Acetyltransferase from wickerhamomyces ciferrii

Patent metadata

Field	Value
Publication number	US-9388439-B2
Application number	US-201314391480-A
Country	US
Kind code	B2
Filing date	Mar 13, 2013
Priority date	Apr 11, 2012
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

The invention relates to novel enzymes that provide acetylated sphingoid bases.

First claim

The invention claimed is:

1. An isolated nucleic acid having a sequence selected from:
a sequence with at least 90% identity to SEQ ID NO: 1 and comprising at least one substitution, addition and/or deletion relative to SEQ ID NO: 1, wherein said sequence encoding a protein which is capable of converting phytosphingosine to triacetylphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A, and a sequence which hybridizes with SEQ ID NO: 1 under stringent conditions,

wherein said stringent conditions include incubation at 68 degrees Celsius in 2× Saline-Sodium Citrate (SSC) Buffer or incubation at 65 degrees Celsius in 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 1 mM Ethylenediaminetetraacetic acid (EDTA) and 250 mM sodium phosphate buffer and washing at 65 degrees Celsius with 0.2×SSC and 0.1% SDS, and
wherein said sequence codes for a protein which is capable of converting phytosphingosine to triacet¥lphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A.

2. An isolated nucleic acid having a sequence selected from:
a sequence with at least 90% identity to SEQ ID NO: 3 and comprising at least one substitution, addition and/or deletion relative to SEQ ID NO: 3, wherein said sequence encoding a protein which is capable of converting phytosphingosine to triacetylphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A; or
a sequence which hybridizes with SEQ ID NO: 3 under stringent conditions, wherein said stringent conditions include incubation at 68 degrees Celsius in 2× Saline-Sodium Citrate (SSC) Buffer or incubation at 65 degrees Celsius in 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 1 mM Eth¥lenediaminetetraacetic acid (EDTA) and 250 mM sodium phosphate buffer and washing at 65 degrees Celsius with 0.2×SSC and 0.1% SDS, and wherein said sequence codes for a protein which is capable of converting phytosphingosine to triacetylphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A.

3. A genetically modified cell, the genetic modification comprising the introduction of one or more expression vectors comprising the nucleic acid sequence of claim 1 or claim 2 resulting in increased expression at least one enzyme comprising:
the polypeptide sequence of SEQ ID NO: 2; or
the polypeptide sequence of SEQ ID NO: 4.

4. The genetically modified cell of claim 3 , wherein said genetic modification results in increased expression of the enzyme of SEQ ID NO: 2 and the enzyme of SEQ ID NO: 4.

5. The genetically modified cell of claim 3 or 4 , selected from Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia ciferrii, Yarrowia lipolytica, Candida albicans, Candida utilis and Ashbya gossypii.

6. A method for the production of sphingoid bases and/or sphingolipids, comprising the steps of:
a) contacting the genetically modified cell of claim 3 with a medium containing a carbon source,
b) culturing the cell under conditions which enable the cell to form sphingoid bases and/or sphingolipids from the carbon source and
c) optionally isolating the sphingoid bases and/or sphingolipids formed.

7. A method for the production of N-acetylated, primary aliphatic amines, comprising the steps of:
A) providing at least one enzyme selected from an enzyme E1 with a polypeptide sequence in which up to 10% of the amino acid residues are modified compared to SEQ ID NO: 2 by substitution, addition and/or deletion, wherein said enzyme E 1 possesses at least 10% of the enzymatic activity of the enzyme set forth in SEQ ID NO: 2, and wherein said enzymatic activity for enzyme E 1 comprises the ability to convert phytosphingosine to triacetylphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A, and
an enzyme E 2 with a polypeptide sequence in which up to 10% of the amino acid residues are modified compared to SEQ ID NO: 4 by substitution, addition and/or deletion, wherein said enzyme E 2 possesses at least 10% of the enzymatic activity of the enzyme set forth in SEQ ID NO: 4, and wherein said enzymatic activity for enzyme E 2 comprises the ability to convert phytosphingosine to triacetylphytosphingosine by transfer of the acetyl residues from three molecules of acetyl coenzyme A,
B) contacting said at least one enzyme with a medium containing a primary aliphatic amine and acetyl CoA, and
C) optionally isolating the acetylated amines formed.

Assignees

• Evonik Industries Ag
• Evonik Degussa Gmbh

Inventors

• Schaffer Steffen
• Farwick Mike
• Andrea Heiko
• Koehler Tim
• Wolff Daniel
• Ter Veld Frank
• Poetsch Ansgar
• Boles Eckhard
• Schorsch Christoph

Classifications

• C12N9/1025 Primary

  Acyltransferases (2.3) · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12P13/02 Primary

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12P13/001

  Amines; Imines · CPC title