IP Library Granted Patent US 9,388,439
Granted Patent B2
US 9,388,439 · App. 14/391,480 · Granted Jul 12, 2016

Acetyltransferase from wickerhamomyces ciferrii

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Quick Facts
Patent No.
US 9,388,439
App. No.
14/391,480
Granted
Jul 12, 2016
Kind
B2
Abstract

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

Claims (21)

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.

Assignments (3)
CHANGE OF NAME Recorded Apr 16, 2020
From: EVONIK DEGUSSA GMBH
To: EVONIK OPERATIONS GMBH
Reel/Frame 052422/0342 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 1, 2015
From: EVONIK INDUSTRIES AG
To: EVONIK DEGUSSA GMBH
Reel/Frame 037174/0982 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 9, 2014
From: SCHAFFER, STEFFEN; FARWICK, MIKE; ANDREA, HEIKO; KOEHLER, TIM; WOLFF, DANIEL; TER VELD, FRANK; POETSCH, ANSGAR; BOLES, ECKHARD; SCHORSCH, CHRISTOPH
To: EVONIK INDUSTRIES AG
Reel/Frame 033921/0612 →