Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Abstract

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering.

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

“…For comparison: the highest yield for BA production (182 mg/L) has been achieved in a S. cerevisiae strain (Czarnotta et al ., ). This strain is the result of multiple optimization rounds and fed‐batch cultivations.…”

“…Typically plant genomes contain from nine to sixteen OSC genes, for example the model plant Arabidopsis thaliana genome contains thirteen OSC gene family enzymes and can produce a diverse array of triterpene skeletons (Vincken et al, 2007;Xue et al, 2012). Among these, a lupeol synthase, At1g78970/LUP1 has been characterized (Herrera et al, 1998), successfully expressed and used for lupeol and BA production in yeasts (Czarnotta et al, 2017;Husselstein-Muller et al, 2001;Lang and Lewandowski, 2016;Zhou et al, 2016).…”

“…To test the feasibility of P. tricornutum as a lupeol production platform, we ectopically expressed two representative OSCs: AtLUS or L. japonicus OSC3 (from now on LjLUS). Both enzymes have been shown to be functional in S. cerevisiae, with AtLUS being the most prevalent in literature (Czarnotta et al, 2017;Sawai et al, 2006;Zhou et al, 2016). The native coding sequences of AtLUS and LjLUS were integrated into the P. tricornutum nuclear genome under the control of the strong fucoxanthin chlorophyll a/c binding protein A (FCPA/LHCF1) promoter, along with zeocin resistance gene marker (ble r ) (SF1, A and B).…”

Enhanced Production and in situ Product Recovery of Fusicocca-2,10(14)-Diene from Yeast

“…A previous report suggested that FCdiene could be readily produced from S. cerevisiae, however, fermentation parameters were not optimized [5,15]. Fermentation parameters such as pH, dissolved oxygen tension (DOT), ethanol, as well as in situ product removal were also shown to strongly influence the production of a triterpenoid, betulinic acid, from S. cerevisiae.…”

Evaluation of pyruvate decarboxylase‐negative Saccharomyces cerevisiae strains for the production of succinic acid

“…One milliliter of culture was mixed with 400 µL of concentrated HCl and 600 µL water to dissolve the CaCO 3 completely. The OD 600 was measured using an Ultrospec 10 cell density meter (Amersham Biosciences, UK) and converted to biomass concentrations using a conversion factor of 0.21 g CDW /L per OD 600 previously determined by Czarnotta et al [28].…”