Triterpenoids represent a diverse group of phytochemicals, widely distributed in the plant kingdom with many biological activities. Recently, the heterologous production of triterpenoids in Saccharomyces cerevisiae has been successfully implemented by introducing various triterpenoids biosynthetic pathways. By engineering related enzymes as well as yeast metabolism, the yield of various triterpenoids is significantly improved from milligram-scale per liter to gram-scale level per liter. This achievement demonstrates that engineering of critical enzymes is considered as a potential strategy to overcome the main hurdles of translation of these potent natural products into industry. Here, we review strategies for designing enzymes to improve the yield of triterpenoids in S. cerevisiae, which is mainly separated into three aspects: 1. elevating the supply of the precursor-2,3-oxidosqualene, 2. optimizing triterpenoid-involved reactions, 3. lowering the competition of the native sterol pathway. And then we provide challenges and prospects on further enhancing the triterpenoid production in S. cerevisiae.

Plant natural products (PNPs) represent a vast and diverse group of natural products, which have wide applications such as emulsifiers in cosmetics, sweeteners in foods, and active ingredients in medicines. Large-scale production of certain PNPs (e.g., artemisinin, taxol) has been implemented by reconstruction of biosynthetic pathways in heterologous hosts. However, unknown biosynthetic pathways greatly restrict wide applications of heterologous production of PNPs of interest. With the rapid development of sequencing and multiomics analysis technologies, huge amounts of omics data, i.e., genomics, transcriptomics, and proteomics, have been deposited in public databases, which is a precious resource for identification of the unknown biosynthetic pathway of PNPs. Herein, we have enumerated the approaches which have been widely used to screen candidate genes involved in the biosynthesis of PNPs of interest. We also discuss recent developments in the characterization of putative genes and elucidation of the complete biosynthetic pathway in heterologous hosts.

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Triterpenoids are a subgroup of terpenoids and have wide applications in the food, cosmetics, and pharmaceutical industries. The heterologous production of various triterpenoids in Saccharomyces cerevisiae, as well as other microbes, has been successfully implemented as these production hosts not only produce the precursor of triterpenoids 2,3-oxidosqualene by the mevalonate pathway but also allow simple expression of plant membrane-anchored enzymes. Nevertheless, 2,3oxidosqualene is natively converted to lanosterol catalyzed by the endogenous lanosterol synthase (Erg7p), causing low production of recombinant triterpenoids. While simple deletion of ERG7 was not effective, in this study, the critical amino acid residues of Erg7p were engineered to lower this critical enzyme activity. The engineered S. cerevisiae indeed accumulated 2,3-oxidosqualene up to 180 mg/L. Engineering triterpenoid synthesis into the ERG7-modified strain resulted in 7.3-and 3-fold increases in the titers of dammarane-type and lupane-type triterpenoids, respectively. This study presents an efficient inducer-free strategy for lowering Erg7p activity, thereby providing 2,3-oxidosqualene for the enhanced production of various triterpenoids.

The quantitative analysis of taurine and edaravone in biological sample is critical in pharmaceutical studies. Although each of them can be individually analyzed by different approaches, concurrent quantification is still a highly challenging task with respect to their great polarity variation and the complex composition of tissue sample. In the present study, to simultaneously determine taurine and edaravone in rat tissue, the sample preparation and chromatographic separation conditions were evaluated and discussed in detail. As for the sample preparation, four kinds of solvent and the volume ratio of the optimal solvent to biological sample were both tested and evaluated based on the chromatographic profile, extraction recovery, and matrix effect (ME). The chromatographic separation was performed in a reverse phase (RP) and two hydrophilic interaction liquid chromatography (HILIC) modes, and the corresponding separation efficiencies were assessed using chromatographic parameters like half-width (W 1/2 ), tailing factor (f t), theoretical plates number (N), and ME. Furthermore, adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated on an Atlantis HILIC silica column according to the resultant chromatographic profiles and peak areas of the analytes. The optimal results were obtained when the biological samples were deproteined by 4-fold volume of methanol/acetonitrile (1:3, v/v) and separated on a HILIC column with a gradient elution of acetonitrile/water containing 0.2 % formic acid and 10 mM ammonium formate. The proposed approach was validated and successfully applied to the parallel determination of the tissue distribution of edaravone and taurine in rat tissues.