inserted into EcoRV site of pT7-Blue T-vector to give plasmid pT7GH.

8-3. Isolation of LCB2 gene free of its own promoter

LCB2 gene free of its own promoter was isolated from plasmid pL2SA by following the procedure in Figure 5. The gene has a size of 2.3 kb. It was inserted into BamHI site of pT7GH to give plasmid ρGAL2.

8-4. Construction of plasmid prACGL2

LCB2 gene of 2.9 kb was isolated from plasmid pGAL2 by following the procedure in Figure 5 and inserted into Eco47III/XbaI site of prHEC1.9F to give plasmid prACGL2.

Plasmid prACGL2 has the Pichia ciferrii ribosomal DNA fragment,

CYH^r(L41), GAPDH promoter gene and LCB2 gene, linked to each other, in this order. Its restriction map is depicted in Figure 5. Plasmid prACGL2 was deposited with Korea Collection of Type Cultures in Taejon on June 25, 1998 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms 14 for the Purposes of Patent Procedure and given an accession number of KCTC- 0498BP.

8-5. Construction of plasmid prHECGL2

When the host cell (e.g., strain of Pichia ciferrii) is transformed with plasmid prACGL2, the resulting transformed cell will carry genes from Pichia ciferrii as well as other undesired regions of the starting bacterial plasmid since the plasmid is treated with only one restriction enzyme for linearization prior to the transformation into the host cell. To avoid this, the present inventors add Pichia ciferrii ribosomal DNA fragment (800 bp) at the downstream of LCB2 gene of plasmid prACGL2. Thus obtained plasmid was designated as prHECGL2. (Figure 6)

Plasmid prHECGL2 has the Pichia ciferrii ribosomal DNA fragment, CYH^r(L41), GAPDH promoter gene, LCB2 and Pichia ciferrii ribosomal DNA fragment, operably linked to each other, in this order. Its restriction map is depicted in Figure 6. Plasmid prHECGL2 was deposited with Korea Collection of Type Cultures in Taejon on August 10, 1998 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and given an accession number of KCTC-0511BP.

When the host cell is transformed with plasmid prHECGL2, the resulting transformed cell carry only genes from Pichia ciferrii by treating the plasmid by proper restriction enzymes to cut both of the Pichia ciferrii ribosomal DNAs. That is to say, when the plasmid prHECGL2 is transformed into Pichia ciferrii, the obtained transformed cell Pichia ciferrii will carry only endogenous genes.

8-6. Transformation Cells of Pichia ciferrii KFCC- 10937 and plasmid prACL2, prACGL2 and prHECGL2 were mixed, respectively and electroporation was performed at voltage 500V, capacity 50μF and resistance 800Ω so as to transform the cells with the 15 plasmid.

8-7. TAPS production by transformed cell cultivation

The transformed cells obtained in the step 8-6 above were cultivated in YGM optimum medium (glycerol 100 g/liter, yeast extract 2 g/liter, KN0₃ 3g/liter, (NH₄)₂S0₄0.5 g/hter, MgS0₄-7H₂0 0.3 g/hter, NaCl 0.5 g/hter, CSL 3 g/liter and LS- 300 1 g/liter) to produce TAPS.

The transformed cells with plasmid prACGL2 (KCTC-0498BP) or with plasmid prHECGL2 (KCTC-051 IBP) according to the present invention exhibited

TAPS production at least 2.1 times and at least 1.5 times greater than the parent strain KFCC-10937 and than the transformed cell with plasmid prACL2 (KCTC-0468BP), respectively.

Free Texts in Sequence Listing

SEQ.ID.NO. 7 is an artificial sequence for PCR primer CYH1.

SEQ.ID.NO. 8 is an artificial sequence for PCR primer CYH4. SEQ.ID.NO. 9 is an artificial sequence for PCR primer No. 3 which is used for isolation of GAPDH promoter gene.

SEQ.ID.NO. 10 is an artificial sequence for PCR primer No. 4 which is used for isolation of GAPDH promoter gene.

SEQ.ID.NO. 11 is an artificial sequence for PCR primer CH-f. SEQ.ID.NO. 12 is an artificial sequence for PCR primer CH-r.

SEQ.ID.NO. 13 is an artificial sequence for PCR primer 18R.

SEQ.ID.NO. 14 is an artificial sequence for PCR primer 26F.

SEQ.ID.NO. 15 is an artificial sequence for PCR primer L2f.

SEQ.ID.NO. 16 is an artificial sequence for PCR primer L2r. SEQ.ID.NO. 17 is an artificial sequence for PCR primer No. 1 which is used 16 for isolation of GAPDH gene.

SEQ.ID.NO. 18 is an artificial sequence for PCR primer No. 2 which is used for isolation of GAPDH gene.

Examples

The present invention will be described in more detail by way of various

Examples, which should not be construed to limit the scope of the present invention.

Example 1 : Isolation of genomic DNA from Pichia ciferrii ATCC 14091.

