Method for producing phytosphingosine or sphinganine

Patent metadata

Field	Value
Publication number	US-11111511-B2
Application number	US-201815898369-A
Country	US
Kind code	B2
Filing date	Feb 16, 2018
Priority date	Aug 24, 2015
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

Abstract

A method for producing an objective substance such as phytosphingosine and sphinganine using yeast is provided. An objective substance is produced by cultivating yeast having an ability to produce the objective substance and modified so that the expression and/or activities of proteins encoded by LCB4 and CKA2 genes are reduced in a culture medium, and collecting the objective substance from cells of the yeast and/or the culture medium.

First claim

We claim:

1. A method for producing an objective substance, the method comprising:
cultivating yeast having an ability to produce the objective substance in a culture medium; and
collecting the objective substance from cells of the yeast and/or the culture medium,
wherein the yeast has been modified so that the expression and/or activities of proteins encoded by LCB4, CKA2, and ORM2 genes are reduced, and
wherein the objective substance is selected from the group consisting of phytosphingosine (PHS) and sphinganine (DHS), and
wherein the culture medium contains cyclodextrin.

2. The method according to claim 1 , wherein the activities of the proteins are reduced by attenuating the expression of the LCB4, CKA2, and ORM2 gene, or by disrupting the LCB4, CKA2, and ORM2 gene.

3. The method according to claim 1 , wherein the activities of the proteins are reduced by deletion of the LCB4, CKA2, and ORM2 genes.

4. The method according to claim 1 , wherein the protein encoded by the LCB4 gene is a protein defined in (A), (B), or (C) mentioned below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 10;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 10 but including substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having sphingoid base kinase activity;
(C) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 10, and having sphingoid base kinase activity.

5. The method according to claim 1 , wherein the protein encoded by the CKA2 gene is a protein defined in (A), (B), or (C) mentioned below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 16;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 16 but including substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having casein kinase 2 activity;
(C) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 16, and having casein kinase 2 activity.

6. The method according to claim 1 , wherein the yeast has further been modified so that the expression and/or activity or activities of one or more proteins selected from proteins encoded by LCB5, ELO3, and CHA1 genes are reduced.

7. The method according to claim 6 , wherein the activity or activities of the one or more proteins are reduced by attenuating the expression of the respective genes encoding the one or more proteins, or by disrupting the respective genes encoding the one or more proteins.

8. The method according to claim 6 , wherein the activity or activities of the one or more proteins are reduced by deletion of the respective genes encoding the one or more proteins.

9. The method according to claim 1 , wherein the yeast has further been modified so that the expression and/or activity or activities of one or more proteins selected from proteins encoded by LCB1, LCB2, TSC10, and SUR2 genes are increased.

10. The method according to claim 9 , wherein the activity or activities of the one or more proteins are increased by increasing the expression of the respective genes encoding the one or more proteins.

11. The method according to claim 9 , wherein the expression of the gene(s) is increased by increasing the copy number of the gene(s), and/or by modifying an expression control sequence of the gene(s).

12. The method according to claim 1 , wherein the phytosphingosine is selected from the group consisting of C16 PHS, C18 PHS, C20 PHS, C18:1 PHS, C20:1 PHS, 4-(hydroxymethyl)-2-methyl-6-tetradecanyl-1,3-oxazinan-5-ol, and 4-(hydroxymethyl)-2-methyl-6-hexadecanyl-1,3-oxazinan-5-ol.

13. The method according to claim 1 , wherein the yeast belongs to the genus Saccharomyces.

14. The method according to claim 1 , wherein the yeast is Saccharomyces cerevisiae.

15. The method according to claim 1 , wherein the yeast is able to produce and accumulate the objective substance in a culture medium or cells of the yeast in an amount larger than that obtainable with a non-modified strain.

Assignees

• Ajinomoto Kk
• Evolva Sa

Inventors

• Schwab Markus
• Babau Maud
• Fischer David
• Hatsch Anaelle
• Tavares Sabina De Andrade Pereira
• Nielsen Curt Aime Friis
• Klein Jens
• Wirdnam Corina Daniela

Classifications

• C12N9/12

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title

• C12Y207/11001

  Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• C12P1/02

  by using fungi · CPC title

• C12Y207/01091

  Sphinganine kinase (2.7.1.91) · CPC title