Pichia ciferrii cells and use thereof

Patent metadata

Field	Value
Publication number	US-9404118-B2
Application number	US-201214238248-A
Country	US
Kind code	B2
Filing date	Jul 23, 2012
Priority date	Aug 18, 2011
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The invention relates to genetically modified Pichia ciferrii cells, to the use thereof and to a method of producing sphingoid bases and sphingolipids.

First claim

The invention claimed is:

1. An genetically modified Pichia ciferrii cell, wherein said genetically modified Pichia ciferrii cell has, compared to its wild type, a reduced activity of an E 1 enzyme encoded by the intron-free nucleic acid sequences selected from the group consisting of
A) SEQ ID NO: 1, and
B) a sequence which is at least 90% identical to SEQ ID NO: 1, wherein said activity of said E 1 enzyme is the catalysis of the reaction 5,10-methylenetetrahydrofolate+L-glycine+H 2 O to tetrahydrofolate+L-serine.

2. The genetically modified Pichia ciferrii cell according to claim 1 , wherein said reduction of enzymatic activity is achieved by modifying a gene comprising the nucleic acid sequence set forth in SEQ ID NO: 1, wherein the modification is selected from the group consisting of insertion of foreign DNA into the gene, deletion of at least a portion of the gene, point mutations in the gene sequence, exposing the gene to the influence of RNA interference, and replacement of a portion of the gene with foreign DNA.

3. The genetically modified Pichia ciferrii cell according to claim 2 , wherein said foreign DNA is a selection marker gene that can be removed without leaving a trace and which leaves a deletion in the target gene.

4. The genetically modified Pichia ciferrii cell according to claim 1 , wherein the Pichia ciferrii cell is obtained from strains selected from the group consisting of
Pichia ciferrii NRRL Y-1031 F-60-10, and
Pichia ciferrii CS.PCΔPro2.

5. The genetically modified Pichia ciferrii cell according to claim 1 , characterized in that the cell further has, compared to its wild type, an increased enzymatic activity of an enzyme E 2 , wherein said enzyme E 2 catalyzes the reaction of sphinganine to phytosphingosine.

6. A method of producing an isolated Pichia ciferrii cell according to claim 1 , said method comprising the steps of:
I) providing an isolated Pichia ciferrii cell, and
II) modifying at least one gene comprising any of the sequences selected from the nucleic acid sequence groups A) and B) specified in claim 1 by insertion of foreign DNA into the gene, deletion of at least parts of the gene, point mutations in the gene sequence, exposing the gene to the influence of RNA interference, and replacement of parts of the gene with foreign DNA.

7. A method of producing sphingoid bases and sphingolipids, said method comprising the steps of
acting the cell according to claim 1 with a medium including a carbon source,
b) culturing the cell under conditions which enable the cell to produce sphingoid bases and sphingolipids from said carbon source, and
c) optionally isolating the sphingoid bases and sphingolipids produced.

8. The genetically modified Pichia ciferrii cell according to claim 2 , wherein said replacement of a portion of the gene with foreign DNA is of the promoter region.

9. The genetically modified Pichia ciferrii cell according to claim 5 , wherein said enzyme E 2 is a sphinganine C4-hydroxylase.

10. The genetically modified Pichia ciferrii cell according to claim 9 , wherein said sphinganine C4-hydroxylase is encoded by SEQ ID NO: 17.

11. The method according to claim 6 , wherein said foreign DNA is DNA coding for a selection marker gene.

12. The method according to claim 6 , wherein said replacement of parts of the gene with foreign DNA is of the promoter region.

13. The genetically modified Pichia ciferrii cell of claim 1 , wherein said intron-free nucleic acid sequences consists of a sequence that is at least 95% identical to SEQ ID NO: 1.

14. The genetically modified Pichia ciferrii cell of claim 13 , wherein said intron-free nucleic acid sequences consists of a sequence that is at least 99% identical to SEQ ID NO: 1.

Assignees

• Köhler Tim
• Schorsch Christoph
• Boles Eckhard
• Andrea Heiko
• Farwick Mike
• Schaffer Steffen
• Evonik Degussa Gmbh

Inventors

• Köhler Tim
• Schorsch Christoph
• Boles Eckhard
• Andrea Heiko
• Farwick Mike
• Schaffer Steffen

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N9/1014

  Hydroxymethyl-, formyl-transferases (2.1.2) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12Y401/02027

  Sphinganine-1-phosphate aldolase (4.1.2.27) · CPC title