Cells of Pichia ciferrii ATCC 14091 grown in YEPD medium (peptone 2%, yeast extract 1% and glucose 2%) by following the method described by Johnston (Johnston et ah, Yeast Genetics, molecular aspects, pp.107-123, IRL Press, 1988) were collected and suspended in SSEM solution (IM sorbitol, lOOmM sodium citrate, 60mM EDTA, lOOmM 2-mercaptoethanol). Novozyme was added to the suspension to a concentration of 0.1 mg/ml, and allowed to react at 37°C for 30 minutes to give protoplast. An equal volume of SDS-TE solution (2% SDS, 50mM EDTA in IM Tris-Cl, pH 8.0) was added and allowed to react at 60°C for 10 minutes to disintegrate cells. To the supernatant obtained by centrifuging at 8,000 rpm for 15 minutes, a twice volume of ethanol was added. The resulting pellets were dissolved in TE solution containing RNase to extract Pichia ciferrii genomic DNA.

Example 2: Isolation of L41 gene from Pichia ciferrii ATCC 14091 Two following primers CYH1 and CYH4 were synthesized.

CYH1 : 5*-CGC GTA GTT AAY GTN CCN AAR AC-3' CYH4 : 5'-GCC TGG CCY TTY TGY TTY TTN TC-3' These two primers are also represented as SEQ. ID. NO. 7 and SEQ. ID. NO. 8, respectively in SEQUENCE LISTING. 17

PCR was performed using the two primers and Pichia ciferrii genomic DNA obtained in Example 1 to give L41 gene fragment of about 300 bp.

This fragment was labeled with DIG-labeling and detection kit (Boehringer Mannheim) by following the manufacturer's manual to give a probe.

Example 3: Sequencing of L41 gene

Genomic DNA of Pichia ciferru obtained in Example 1 was digested with various restriction enzymes, and the digestion products were subjected to 0.9% agarose gel electrophoresis and transferred to Nytran^R membrane (Schleicher & Schuell) followed by hybridization with a hybridization solution (5X SSC, 0.1% N- laurylsarcosine, 0.02% SDS, 2% blocking agent and 30% formaldehyde) using probes obtained in Example 2. The hybridization was carried out at 42°C for 6 hours according to the manufacturer's manual prepared by Boehringer Mannheims.

Antibodies coupled to alkaline phosphatase were introduced and BCIP and X- phosphate were added. Violet-stained band was observed. Band is detected at 1.9 kb size of genomic DNA treated with EcoRI. This

DNA fragment (1.9 kb) was collected and linked to EcoRI site of plasmid pBluescript KS+ (Stratagene) and transformed into E. coli DH5α to establish a library.

This library was repeatedly subjected to Southern blot analysis to isolate 1.9 kb gene fragment containing L41 gene(plasmid pCYH1.9). Nucleotide sequencing was performed for this plasmid using Automatic sequencer Model 373 A (Applied Biosystem). The result is represented as SEQ. ID. NO. 1 together with its putative amino acid sequence (SEQ. ID. NO. 2) in SEQUENCE LISTING. This sequence was submitted on March 7, 1998 to GenBank under accession number of AF053457.

L41 gene of Pichia ciferrii ATCC 14091 consists of 737 base pairs including 419 bp intron. The amino acid residue 56 is proline.

Example 4: Construction of plasmid pCYH1.9^r in which proline (aa 56) is replaced 18 by glutamine.

Two primers CH-f and CH-r were prepared:

Primer CH-f : GGT CAA ACC AAA CCA GTT TTC

Primer CH-r : ATG GAA AAC TTG TTT GGT TTG ACC These two primers are also represented as SEQ .ID. NO. 11 and SEQ. ID. NO.

12, respectively in SEQUENCE LISTING.

Combinations of universal primer and primer CH-r, and of reverse primer and primer CH-f were employed for PCR using Pfu DNA polymerase and pCYHl.9 as a template to obtain two PCR products of 1.2 kb and 0.7 kb. PCR was performed again using the two PCR products (1.2 kb and 0.7 kb), and universal and reverse primers to obtain plasmid pCYH1.9^r in which proline (aa 56) is replaced by glutamine.

Example 5: Construction of plasmid prDX9.0 containing ribosomal DNA fragment Two following primers 18R and 26F were synthesized.

Primer 18R : 5'-CAA TAA TTG CAA TGC TCT ATC CCC AGC ACG-3' Primer 26F : 5'-GGA TAT GGA TTC TTC ACG GTA ACG TAA CTG-3' These two primers are also represented as SEQ. JO. NO. 13 and SEQ. ID. NO.

14, respectively in SEQUENCE LISTING. PCR was performed using the two primers and Pichia ciferru genomic DNA obtained in Example 1 to give a PCR product of 6.0 kb. This PCR product was labeled in the same manner as in Example 2 to give a probe.

When the probe is subjected to Southern blot analysis with Pichia ciferru genomic DNA, band is detected at 9 kb size of genomic DNA treated with Xhol. This DNA fragment (9 kb) was collected and inserted into plasmid pBluescript KS+

(Stratagene) and its library was established. This library was repeatedly subjected to

Southern blot analysis to isolate 9 kb gene fragment containing ribosomal DNA

(plasmid ρrDX9.0). 19

Nucleotide sequencing was performed for this plasmid using Automatic sequencer Model 373A (Applied Biosystem). The results including the location and orientation of 26S, 18S, 5.8S and 5S ribosomal gene are shown in Figure 1.

Example 6: Construction of plasmid prXHNC

Plasmid prDX9.0 obtained in Example 5 was treated with Xbal to obtain 3.5 kb ribosomal DNA, which is then inserted into plasmid pBluescript KS+. The resulting recombinant plasmid is treated with Hpal/Ncol/Klenow to remove 1.6 kb ribosomal DNA and linked to 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI/Klenow, to obtain plasmid prXHNC.

