Increased production of ginsenosides through improvement of protein-folding machinery of yeast

Patent metadata

Field	Value
Publication number	US-11046990-B2
Application number	US-201816057617-A
Country	US
Kind code	B2
Filing date	Aug 7, 2018
Priority date	Aug 9, 2017
Publication date	Jun 29, 2021
Grant date	Jun 29, 2021

Abstract

The present invention relates to recombinant yeast, in which the productivity of ginsenoside is enhanced by overexpressing CPR5, PDI1, or ERO1 in yeast having the productivity of ginsenosides; a method for preparing the yeast; and a method for producing ginsenosides using the yeast.

First claim

The invention claimed is:

1. A recombinant yeast for producing a ginsenoside or a precursor thereof, wherein the recombinant yeast expresses a polynucleotide that encodes a protein involved in protein folding having at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO:3,
wherein the protein involved in protein folding has Cyclosporin-sensitive Proline Rotamase 5 (CPR5)-like activity, or ER Oxidation or Endoplasmic Reticulum Oxidoreductin (ERO1)-like activity;
wherein the recombinant yeast has elevated expression levels of the protein involved in protein folding in comparison to endogenous expression levels of the protein; and
wherein the recombinant yeast comprises ginsenoside synthesizing genes encoding the proteins of HMG-CoA reductase (tHMG1), Panax ginseng squalene epoxidase (PgSE), Panax ginseng dammarenediol-ll synthase (PgDDS), Panax ginseng cytochrome P450 CYP716A47 (PgPPDS), and Panax ginseng NADPH-cytochrome P450 reductase (PgCPR).

2. The recombinant yeast of claim 1 , wherein the polynucleotide has at least 99% sequence homology to SEQ ID NO:1 and the protein involved in protein folding has CPR5-like activity.

3. The recombinant yeast of claim 1 , wherein the polynucleotide has at least 99% sequence homology to SEQ ID NO:3 and the protein involved in protein folding has ERO1-like activity.

4. The recombinant yeast of claim 1 , wherein the yeast is selected from the group consisting of S. cerevisiae, S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, S. turicensis, S. unisporus, S. uvarum , and S. zonatus.

5. The recombinant yeast of claim 1 , wherein the expression levels of tHMG1, PgSE, PgDDS, PgPPDS, and PgCPR are elevated in comparison to their endogenous expression levels.

6. The recombinant yeast of claim 1 , wherein the precursor is squalene or 2,3-oxidosqualene.

7. The recombinant yeast of claim 1 , wherein a vector comprises the polynucleotide and expression of the polynucleotide is driven by a PGK1 promoter.

8. The recombinant yeast of claim 1 , wherein the recombinant yeast comprises ginsenoside synthesizing genes encoding proteins having the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

9. The recombinant yeast of claim 4 , wherein the recombinant yeast is S. cerevisiae.

10. The recombinant yeast of claim 9 , wherein the recombinant yeast has been recombinantly modified to express tHMG1, PgSE, PgDDS, PgPPDS, and PgCPR proteins from vector(s) comprising ginsenoside synthesizing genes.

11. The recombinant yeast of claim 10 , wherein the expression of the ginsenoside synthesizing genes is driven by a Glycerol-3-Phosphate Dehydrogenase (GPD1) promoter.

12. The recombinant yeast of claim 11 , wherein expression of the polynucleotide results in about a 5-fold increase in production of the ginsenoside in comparison to non-expression of the polynucleotide.

13. A method for preparing recombinant yeast with an enhanced productivity of ginsenosides compared to parent yeast, comprising transforming the recombinant yeast cell with a polynucleotide having the sequence of SEQ ID NO:1 and/or SEQ ID NO:3 such that expression of a protein encoded by the polynucleotide is increased, and wherein the protein has CPR5-like activity or ERO1-like activity in a ginsenoside-producing yeast strain relative to its endogenous expression levels.

14. The method of claim 13 , wherein the ginsenoside-producing yeast strain is one or more selected from the group consisting of Panax ginseng dammarenediol-ll synthase (PgDDS), Panax ginseng cytochrome P450 CYP716A47 (PgPPDS), Panax ginseng NADPH-cytochrome P450 reductase (PgCPR), S. cerevisiae HMG-CoA reductase (tHMG1), and Panax ginseng squalene epoxidase (PgSE).

15. A method for preparing recombinant yeast with an enhanced productivity of ginsenoside precursors compared to parent yeast, comprising increasing the expression level of a protein encoded by the sequence of SEQ ID NO:1 and/or 3, and wherein the protein has CPR5-like activity or ERO1-like activity in a ginsenoside precursor-producing yeast strain relative to its endogenous expression levels.

16. The method of claim 15 , wherein the ginsenoside precursor comprises squalene or 2,3-oxidosqualene.

17. A method for producing ginsenoside or a precursor thereof, comprising culturing the recombinant yeast of any one of claim 1 .

Assignees

• Intelligent Synthetic Biology Ct
• Korea Res Inst Chemical Tech

Inventors

• Kim Sun-Chang
• Lee Ju Young
• Jang In Seung
• Kim Seohyun
• Jegal Jong Geon

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y101/01034

  Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34) · CPC title

• C12Y108/03002

  Thiol oxidase (1.8.3.2) · CPC title

• C12N9/0071

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

• C12N9/0095

  acting on iron-sulfur proteins as donor (1.18) · CPC title