Plasmid for gene expression in pichia ciferrii and transformation method using the same

• US 6,638,735 B1
• Filed: 07/17/2001
• Issued: 10/28/2003
• Est. Priority Date: 05/07/1998
• Status: Expired due to Fees

• Current Assignee: Doosan Corporation, Korea Advanced Institute of Science and Technology
• Original Assignee: Doosan Corporation, Korea Advanced Institute of Science and Technology
• Inventors: Park, Chang Seo, Kang, Hyun Ah, Choi, Eui Sung, Sohn, Jung Hoon, Bae, Jung Hoon, Rhee, Sang Ki
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Nguyen, Quang
• Application Number:
  US09/674,826
• Time in Patent Office: 833 Days
• Field of Search: 435/320.1, 435/69.1, 435/70.1, 435/71.1, 435/455, 435/461, 435/254.23, 435/255.51, 435/255.6, 536/23.11, 536/23.7, 536/23.21, 536/24.2, 536/23.2
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/39 from yeasts

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, CYH^r 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.

Claims

1. An isolated L41 gene represented as SEQ ID NO:

1 and coding for a Pichia ciferrii ribosomal protein.

2. An expression cassette for Pichia ciferrii comprising:

(a) a Pichia ciferrii ribosomal DNA, which is operably linked to

(b) a modified L41 gene of SEQ ID NO;

1, wherein its encoded amino acid proline at residue 56 is replaced with glutamine, and

(c) a desired structural gene, said modified L41 gene being operably linked to said desired structural gene.

3. The expression cassette according to claim 2, wherein said Pichia ciferrii ribosomal DNA is a non-transcribed region comprising the about 1.4 kb fragment of HindIII/EcoRI digestion product thereof.

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

3.

5. The expression cassette according to claim 3, wherein said desired structural gene is the 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 plasmid prACL2 (KCTC-0496BP) having the restriction map in FIG. 3.

7. A process for producing a protein product coded by a desired structural gene comprising the step of transforming a Pichia ciferrii cell with a plasmid having the expression cassette of claim 2, wherein the protein product is expressed in said Pichia ciferrii cell.

8. The process according to claim 7, wherein Pichia ciferrii is transformed by electroporation under conditions of the voltage of about 500V, the capacity of about 50 μ

F, and the resistance of about 800Ω

.

9. A Pichia ciferrii 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 phytosphingosine comprising the steps of

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

(b) recovering tetraacetyl phytosphingosine therefrom.

15. A process for producing tetraacetyl phytosphingosine comprising the steps of

(a) cultivating the transformed Pichia ciferrii cell of claim 12 in a suitable cultivation medium;

and

(b) recovering tetraacetyl phytosphingosine therefrom.

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

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

and

(b) recovering tetraacetyl phytosphingosine therefrom.

17. An isolated LCB2 gene represented as SEQ ID NO:

3 and coding for Pichia ciferrii serine palmitoyl transferase.

18. An expression cassette for Pichia ciferrii comprising:

(a) a Pichia ciferrii ribosomal DNA, which is operably linked to

(b) a modified L41 gene encoding for the polypeptide consisting of amino acid residues of SEQ ID NO;

2, wherein its encoded amino acid proline at residue 56 is replaced with glutamine, and

(c) a desired structural gene, said modified L41 gene being operably linked to said desired structural gene.