Example 7: Construction of plasmid prEHC

Plasmid prDX9.0 obtained in Example 5 was treated with EcoRI to obtain 3.8 kb ribosomal DNA, which is then inserted into plasmid pBluescript KS+. The resulting recombinant plasmid is treated with Hpal to remove 2 kb ribosomal DNA and linked to 1.9 kb CYH^r gene, which was obtained by digesting plasmid pC YH 1.9^r in Example 4 with EcoRI/Klenow, to obtain plasmid prEHC.

Example 8: Construction of plasmid prCEX

Plasmid prDX9.0 obtained in Example 5 was treated with EcoRI/Xbal to obtain 1.1 kb ribosomal DNA. 1.9 kb C YΗ gene, which was obtained by digesting plasmid pCYHl^ in Example 4 with EcoRI/Klenow, was linked to EcoRI site of the 1.1 kb ribosomal DNA to give plasmid prCEX.

Example 9: Construction of plasmid prCRX

Plasmid prDX9.0 obtained in Example 5 was treated with EcoRV/Xbal to obtain 1.3 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI/Klenow, was linked to EcoRV site of 20 the 1.3 kb ribosomal DNA to give plasmid prCRX.

Example 10: Construction of plasmid prXCH

Plasmid prDX9.0 obtained in Example 5 was treated with Xbal to obtain 3.5 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI/Klenow, was linked to Hpal site of the 3.5 kb ribosomal DNA to give plasmid prXCH.

Example 11: Construction of plasmid prXCE Plasmid prDX9.0 obtained in Example 5 was treated with Xbal to obtain 3.5 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYHl^ in Example 4 with EcoRI, was linked to EcoRI site of the 3.5 kb ribosomal DNA to give plasmid prXCE.

Example 12: Construction of plasmid prXHC 1.9

Plasmid prDX9.0 obtained in Example 5 was treated with Xhol/Hindlll to obtain 1.6 kb ribosomal DNA. A plasmid containing this 1.6 kb ribosomal DNA was digested with HmdIII/Klenow to give ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI, was linked to EcoRI site of the Hindlll/Klenow digestion product to give plasmid prXHC 1.9.

Example 13: Construction of plasmid prAC1.9

Plasmid prDX9.0 obtained in Example 5 was treated with Hindlll/EcoRV to obtain 0.6 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI/Klenow, was linked to EcoRV site of the 0.6 kb ribosomal DNA to give plasmid prAC1.9. 21

Example 14: Construction of plasmid prEC1.9

Plasmid prDX9.0 obtained in Example 5 was treated with Hindlll/EcoRI to obtain 1.4 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYHl^ in Example 4 with EcoRI, was linked to EcoRI site of the 1.4 kb ribosomal DNA to give plasmid prXCH.

Example 15: Construction of plasmid prHEC1.9F

Plasmid prDX9.0 obtained in Example 5 was treated with Hindlll/EcoRI to obtain 1.4 kb ribosomal DNA. 1.9 kb CYH^r gene, which was obtained by digesting plasmid pCYH1.9^r in Example 4 with EcoRI/Klenow, was linked to EcoRV site of the 1.4 kb ribosomal DNA to give plasmid prHEC1.9F.

Example 16: Construction of plasmid prHEC1.9R

The procedure in Example 15 was repeated except that the insertion orientation of C YH ^r was opposite to that of Example 5 to obtain plasmid prHEC 1.9R

Example 17: Transformation efficiency of plasmids.

The method described by Klass and Peter (Klass & Peter, Curr. Genet., 25, 305, 1994) was followed. That is to say, cells of Pichia ciferrii KFCC-10937 grown in YEPD medium (100 ml) to OD_600nm of 1.5 were collected by centrifugation and dispersed in 50 mM phosphate buffer (pH 7.5; 40 ml), to which 25 mM DTT was added, at 37°C for 15 minutes. The mixture was washed twice with ice-cooled stabilization solution (100 ml; 270 mM sucrose, 10 mM Tris-Cl (pH 7.5), 1 mM MgCl₂) and suspended in 1 ml of stabilization solution.

To the resulting suspension (50 microliters), 5 microliters of the solution of respective plasmids obtained in Examples 6 - 16 are added and allowed to stand on ice for 10 minutes. Then, the solution was transferred to 0.2 mm electroporation cuvettes (Bio-Rad). Electroporation was carried out using Gene-pulser II (Bio-Rad) 22 at 500V, 50μF and 800Ω, and the electroporation product was suspended into 0.5 mL of stabilization solution. After adding 2 mL of YEPD medium, the cultivation was carried out at 25°C for 5 hours. Then the culture broth was plated on YEPD solid medium, to which 10 μg/mL of cycloheximide was added, at 25°C for 4 - 5 days. The number of transformed cells was counted and transformation efficiency was shown in Table 2.

Table 2

Plasmid No. of transformed Plasmid No. of transformed cell per μg cell per μg prXHNC 276 prEHC 250 prCEX 194 prCRX 226 prXCH 314 prXCE 134 prAC1.9 1574 prXHC1.9 1287 prEC1.9 983 prHC1.9F 1760 prHEC 1.9R 54

The results in Table 2 reveals that the site of ribosomal DNA where CYH^r gene is inserted and the transcription direction of C YH^r gene are closely related with the transformation efficiency. Plasmids prAC1.9 and prHEC 1.9F in which the non- transcribed region between 5S and 26S ribosomal RNA structural genes is used as an insertion site has the highest transformation efficiency. And, prHEC 1.9R in which the transcription direction of 5S RNA gene is opposite to that of CYH^r shows significantly decreased transformation efficiency.

