Exploration of In Situ Extraction for Enhanced Triterpenoid Production by Saccharomyces cerevisiae

Abstract

Plant-derived triterpenoids are in high demand due to their valuable applications in cosmetic, nutraceutical, and pharmaceutical industries. To meet this demand, microbial production of triterpenoids is being developed for large-scale production. However, a prominent limitation of microbial synthesis is the intracellular accumulation, requiring cell disruption during downstream processing. Destroying the whole-cell catalyst drives up production costs and limits productivity and product yield per cell. Here, in situ product extraction of triterpenoids into a second organic phase was researched to address this limitation. An organic solvent screening identified water-immiscible isopropyl myristate as a suitable in situ extractant, enabling extraction of up to 90% of total triterpenoids from engineered Saccharomyces cerevisiae. Combining isopropyl myristate and β-cyclodextrins improved extraction efficiency. In a first configuration, repeated batch fermentation with sequential product extraction and cell recycling resulted in 1.8 times higher production than a reference fermentation without in situ product extraction. In the second configuration, yeast cells were in contact with the second organic phase throughout a fed-batch fermentation to continuously extract triterpenoids. This resulted in 90% product extraction and an extended production phase. Further improvement of triterpenoid production was not achieved due to microbial host limitations uncovered through omics analyses.

Keywords: betulinic acid; biocompatible solvent; in situ extraction; metabolic engineering; organic solvent; plant natural products; process development; secondary metabolites; triterpenoids; yeast.

FIGURE 1

Schematic overview of cytosolic mevalonate (blue) and farnesyl diphosphate synthase (FPP) (red) pathways. FPP is converted to squalene by Erg9p, which is then converted into yeast native ergosterol pathway (yellow). 2,3‐Oxidosqualene also serves as the common precursor for heterologous biosynthesis of betulinic acid (green). CPR, cytochrome P450 reductase; CYP, cytochrome P450 monooxygenase; Erg10p, acetyl‐CoA C‐acetyltransferase; Erg11p, lanosterol 14‐α‐demethylase; Erg12p, mevalonate kinase; Erg13p, HMG‐CoA synthase; Erg19p, mevalonate pyrophosphate decarboxylase; Erg1p, squalene epoxidase; Erg20p, farnesyl pyrophosphate synthetase; Erg24p, C‐14 sterol reductase; Erg25p, sterol C‐4 methyloxydase; Erg26p, sterol C‐3 dehydrogenase; Erg27p, sterol C‐3 ketoreductase; Erg2p, sterol isomerase; Erg3p, sterol desaturase; Erg4p, sterol reductase; Erg5p, sterol desaturase; Erg6p, delta 24‐sterol C‐methyltransferase; Erg7p, lanosterol synthase; Erg8p, phosphomevalonate kinase; Erg9p, squalene synthase; Hmg1/2, HMG‐CoA reductase; OEW, lupeol synthase. Adapted and modified from Czarnotta et al. (2017).

FIGURE 2

Ratio of intracellular triterpenoids in treated cells and the untreated control [%] (A), and viability [%] of cells treated with solvents (B). All cell suspensions were incubated with 20 vol. % solvent for 2 h, except for the control.

FIGURE 3

Headspace CO₂ volume concentration during the cultivation of S. cerevisiae BA6∆ in the presence of (A) 4 vol. % ethyl decanoate (blue), (B) 4 vol. % methyl decanoate (red), and (C) 4 vol. % IPM (green). For each approach, a control batch without solvent (black) was carried out. In approach containing ethyl decanoate (A), CO₂ increased after 40 h, indicating ethyl decanoate being used as carbon source by the cells. In approach containing methyl decanoate (B), cell growth is slower than the control approach, showing the negative impact of methyl decanoate on cell metabolism. In approach with IPM (C), the CO₂ course is similar to the control, indicating no negative impact of IPM on cell metabolism. Representative data sets of two biological replicates are shown.

FIGURE 4

Distribution of intracellular and extracted triterpenoids in [%] after 2, 5, 18, and 24 h extraction with 150 vol. % IPM (A) and 150 vol. % IPM plus 8 mM β‐CD (B), showing that addition of β‐CD allows a faster extraction of triterpenoid from the cell into the organic phase. Two biological replicates per each time point were measured (shown by (I) and (II)). Error bars refer to two technical replicates.

FIGURE 5

Sequential batch fermentations with and without in situ extraction. Shown are the sum of total triterpenoids (intracellular (pellet) and extracellular (OP)) which is higher in the in situ extraction approach (grey), than in the control approach (black) (A), cell viability, which was similar in both in situ extraction approach (grey) and control (black) (B), and product yield in both approaches, which was slightly higher in in situ extraction approach than in the control (C) over six sequential batch runs. For the in situ extraction approach, the product distribution between pellet and OP [%] was determined (D). Error bars show the standard deviation of three biological replicates.

FIGURE 6

Ethanol‐pulsed fed‐batch fermentation: Concentration profiles of triterpene and sterol products (top) and extracellular substrates and products as well as cell viability (bottom) for the control (A, C) and the fermentation with in situ extraction (B, D) shows no difference in product titre. The dashed line indicates the start of in situ extraction. (A and B) Concentrations of betulinic acid (BA), betulin (B), betulin aldehyde (BetAld), lupeol (Lup), ergosterol (Erg), and squalene (SQ) and total triterpenoid concentration (B, BetAld, BA). (C and D) Concentrations of glucose, ethanol, glycerol, acetate, and CDW and cell viability. Data from a single experiment are shown.

FIGURE 7

Glucose‐pulsed fed‐batch fermentation: Concentration profiles of triterpene and sterol products (top) and extracellular substrates and products as well as cell viability (bottom) for the control (A, C) and the fermentation with in situ extraction (B, D) shows no difference in product titre. The dashed line indicates the start of in situ extraction. (A and B) Concentrations of betulinic acid (BA), betulin (B), betulin aldehyde (BetAld), lupeol (Lup), ergosterol (Erg), and squalene (SQ) and the total triterpenoid concentration. (C and D) Concentrations of glucose, ethanol, glycerol, acetate, and CDW, and cell viability. Data from a single experiment are shown. Glucose pulses were added when a rapid increase in the DO signal (Figure S8) indicated carbon source depletion. This time point is displayed in Figures C and D with an assumed glucose concentration of zero. In contrast, the glucose concentration after pulse was determined by HPLC‐RI.

FIGURE 8

Distribution of the triterpenoids betulinic acid (BA), betulin (B), and betulin aldehyde (BetAld) in pellet and organic phase in ethanol‐pulsed (A) and glucose‐pulsed (B) fed‐batch with application of 150 vol. % IPM and 8 mM β‐CD for in situ extraction. After start of in situ extraction, indicated by the arrows, the triterpenoids were detected in the organic phase, showing the successful extraction of triterpenoids into the organic phase. Data from a single experiment are shown, error bars represent the standard deviation of two technical replicates.

FIGURE 9

Schematic illustration of the gene cassette encoding cytochrome P450 monooxygenase (CYP) and lupeol synthase (OEW), with the respective promoters pTEF1 and pPGK1 and terminators tADH1 and tCYC1(A), the marker gene URA3 and the promoter pURA3 with the loxP sites, the TY sites TY4/5 and TY4/3. Annotation of genetic components integrated into chromosome X of S. cerevisiae BA6∆ at the beginning (B), and at the end (C) of ethanol‐pulsed fed‐batch cultivation as well as at the beginning (D) and end (E) of glucose‐pulsed fed‐batch based on results of the genome sequencing. In all approaches, only one or parts of the plasmid were found.