Triterpenoid production with a minimally engineered Saccharomyces cerevisiae chassis

Abstract

Triterpenoids, one of the most diverse classes of natural products, have been used for centuries as active ingredients in essential oils and Chinese medicines and are of interest for many industrial applications ranging from low-calorie sweeteners to cosmetic ingredients and vaccine adjuvants. However, not only can the extraction from plant material be cumbersome due to low concentrations of the specific triterpenoid, but concerns are also increasing regarding the sustainability of wild plant harvest while meeting market demands. The alternative is to produce triterpenoids with engineered microbes. Here, we present a generally applicable strategy for triterpenoid production in the yeast Saccharomyces cerevisiae based on a modified oxidosqualene cyclase Erg7. The modification reduces the flux into the sterol pathway while increasing the precursor supply for triterpenoid production. The minimally engineered strain was exploited for the exemplary production of the lupane triterpenoids betulin, betulin aldehyde, and betulinic acid at a total titer above 6 g/L, the highest reported so far. To further highlight the chassis concept, squalene, oleanane- and dammarane-type triterpenoids were synthesized to titers at a similar gram scale. We propose the developed baker's yeast as a host for the thousands of triterpenoid synthesis pathways from plants, reducing the pressure on the natural resources.

Bioprocess optimization enables enhanced protopanaxadiol production in yeast

“…37,38 The engineered yeast BA6Δ for betulin synthesis was modified from S. cerevisiae Simo1575. 16 PPD and DM concentration were determined with an HPLC system (UltiMate 3000 HPLC system, Thermo Fisher Scientific, USA) coupled to a Dionex Corona Veo Charged Aerosol Detector. The column was an EC150/4.6 NUCLEODUR C18 gravity column (Macherey-Nagel GmbH and Co. KG, Germany), the mobile phase was Acetonitrile:0.2 % (v/v) formic acid, the column temperature was set to 40 ℃, the flow rate was programmed to 1.2 mL/min, the injection volume was set to 5 µL, the runtime was 23 min.…”

“…37,38 The engineered yeast BA6Δ for betulin synthesis was modified from S. cerevisiae Simo1575. 16 Standard WM8+ medium included 50 g/L glucose, salt solution, 10 g/L glutamate, trace elements solution, vitamins solution, verduyn-trace elements solution, and verduyun-vitamins solution. In addition, the salt solution includes 0.25 g/L (NH 4 )H 2 PO 4 , 2.8 g/L NH 4 Cl, 0.25 g/L MgCl 2 ·6H 2 O, 0.1 g/L CaCl 2 ·2H 2 O, 2 g/L KH 2 PO 4 , 0.55 g/L MgSO 4 ·7H 2 O, and 0.075 g/L C 6 H 12 O 6 .…”

“…The engineered yeast 102-tm-reerg7-2PP for PPD synthesis was modified from S. cerevisiae CEN.PK 102-5B and originated from a previous study. 16,36 S. cerevisiae BA6Δ (unpublished) is a pah1 deletion mutant of BA6, generated through replacement of PAH1 with a hygromycin resistance cassette (YMR165CΔ:hphMX). 37,38 The engineered yeast BA6Δ for betulin synthesis was modified from S. cerevisiae Simo1575.…”

“…13 Triterpenoids are either synthesized from squalene or 2,3-oxidosqualene, which are both intermediates of sterol synthesis in yeast. 5,16 The synthesis of ginsenosides by engineered yeasts from renewable resources is a promising strategy to address the increasing demand for this interesting class of molecules. 5 The microbial biosynthesis of ginsenosides has several advantages, including rapid growth, controllable culture conditions, space efficiency, sustainable production, environmentally friendly, and well-developed genetic manipulation technology.…”