Method for transformation of stramenopile

Patent metadata

Field	Value
Publication number	US-2016289689-A1
Application number	US-201514711075-A
Country	US
Kind code	A1
Filing date	May 13, 2015
Priority date	Oct 1, 2010
Publication date	Oct 6, 2016
Grant date	—

Abstract

To provide a transformation method for producing a stramenopile organism having an improved unsaturated fatty acid production capability by disrupting a gene of the stramenopile organism or inhibiting the expression of the gene in a genetically engineering manner. [Solution] A method for transforming a stramenopile organism, which comprises disrupting a gene of the stramenopile organism or inhibiting the expression of the gene in a genetically engineering manner, and which is characterized in that the stramenopile organism is selected from Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum and Parietichytrium sp. and the gene to be disrupted or of which the expression is to be inhibited is a gene associated with the biosynthesis of a fatty acid.

First claim

1 . A method for transforming Stramenopile, the method comprising disrupting a stramenopile gene and/or inhibiting expression thereof by genetic engineering in microorganisms belong to Stramenopile, wherein the microorganisms are selected from the group consisting of Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210.

2 . The method according to claim 1 , wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

3 . The method according to claim 2 , wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

4 . The method according to claim 3 , wherein the fatty acid chain elongase is a C20 elongase.

5 . The method according to claim 3 , wherein the fatty acid desaturase is a Δ12 desaturase.

6 . The method according to claim 5 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

7 . The method according to claim 6 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

8 . The method according to claim 7 , further comprising introducing a gene associated with fatty acid desaturase.

9 . The method according to claim 8 , wherein the gene associated with fatty acid desaturase is an ω3 desaturase.

10 . A method for modifying the fatty acid composition of a stramenopile, the method comprising disrupting a stramenopile gene and/or inhibiting expression thereof by genetic engineering in microorganisms, wherein the microorganisms are selected from the group consisting of Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210.

11 . The method according to claim 10 , wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

12 . The method according to claim 11 , wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

13 . The method according to claim 12 , wherein the fatty acid chain elongase is a C20 elongase.

14 . The method according to claim 12 , wherein the fatty acid desaturase is a Δ12 desaturase.

15 . The method according to claim 14 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

16 . The method according to claim 15 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

17 . The method according to claim 16 , further comprising introducing a gene associated with fatty acid desaturase.

18 . The method according to claim 17 , wherein the gene associated with fatty acid desaturase is an 3 desaturase.

19 . A method for highly accumulating a fatty acid in a stramenopile, wherein the method uses the method of claim 18 .

20 . The method according to claim 19 , wherein the fatty acid is an unsaturated fatty acid.

21 . The method according to claim 20 , wherein the unsaturated fatty acid is an unsaturated fatty acid of 18 to 22 carbon atoms.

22 . A fatty acid obtained from the stramenopile in which the fatty acid is highly accumulated by using the method of claim 21 .

23 . A stramenopile transformed for the modification of the fatty acid composition through disruption of its gene and/or inhibition of expression thereof by genetic engineering in microorganisms, wherein the microorganisms are selected from the group consisting of Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210.

24 . The stramenopile according to claim 23 , wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

25 . The stramenopile according to claim 24 , wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

26 . The stramenopile according to claim 25 , wherein the fatty acid chain elongase is a C20 elongase.

27 . The stramenopile according to claim 25 , wherein the fatty acid desaturase is a Δ12 desaturase.

28 . The stramenopile according to claim 27 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

29 . The stramenopile according to claim 28 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

30 . The stramenopile according to claim 29 , further comprising introducing a gene associated with fatty acid desaturase is introduced.

31 . The stramenopile according to claim 30 , wherein the gene associated with fatty acid desaturase is an ω3 desaturase.

32 . The method according to claim 1 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

33 . The method according to claim 32 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

34 . The method according to claim 2 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

35 . The method according to claim 34 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

36 . The method according to claim 3 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

37 . The method according to claim 36 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

38 . The method according to claim 4 , wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

39 . The method according to claim 38 , wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

Assignees

• Kyushu Univ Nat'L Univ Corp
• Univ Miyazaki
• Konan Gakuen
• Nippon Suisan Kaisha Ltd

Inventors

• Sakaguchi Keishi
• Hamaguchi Rie
• Matsuda Takanori
• Ito Makoto
• Nagano Naoki
• Hayashi Masahiro
• Honda Daisuke
• Okita Yuji
• Sugimoto Shinichi

Classifications

• C12N9/1029

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

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2310/11

  Antisense · CPC title

• C12N15/895

  using biolistic methods · CPC title