Marker composition for selecting living modified organism, living modified organism, and transformation method

Patent metadata

Field	Value
Publication number	US-11549117-B2
Application number	US-201816612918-A
Country	US
Kind code	B2
Filing date	May 11, 2018
Priority date	May 11, 2017
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

A marker composition for selecting a living modified organism allows transformation and the production of a target product without antibiotics or antibiotic resistance genes. The marker composition for selecting a living modified organism may basically prevent problems caused by the use of antibiotics and antibiotic resistance genes and produce a target product at a high yield.

First claim

The invention claimed is:

1. A method of transforming an organism, the method comprising:
attenuating or deleting at least one of the genes encoding enzymes in an isopentenyl diphosphate or dimethylallyl diphosphate synthetic pathway of an organism to be transformed;
preparing a recombinant plasmid into which (i) a selection marker gene comprising the at least one of the genes encoding the enzymes or a complementary gene thereof and (ii) a target product gene comprising a gene encoding an enzyme in a target product synthetic pathway synthesizing a target product other than the isopentenyl diphosphate or the dimethylallyl diphosphate are introduced; and
transforming the organism with the recombinant plasmid.

2. The method of claim 1 , wherein the transformation comprises culturing the organism in a medium that is free of an antibiotic.

3. The method of claim 1 , wherein the isopentenyl diphosphate or dimethylallyl diphosphate synthetic pathway is a methylerythritol 4-phosphate (MEP) pathway or a mevalonate (MVA) pathway.

4. The method of claim 1 , wherein the at least one of the genes encoding the enzymes is a gene encoding one or more enzymes selected from the group consisting of 1-dioxy-D-xylulose-5-phosphate (DXP) synthase, DXP reductoisomerase, 2-C-methyl-D-erythritol-4-phosphate (MEP) cytidyltransferase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) synthase, 4-hydroxy-3-methyl- 2-butenyl diphosphate (HMBPP) synthase, HMBPP reductase, acetoacetyl-CoA synthase, 3-hydroxyl-3-methylglutary-CoA (HMG-CoA) synthase, HMG-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase and isopentenyl pyrophosphate (IPP) isomerase.

5. The method of claim 1 , wherein the attenuated or deleted gene is a gene encoding enzymes in the methylerythritol 4-phosphate (MEP) pathway.

6. The method of claim 5 , wherein the attenuated or deleted gene is a gene encoding at least one of DXP synthase and DXP reductoisomerase.

7. The method of claim 1 , wherein the attenuated or deleted gene is a gene encoding enzymes in the methylerythritol 4-phosphate (MEP) pathway, and the complementary gene is a gene encoding at least one of enzymes in the mevalonate (MVA) pathway.

8. The method of claim 7 , wherein the complementary gene is a gene encoding acetoacetyl-CoA synthase, 3-hydroxyl-3-methylglutary-CoA (HMG-CoA) synthase, HMG-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase or isopentenyl pyrophosphate (IPP) isomerase.

9. The method of claim 1 , wherein the attenuated or deleted gene is a gene encoding at least one of enzymes in the mevalonate (MVA) pathway.

10. The method of claim 1 , wherein the target product synthetic pathway is selected from the group consisting of isoprenoid, santalene, bisabolol and retinol synthetic pathways.

11. The method of claim 1 , wherein the attenuating or deleting comprises attenuating or deleting at least two genes, and the preparing comprises preparing two plasmids comprising a gene encoding the same enzyme as the attenuated or deleted gene, respectively.

12. The method of claim 9 , wherein the complementary gene is a gene encoding 1-dioxy-D-xylulose-5-phosphate (DXP) synthase, DXP reductoisomerase, 2-C-methyl-D-erythritol-4-phosphate (MEP) cytidyltransferase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) synthase, 4-hydroxy-3-methyl- 2-butenyl diphosphate (HMBPP) synthase, or HMBPP reductase.

13. The method of claim 1 , wherein the target product synthetic pathway is selected from the group consisting of santalene, bisabolol and retinol synthetic pathways.

14. A method of transforming an organism, the method comprising:
attenuating or deleting at least one of the genes encoding enzymes in a methylerythritol 4-phosphate (MEP) pathway of an organism to be transformed;
preparing a recombinant plasmid into which (i) a selection marker gene comprising the at least one of the genes encoding the enzymes and (ii) a target product gene comprising a gene encoding an enzyme in a target product synthetic pathway synthesizing a target product other than the isopentenyl diphosphate or the dimethylallyl diphosphate are introduced; and
transforming the organism with the recombinant plasmid.

15. The method of claim 14 , wherein the at least one of the genes encoding the enzymes is a gene encoding one or more enzymes selected from the group consisting of 1-dioxy-D-xylulose-5-phosphate (DXP) synthase, DXP reductoisomerase, 2-C-methyl-D-erythritol-4-phosphate (MEP) cytidyltransferase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) synthase, 4-hydroxy-3-methyl- 2-butenyl diphosphate (HMBPP) synthase, and HMBPP reductase.

16. The method of claim 14 , wherein the attenuated or deleted gene is a gene encoding at least one of DXP synthase and DXP reductoisomerase.

17. The method of claim 14 , wherein the target product synthetic pathway is selected from the group consisting of isoprenoid, santalene, bisabolol and retinol synthetic pathways.

18. The method of claim 14 , wherein the attenuating or deleting comprises attenuating or deleting at least two genes, and the preparing comprises preparing two plasmids comprising a gene encoding the same enzyme as the attenuated or deleted gene, respectively.

19. The method of claim 14 , wherein the target product synthetic pathway is selected from the group consisting of santalene, bisabolol and retinol synthetic pathways.

20. A method of transforming an organism, the method comprising:
attenuating or deleting at least one of the genes encoding enzymes in a mevalonate (MVA) pathway of an organism to be transformed;
preparing a recombinant plasmid into which (i) a selection marker gene comprising the at least one of the genes encoding the enzymes and (ii) a target product gene comprising a gene encoding an enzyme in a target product synthetic pathway synthesizing a target product other than the isopentenyl diphosphate or the dimethylallyl diphosphate are introduced; and
transforming the organism with the recombinant plasmid.

21. The method of claim 20 , wherein the at least one of the genes encoding the enzymes is a gene encoding one or more enzymes selected from the group consisting of acetoacetyl-CoA synthase, 3-hydroxyl-3-methylglutary-CoA (HMG-CoA) synthase, HMG-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase and isopentenyl pyrophosphate (IPP) isomerase.

22. The method of claim 20 , wherein the target product synthetic pathway is selected from the group consisting of santalene, bisabolol and retinol synthetic pathways.

Assignees

• Nat Univ Gyeongsang Iacf

Inventors

• Kim Seon Won
• Han Hyo Jung
• Kim Sin Young
• Park Ji Bin

Classifications

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/746

  for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus) · CPC title

• C12P5/002

  cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00) · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title