Short CommunicationEnhancing oleanolic acid production in engineered Saccharomyces cerevisiae
Graphical abstract
Introduction
Oleanolic acid (OA) is an important plant-derived triterpenoid with various biological activities, including anti-inflammation, anti-viral, anti-tumor and hepatoprotective effect (Pollier & Goossens, 2012). Thus, OA has been widely used in agriculture, food, cosmetics and pharmacy industry (Liu, 2005). Currently, OA is primarily manufactured by extraction from plants while the OA content in plants is low and generally needs a long period (Xia et al., 2011). Compared to plants, microbes exhibit several advantages in producing natural chemicals such as fast-growing, land-saving and controllable culture conditions. With the development of synthetic biology, various valuable plant-derived terpenoids have been produced in microbial cell factories, such as artemisinic acid (Paddon et al., 2013), taxadiene (Zhou et al., 2015), β-amyrin (Liu et al., 2014) and glycyrrhetinic acid (Zhu et al., 2018).
Recently, the biosynthetic pathway of OA has been decoded (Fig. 1). It is generated by condensing six isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which can be generated through the mevalonate (MVA) pathway. Based on the endogenous MVA pathway, heterologous production of OA is accomplished by introducing three heterogenous genes (bAS, CYP716A12 and CPR) into Saccharomyces cerevisiae, but the production was only 71.0 mg/L, which was too low for industrial production (Dai et al., 2014). Although many strategies have been tried to enhance its production, few of them are proven to be useful.
Recently, it has been reported that the electron transfer efficiency mediated by CPR may be a key issue of triterpenoids production in heterogeneous hosts (Zhu et al., 2018). The uncoupling of CYP450 and CPR would generate reactive oxygen, which could cause cell damage, thus decreasing the production of triterpenoids. Herein, we hypothesize that the low production of OA may be due to the mismatch of redox between selected CYP450 and CPR. Furthermore, the expression level of key genes may be not high enough either. Thus, we first optimized oxidation-reduction system to improve OA production by testing four CPRs to couple with CYP716A12. Then, the transcriptional level of key genes were improved by the overexpression under GAL promoters. Although GAL promoters are strong promoters in yeast, they are inhibited by GAL80 under glucose condition (Bhat & Iyer, 2009). In addition, galactose, the inducer of GAL promoters, can be metabolized through galactokinase (GAL1) (Sellick et al., 2008) that deeply reduces its utilization efficiency. To solve these issues, genes GAL80 and GAL1 were knocked out to improve the transcriptional level of key genes under glucose condition. Finally, the truncated HMG-CoA reductase, squalene synthase and squalene epoxidase were overexpressed to enhance the precursor supply. These strategies improved the production of OA up to 186.1 ± 12.4 mg/L in flask shake. Combined with fermentation optimization, the final oleanolic acid production was 606.9 ± 9.1 mg/L which was 7.6-fold higher than the reported maximum production. To our knowledge, this is the highest titer of OA in microbial cell factories, which dramatically promotes the industrialization process of OA production.
Section snippets
Strains, media and cell cultivation
S. cerevisiae strain JDY52 (MATa his3Δ200 leu2Δ0 lys2Δ0 trp1Δ63 ura3Δ0 met15Δ0) derived from S288C, was used as the parent strain. SD medium was used for selection of engineered strains. Engineered strains were fermented at 30 °C in YPDG medium. Escherichia coli TOP10 cells were used for transformation and plasmid DNA extraction. Strains were cultivated at 37 °C in LB medium with 100 mg/L ampicillin.
Plasmids and strains construction
The Glycyrrhiza glabra β-amyrin synthase gene (GgbAS) (GenBank: AB037203), Medicago truncatula
Engineering S. cerevisiae for the synthesis of OA
The biosynthesis pathway of OA in plants can be divided into three parts: (1) forming the core structure 2,3-oxidosqualene, (2) forming the pentacyclic triterpenoids backbone, β-amyrin, (3) CYP450 and CPR adding the functional groups at the C-28 position of β-amyrin. S. cerevisiae was chosen as the chassis host for producing OA based on the endogenous 2,3-oxidosqualene biosynthesis pathway. To construct the entire pathway of OA biosynthesis, GgbAS, MtCYP716A12 and AtCPR1 were introduced into
Conclusion
In this work, plant-derived OA production in engineered S. cerevisiae has been effectively boosted via pairing the MtCYP716A12 with MtCPR to optimize the oxidation-reduction system. In addition, reconstructing galactose regulatory network also improved the transcriptional level of key genes and enhanced OA production. Under these metabolic strategies, OA titer was increased up to 186.1 ± 12.4 mg/L in flask shake. Combined with fermentation optimization, the final OA production was
Acknowledgements
The authors kindly acknowledge financial support from the National Science Foundation for Distinguished Young Scholars of China (21425624) and the National Natural Science Foundation of China (21506011, 21606018 and 21476026).
References (11)
Oleanolic acid and ursolic acid: research perspectives
J. Ethnopharmacol.
(2005)- et al.
Enhanced pathway efficiency of Saccharomyces cerevisiae by introducing thermo-tolerant devices
Bioresour. Technol.
(2014) - et al.
Oleanolic acid
Phytochemistry
(2012) - et al.
Galactose metabolism in yeast-structure and regulation of the Leloir pathway enzymes and the genes encoding them
Int. Rev. Cell. Mol. Biol.
(2008) - et al.
Boosting 11-oxo-β-amyrin and glycyrrhetinic acid synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction system from legume plants
Metab. Eng.
(2018)