Based on these results, plasmid prHEC 1.9F was selected.

Example 18: Selection of transformation conditions

In order to establish the optimum conditions of transformation, linear prHEC 1.9 obtained after treating with Apal/Scal was transformed by following the 23 procedure in Example 17 except that the voltage was changed to 500, 600 or 700V and the resistance to 100, 200, 300, 400, 500, 600, 700 or 800Ω while keeping the capacity of 50μF. The results are shown in Table 3.

Table 3

Voltage (V) Resistance (Ω) No. oftransformed cell per μg

100 0

200 82

300 1065

500 400 1770

500 2890

600 4250

700 6300

800 6850

100 0

200 210

300 1488

600 400 2644

500 5250

600 6750

700 6500

800 4740

100 18

200 620

700 300 2360

400 4400

500 3840

600 2640

24

The results in Table 3 confirm that the optimum transformation conditions are capacity 50μF, voltage 500V and resistance 800Ω.

Example 19: Analysis of transformed cell by Southern blot analysis

Each of transformed cells selected in Example 17 was inoculated into YEPD medium to which 5 μg/mL of cycloheximide was added and subjected to cultivation while agitation at 25°C for 18 - 20 hours, followed by centrifugation to collect cell pellets. Thus obtained cells were placed in 1.5 mL tubes. Cells were suspended in 30μL of STES solution (0.5M NaCl, 0.01M EDTA, 1% SDS in 0.2M Tris-Cl, pH 7.6) and 0.8 volumes of glass beads (diameter 0.4 mm) were added thereto. The mixtures were stirred for 5 minutes, 200 μL of TE buffer (ImM EDTA in lOmM Tris-Cl, pH 8.0) and 200 μL of phenol/chloroform/isoamylalcohol (25:24:1) were added. The resulting mixtures were stirred for 2 minutes and centrifuged at 12,000 rpm. 2.5 Volumes of ethanol was added to the supernatant to precipitate genomic DNA and dried. Genomic DNA (2 - 3 μg) was dissolved into 50 μL of distilled water, treated with EcoRI, and subjected to electrophoresis on 0.8% agarose gel. Southern blot analysis was carried out by using L41 gene in Example 2 as a probe to detect bands. It was observed that 4 - 5 copies of L41 gene are carried on genomic DNA of all transformed cells.

Example 20 : Construction of plasmid prACL2

(1) Isolation of LCB2 gene from Pichia ciferrii ATCC 14091.

Cells of Saccharomyces cerevisiae grown in YEPD medium (peptone 2%, yeast extract 1% and glucose 2%) by following the method described by Johnston (Johnston et al, Yeast Genetics, molecular aspects, pp.107-123, IRL Press, 1988) were collected and genomic DNA was isolated therefrom.

Two following primers L2f and L2r were synthesized. 25

Primer L2f : 5'-ATG AGT ACT CCT GCA AAC TA-3'

Primer L2r : 5'-TAA CAA AAT ACT TGT CGT CC-3'

These two primers are also represented as SEQ. ID. NO. 15 and SEQ. ID. NO. 16, respectively in SEQUENCE LISTING. PCR was performed using the two primers and the Saccharomyces cerevisiae genomic DNA obtained in the above to give LCB2 gene fragment of about 1680 bp. The Sail fragment of 913bp was labeled with DIG-labeling and detection kit (Boehringer Mannheim) by following the manufacturer's manual to give a probe.

Genomic DNA of Pichia ciferrii obtained in Example 1 was digested with various restriction enzymes, BamHI, EcoRI, EcoRV, Hindlll, PstI or Sail, and the digestion products were subjected to electrophoresis in TAE buffer and transferred to Nytran^R membrane (Schleicher & Schuell) followed by Southern blot analysis.

The Southern blot analysis was carried out using a hybridization solution (5X SSC, 0.1%N-laurylsarcosine, 0.02% SDS, 2% blocking agent and 30% formamide) at 42°C for 6 hours according to the manufacturer^' s manual prepared by Boehringer Mannheims.

Antibodies coupled to alkaline phosphatase were introduced and BCIP and X- phosphate were added. Violet color-stained bands were observed.

Band is detected at 12 kb size of genomic DNA treated with HindTII. This DNA fragment(12kb) was collected and inserted into plasmid pBluescript

KS+(Stratagene) and transformed into E. coli DH5cc to establish a library.

This library was repeatedly subjected to Southern blot analysis to isolate 3.0 kb Sacl/Afllll fragment containing LCB2 gene(plasmid pL2SA).

Restriction map and sequencing of Pichia ciferrii LCB2 gene are shown in Figure 2. The nucleotide sequence is represented as SEQ. ID. NO. 3 together with its putative amino acid sequence (SEQ. ID. NO. 4) in SEQUENCE LISTING. The nucleotide sequence was submitted on March 7, 1998 to GenBank under accession number of AF053456. 26

LCB2 gene of Pichia ciferrii ATCC 14091 consists of 1688 base pairs without intron, and its putative amino acid sequence shows a high homology to that of Saccharomyces cerevisiae.

