Triterpenoid sapogenin production in plant and microbial cultures

Patent metadata

Field	Value
Publication number	US-9994883-B2
Application number	US-201314400527-A
Country	US
Kind code	B2
Filing date	May 13, 2013
Priority date	May 11, 2012
Publication date	Jun 12, 2018
Grant date	Jun 12, 2018

Abstract

The disclosure relates to a method for enhancing the biosynthesis and/or secretion of sapogenins in the culture medium of plant and microbial cell cultures. Further, the disclosure also relates to the identification of novel genes involved in the biosynthesis of sapogenin intermediates, as well as to novel sapogenin compounds.

First claim

The invention claimed is:

1. A method for the intracellular production and extracellular secretion of triterpenoid sapogenins in a genetically engineered yeast culture, the method comprising:
incubating genetically engineered yeast cells that express an exogenous regulatory or biosynthetic enzyme of the sapogenin biosynthesis pathway in a culture medium comprising randomly methylated cyclodextrins, hydroxypropylated cyclodextrins, and/or beta-cyclodextrins at a concentration such that the cyclodextrins increase the amount of the produced triterpenoid sapogenin secreted from the eukaryotic genetically engineered yeast cells into the culture medium in comparison to a culture medium lacking said cyclodextrins where the biosynthetic enzyme is an oxidosqualane cyclase and/or a cytochrome P450.

2. The method according to claim 1 , wherein the yeast cells are Saccharomyces cells, Schizosaccharomyces cells, Pichia cells, Yarrowia cells, Hansenula cells, Kluyveromyces cells, or Candida cells.

3. The method according to claim 1 , wherein the genetically engineered yeast cells are deficient in expression and/or activity of an enzyme involved in endogenous sterol synthesis.

4. The method according to claim 1 , wherein the culture medium comprises cyclodextrins selected from the group consisting of randomly methylated cyclodextrins and hydroxypropylated cyclodextrins.

5. The method according to claim 1 , wherein the culture medium comprises β-cyclodextrin.

6. The method according to claim 1 , wherein the cyclodextrin concentration in the culture medium is less than 25 mM.

7. The method according to claim 1 , wherein cyclodextrins are added to the culture medium at different consecutive time points.

8. The method according to claim 1 , further comprising: extracting sapogenins from the culture medium.

9. The method according to claim 1 , wherein the yeast cells are genetically engineered to overexpress an oxidosqualene cyclase and/or a cytochrome P450.

10. The method according to claim 6 , wherein the cyclodextrin concentration is less than 10 mM.

11. The method according to claim 10 , wherein the cyclodextrin concentration is between 2 mM and 7 mM.

Assignees

• Vib Vzw
• Univ Gent

Inventors

• Goossens Alain
• Moses Tessa
• Pollier Jacob
• Almagro Romero Lorena

Classifications

• C07C2603/52

  containing five condensed rings · CPC title

• C07D307/00

  Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom · CPC title

• C12Y504/99

  transferring other groups (5.4.99) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C07C49/727

  a keto group being part of a condensed ring system · CPC title