(2) Construction of plasmid prACL2

5 Plasmid prHEC 1.9F obtained in Exmaple 15 was digested with

Eco47III/BamHI to give a linear one. On the other hand, plasmid pL2SA obtained in (1) above was treated with HindHI, Klenow and BamHI, in this order, to give a 3.0 kb fragment of LCB2 gene. This 3.0 kb fragment was inserted to the above linear plasmid to give plasmid prACL2. (Figure 3)

10 Plasmid prACL2 has the Pichia ciferrii ribosomal DNA fragment, CYH^r(L41) and LCB2 gene, linked to each other, in this order. Plasmid prACL2 was deposited with Korea Collection of Type Cultures in Taejon on May 4, 1998 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and given an accession number of KCTC-

15 0468BP.

Example 21: Transformation

Plasmid prACL2 was linearized with Apal and transformed into the mutant Pichia ciferrii KFCC- 10937 by following the procedure in Example 17. 10² - 10³ colonies per μg of plamid were formed. Twelve largest colonies were selected.

20 Each of transformed cells was inoculated into YEPD medium to wich 5 μg/mL of cycloheximide was added and subjected to cultivation while agitation at 25°C for 18 - 20 hours, followed by centrifugation to collect cell pellets. Thus obtained cells were placed in 1.5 mL tubes. Cells were suspended in 30μL of STES solution (0.5M NaCl, 0.01M EDTA, 1% SDS in 0.2M Tris-Cl, pH 7.6) and 0.8 volumes of glass

25 beads (diameter 0.4 mm) were added thereto. The mixtures were stirred for 5 minutes, 200 μL of TE buffer (ImM EDTA in lOmM Tris-Cl, pH 8.0) and 200 μL 27 of phenol chloroform/isoamylalcohol (25:24: 1) were added. The resulting mixtures were stirred for 2 minutes and centrifuged at 12,000 rpm. 2.5 Volumes of ethanol was added to the supernatant to precipitate genomic DNA and dried.

Genomic DNA (2 - 3 μg) was dissolved into 50 μL of distilled water, treated with EcoRI, and subjected to electrophoresis on 0.8% agarose gel. Southern blot analysis was carried out by using L41 gene in Example 2 as a probe to detect bands.

It was observed that 5 - 10 copies of L41 gene are carried on genomic DNA of all transformed cells. One of them is picked up and named as 'Transformed cell 1.'

Comparative Example 1 : TAPS production by strain KFCC- 10937 cultivation The parent strain KFCC-10937 was cultivated in lOOmL of YGM optimum medium (glycerol 100 g/liter, yeast extract 2 g/liter, KN0₃ 3g/liter, (NH₄)₂S0₄ 0.5 g/hter, MgS0₄-7H₂0 0.3 g/liter, NaCl 0.5 g/liter, CSL 3 g/liter and LS-300 1 g/liter) at 25°C and 250 rpm for 4 days. After standing at 25 C for 2 days, 4 volumes of mixed solvent of chlorofoim/methanol (1:1) was added to separate phases and extract TAPS.

TAPS was analyzed on HPLC using ELSD(Electron Light Scanning Detector). As solvent, a mixture of iso-octane and THF/formic acid (100: 1.5) varying its ratio 9: 1, 7:3 and then 9: 1 was used.

Example 22 : TAPS production by Transformed cell 1 Transformed cell 1 obtained in Example 20 was cultivated in the same condition and manner as in Comparative Example 1 to produce TAPS.

TAPS productions in Comparative Example 1 and Example 22 are shown in Table 4. 28

Table 4

KFCC-10937 Transformed cell 1

Doubling Time (hr) 1.5 1.5

Biomass concentration (g/L) 41.6 43.6

Amount of TAPS (mg/L) 5206 7444

TAPS specific yield (mg/gdw*) 125.1 170.7

Volume productivity (mg TAPS/L/hr) 54.2 77.5

* gdw = dry weight (g)

The results in Table 4 affirmed that the transformation of Pichia ciferrii with the expression cassette according to the present invention makes it possible to maximize the integration of the desired gene onto the chromosome of the host.

Example 23: Preparation of probe for GAPDH gene cloning

Two following primers (Primer No. 1 and No. 2) were synthesized.

Primer 1 : 5'-ATG GTT AGA GTT GCT ATT AAC G-3' Primer 2 : 5'-AAG CCT TGG CAA TGT GTT CAA-3'

These two primers also represented as SEQ. ID. NO. 17 and SEQ. ID. NO. 18, respectively in SEQUENCE LISTING.

PCR was performed using the two primers to isolate 1 kb GAPDH gene from Saccharomyces cerevisiae 2805 (provided by courtesy of RB.Wickner at NIH) . The gene was inserted into EcoRV site of plasmid pT7-Blue T-vector (Novagen) to obtain plasmid pT7-SGH.

Plasmid pT7-SGH was digested with Xbal and Sail to give 0.9 kb gene fragment, which was labeled with DIG-labeling and detection kit (Boehringer Mannheim) by following the manufacturer' s manual to give a probe. 29

Example 24: Isolation of GAPDH gene from Pichia ciferrii

Genomic DNA of Pichia ciferrii obtained in Example 1 was digested with various restriction enzymes, BamHI, EcoRI, EcoRV, Hindlll, PstI or Sail, and the digestion products were subjected to electrophoresis on 0.9% agarose gel and

5 transferred to Nytran^R membrane (Schleicher & Schuell) followed by Southern blot analysis using the probe obtained in Example 23.

The Southern blot analysis was carried out using a hybridization solution (5X SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 2% blocking agent and 30% formamide) at 42°C for 6 hours according to the manufacturer's manual prepared by Boehringer 10 Mannheim.

Antibodies coupled to alkaline phosphatase were introduced and BCIP and X- phosphate were added. Violet color-stained bands were observed at about 6 kb sized fragment of the genomic DNA treated with HindTII/EcoRI.

This DNA fragment (about 6 kb) was collected and inserted into plasmid 15 pBluescript KS+ (Stratagene) and transformed into E. coli DH5α to establish a library. This library was repeatedly subjected to Southern blot analysis to isolate 6.0 kb Afllll/Hindlll fragment containing GAPDH gene(plasmid pGH2.2).

The restriction map and sequencing of GAPDH gene of Pichia ciferrii ATCC

14091 are shown in Figure 4. The nucleotide sequence is represented as SEQ. ID.

20 NO. 5 together with its putative amino acid sequence (SEQ. ID. NO. 6) in

SEQUENCE LISTING. This sequence was submitted on March 7, 1998 to GenBank under accession number of AF053300.

Pichia ciferrii ATCC 14091 GAPDH gene comprises 1004 base pairs and has no intron. The nucleotide sequence and the putative amino acid sequence show 25 69.3% and 76.2% homology to that from Saccharomyces cerevisiae, respectively.

Example 25: Isolation of Pichia ciferrii GAPDH promoter gene

Two following primers (Primer No. 3 and No. 4) were synthesized. 30

Primer 3 : 5'-GAT ATC TAC ATA CAA TTG ACC CAT AG-3'

Primer 4 : 5'-GGA TCC TTA ATT ATT TGT TTG TTT-3'

These two primers are also represented as SEQ. ID. NO. 9 and SEQ. ID. NO.

10, respectively in SEQUENCE LISTING. PCR using plasmid pGH2.2 obtained in Example 24 and the two primers

(primer Nos. 3 and 4) was performed to isolate Pichia ciferrii GAPDH promoter gene

(600 bp). This gene was inserted into EcoRV site of pT7-Blue T-vector to give plasmid pT7GH.

Example 26: Isolation of LCB2 gene free of its own promoter Plasmid pL2SA obtained in Example 20 was treated with Afllll to give promoter-free LCB2 gene (2.3 kb). This 2.3 kb gene was treated with Klenow and inserted into plasmid pBluescript KS+ treated with BamHI/Klenow to give plasmid pL2B2.3. (Figure 5)

Plasmid pL2B2.3 was digested with BamHI to give 2.3 kb LCB2 gene, which was inserted to BamHI site of plasmid pT7GH in Example 4 to give pGAL2.

Example 27: Construction of exression cassette

Plasmid prHEC 1.9F obtained in Example 15 was linearized by treating with Eco47III and Xbal. GAPDH ρromoter/LCB2 gene (2.9 kb) obtained by treating plasmid pGAL2 (Examlple 26) with EcoRV and Xbal was inserted to the linear plasmid prHEC1.9F to give prACGL2. (Figure 5)

Plasmid prACGL2 has the Pichia ciferrii ribosomal DNA fragment, CYH^r(L41), GAPDH promoter gene and LCB2 gene, linked to each other, in this order. Plasmid prACGL2 was deposited with Korea Collection of Type Cultures in Taejon on June 25, 1998 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and given an accession number of KCTC-0498BP. 31

Plasmid pCYH1.9^r obtained in Example 4 was linearized by treating with Eco47III and Xbal. GAPDH promoter/LCB2 gene (2.9 kb) obtained by treating plasmid pGAL2 (Examlple 26) with EcoRV and Xbal was inserted to the linear plasmid pCYH1.9^r to give pCHGL2. (Figure 6). This plasmid was treated with EcoRV/Xbal/Klenow to give 4.6 kb CYH7GAPDH promoter/LCB2 gene which is then inserted to EcoRV site of 1.4 kb ribosomal DNA, which was obtained by treating plasmid prDX9.0 (Example 5) with Hindlll/EcoRI, to give plasmid prHECGL2. (Figure 6)

Plasmid prHECGL2 has a structure that it contains additional Pichia ciferru ribosomal DNA fragment (800 bp) linked at downstream of LCB2 gene of plasmid prACGL2. That is to say, plasmid prHECGL2 contains Pichia ciferrii ribosomal

DNA fragment, COT(L41), GAPDH promoter gene, LCB2 and Pichia ciferrii ribosomal DNA fragment, operatively linked to each other, in this order.

Plasmid prHECGL2 was deposited with Korea Collection of Type Cultures in Taejon on August 10, 1998 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and given an accession number of KCTC-051 IBP.

Plasmid prHECGL2 is distinguished from and advantagous over plasmid prACGL2 that the former introduces only Pichia ciferrii endogenous genes onto the chromosome of Pichia ciferrii transformed cell while the latter introduces Pichia ciferrii endogenous genes together with bacterial genes (originated from plasmid pBluescript KS+) when they are used to transform Pichia ciferrii.

Example 28: Transformation

Plasmid prACGL2 (Example 27) and prHECGL2 (Example 27) were linearized with Apal and Apal/Scal, respectively. Then, these linear plasmids were transformed into Pichia ciferrii KFCC- 10937 by following the procedure in Example

17. 10³ colonies per μg of plasmid were formed. Four largest colonies were selected 32 for respective plasmid-transformed cells.

Each of transformed cells was inoculated into YEPD medium to wich 5 μg/mL of cycloheximide was added and subjected to cultivation while agitation at 25°C for 18 - 20 hours, followed by centrifugation to collect cell pellets.Thus obtained cells were placed in 1.5 mL tubes. Cells were suspended in 30μL of STES solution (0.5M NaCl, 0.01M EDTA, 1% SDS in 0.2M Tris-Cl, pH 7.6) and 0.8 volumes of glass beads (diameter 0.4 mm) were added thereto. The mixtures were stirred for 5 minutes, 200 μL of TE buffer (ImM EDTA in lOmM Tris-Cl, pH 8.0) and 200 μL of phenol/chloroform/isoamylalcohol (25:24: 1) were added. The resulting mixtures were stirred for 2 minutes and centrifuged at 12,000 rpm. Two and half (2.5) volumes of ethanol was added to the supernatant to precipitate genomic DNA and dried.

Genomic DNA (2 - 3 μg) was dissolved into 50 μL of distilled water, treated with Hindlll, EcoRI or Xbal, and subjected to electrophoresis on 0.8% agarose gel. Southern blot analysis was carried out by using DIG-labeled L41 gene, DIG-labeled LCB2 gene or DIG-labeled GAPDH promoter gene as a probe to detect bands(Figure 7). It was observed that about 4 - 5 copies of the genes are carried on genomic DNA of all transformed cells. Each one of the respective prACGL2-transformed and prHECGL2-transformed cells is picked up and named as 'Transformed cell 2' and 'Transformed cell 3,' respectively.

Example 29 : TAPS production by Transformed cell 2

Transformed cell 2 obtained in Example 28 was cultivated in the same condition and manner as in Comparative Example 1 to produce TAPS. TAPS production is shown in Table 5. 33

Table 5

Transformed cell 2 (prACGL2)

Doubling Time (hr) 1.5

Biomass concentration (g/L) 40.1

Amount of TAPS (mg/L) 10933

TAPS specific yield (mg/gdw*) 272.6

Volume productivity (mg TAPS/L/hr) 113.9

* gdw = dry weight (g).

Example 30 and Comparative Example 2 : TAPS production by Transformed cell 3 Transformed cell 2 obtained in Example 28 or parent strain of Pichia ciferrii was cultivated in YMGL medium (yeast extract 3 g/L, malt extract 3 g/L, glycerol 30 g/L) under the same conditions as that in Comparative Example 1 to produce TAPS. TAPS productions are shown in Table 6.

Table 6

Parent Strain Transformed cell 3 (KFCC- 10937) (prHECGL2)

TAPS in mg/mL 0.200 0.420

TAPS in g/g cell 0.012 0.027

The results in Table 6 reveal that the absolute production of TAPS in YMGL medium decreases when compared to that in YGM optimum medium, and that the TAPS production by transformed cell 3 is 2.1 times greater than that of the parent strain.

In summary, the present invention has following advantages:

(1) The expression cassette of the present invention allows a maximized 34 integration of desired genes into chromosome of host Pichia ciferrii cells.

(2) Plasmid prACL2 according to the present invention has 0.6 kb Pichia ciferrii ribosomal DNA fragment, CYH^r(L41) and LCB2 gene coding for serine palmitoyl transferase, operatively linked to each other, in this order. It shows a good efficiency of transformation into host cells and the resulting transformed cells show at least 1.3 times greater TAPS production than the parent strain.

(3) Introduction of GAPDH promoter gene into the expression cassette according to the present invention allows a further increase in the expression of the desired gene in the transformed cell. (4) Plasmid prACGL2 according to the present invention has 0.6 kb Pichia ciferrii ribosomal DNA fragment, CYH^r(L41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter gene and LCB2 gene, and plasmid prHECGL2 has further Pichia ciferrii ribosomal DNA fragment of 800 bp at the downstream of the LCB2 gene in plasmid prACGL2. These plasmids show an excellent transformation efficiency as well as show TAPS production at least 2.1 times greater than the parent strain KFCC-10937 due to an increased expression of LCB2 gene by action of GAPDH promoter.

Although preferred embodiments of the present invention have been described in detail herein above, it should be clearly understood that many variations and/or modifications of the basic inventive concepts herein taught which may appear to those skilled in the art will still fall within the spirit and scope of the present invention as defined in the appended claims.

Claims

35CLAIMS

1. A L41 gene represented as SEQ. ID. NO. 1 and coding for a Pichia ciferrii ribosomal protein.

2. An expression cassette for a desired gene in Pichia ciferrii comprising: (a) a Pichia ciferrii ribosomal DNA, which is operably linked to

(b) a CYH^r gene which is responsible for resistance to cycloheximide and is operably linked to

(c) a desired structural gene.

3. An expression cassette according to claim 2, wherein said Pichia ciferrii ribosomal DNA is a non-transcribed region comprising an about 1.4 kb fragment of

Hindlll/EcoRI digestion product thereof.

4. An expression cassette according to claim 2, wherein said desired gene is a LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as SEQ. ID. NO.3.

5. An expression cassette according to claim 3, wherein said desired gene is a LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as SEQ. ID. NO.3.

6. A plasmid comprising the expression cassette according to claim 5 which is a plasmid prACL2 (KCTC-0468BP) having a restriction map in Figure 3.

7. A process for producing a protein coded by a desired gene comprising the step of fransforming a Pichia ciferrii cell with a plasmid having the expression cassette of 36 claim 2.

8. A process according to claim 7, wherein Pichia ciferru is transformed by an electroporation under conditions of a voltage of about 500V, a capacity of about 50μF and a resistance of about 800Ω.

9. A Pichia ciferru cell transformed with a vector comprising the expression cassette of claim 2.

10. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 3.

11. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 4.

12. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 5.

13. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 6.

14. A process for producing tetraacetyl phytospingosine comprising the steps of

(a) cultivating the transformed Pichia ciferru cell of claim 11 in a suitable cultivation medium; and

(b) recovering tetraacetyl phytospingosine therefrom.

15. A process for producing tetraacetyl phytospingosine comprising the steps of (a) cultivating the transformed Pichia ciferrii cell of claim 12 in a suitable 37 cultivation medium; and

(b) recovering tetraacetyl phytospingosine therefrom.

16. A process for producing tetraacetyl phytospingosine comprising the steps of

(a) cultivating the transformed Pichia ciferrii cell of claim 13 in a suitable cultivation medium; and

(b) recovering tetraacetyl phytospingosine therefrom.

17. A LCB2 gene represented as SEQ. ID. NO. 3 and coding for Pichia ciferru serine palmitoyl transferase.

18. A GAPDH gene represented as SEQ. ID. NO. 5 and coding for Pichia ciferrii glyceraldehyde-3 -phosphate dehydrogenase

19. A promoter gene for the gene GAPDH of claim 18 and covering the sequence of nucleotide 1 to nucleotide 906.

20. An expression cassette for a desired gene in Pichia ciferrii comprising:

(a) a Pichia ciferrii ribosomal DNA, which is operably linked to (b) a CYH^r gene which is responsible for resistance to cycloheximide and is operably linked to

(c) a GAPDH promoter gene of claim 19 operably linked to

(d) a desired structural gene.

21. An expression cassette according to claim 20, wherein said Pichia ciferru ribosomal DNA is a non-transcribed region comprising an about 1.4 kb fragment of

HindTII/EcoRI digestion product thereof. 38

22. An expression cassette according to claim 20, wherein said desired gene is a LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as SEQ. ID. NO.3.

23. An expression cassette according to claim 21, wherein said desired gene is a LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as

SEQ. ID. NO.3.

24. A plasmid comprising the expression cassette according to claim 23 which is a plasmid prACL2 (KCTC-0498BP) having a restriction map in Figure 5.

25. An expression cassette for a desired gene in Pichia ciferrii comprising: (a) a Pichia ciferru ribosomal DNA, which is operably linked to

(b) a CYH^r gene which is responsible for resistance to cycloheximide and is operably linked to

(c) a GAPDH promoter gene of claim 19 operably linked to

(d) a desired structural gene operably linked to (e) an about 800 bp Pichia ciferrii ribosomal DNA fragment.

26. An expression cassette according to claim 25, wherein said Pichia ciferrii ribosomal DNA is a non-transcribed region comprising an about 1.4 kb fragment of HindTII/EcoRI digestion product thereof.

27. An expression cassette according to claim 25, wherein said desired gene is a LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as

SEQ. ID. NO.3.

28. An expression cassette according to claim 26, wherein said desired gene is a 39

LCB2 gene coding for Pichia ciferrii serine palmitoyl transferase and represented as SEQ. ID. NO.3.

29. A plasmid comprising the expression cassette according to claim 28 which is a plasmid prACL2 (KCTC-051 IBP) having a restriction map in Figure 6.

5 30. A process for producing a protein coded by a desired gene comprising the steps of linearizing a plasmid having the expression cassette of claim 25 with Apal and Seal and tiansforming a Pichia ciferrii cell with thus treated plasmid so that exogenous gene from organism origins other than Pichia ciferrii shall not be integrated onto Pichia ciferrii chromosome.

10 31. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 20.

32. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 21.

33. A Pichia ciferrii cell transformed with a vector comprising the expression 15 cassette of claim 22.

34. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 23.

35. A Pichia ciferrii cell transformed with the plasmid of claim 24.

36. A Pichia ciferrii cell transformed with a vector comprising the expression 20 cassette of claim 25. 40

37. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 26.

38. A Pichia ciferru cell transformed with a vector comprising the expression cassette of claim 27.

39. A Pichia ciferrii cell transformed with a vector comprising the expression cassette of claim 28.

40. A Pichia ciferru cell transformed with the plasmid of claim 29.

41. A process for producing tetraacetyl phytospingosine comprising the steps of

(a) cultivating the transformed Pichia ciferrii cell of claim 35 in a suitable cultivation medium; and

(b) recovering tetraacetyl phytospingosine therefrom.

42. A process for producing tetraacetyl phytospingosine comprising the steps of

(a) cultivating the transformed Pichia ciferrii cell of claim 40 in a suitable cultivation medium; and (b) recovering tetraacetyl phytospingosine therefrom.