Improving the production of squalene-type triterpenoid 2,3;22,23-squalene dioxide by optimizing the expression of CYP505D13 in Saccharomyces cerevisiae

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The efficient bioproduction of squalene-type triterpenoids (STs) has attracted considerable attention due to their significant biological activities. In a previous study, we constructed a recombinant Saccharomyces cerevisiae capable of producing three STs; 4,8-dihydroxy-22,23-oxidosqualene (ST-1), 8-hydroxy-2,3;22,23-squalene dioxide (ST-2), and 2,3;22,23-squalene dioxide (ST-3). Here, we first evaluated the effects of these STs on the growth of human non-small cell lung cancer (NSCLC) cells, and found that ST-3 exhibited the greatest potency compared to the other two STs. To further enhance the bioproduction of ST-3, we adopted a tunable system to balance the expression of the Ganoderma lucidum cytochrome P450 gene CYP505D13 in S. cerevisiae, which significantly improved the ST-3 production titer. The most effective strain produced 78.61 mg/L of ST-3 after 62 h fermentation, which was 6.43 times higher than that of our previous study. The present study demonstrated that ST-3 effectively inhibits the proliferation of NSCLC cells, and provides insight into its efficient bioproduction.

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Strains and culture

Escherichia coli DH5α (Tiangen Biotech, Beijing, China) was used as the cloning host. S. cerevisiae YL-T3 (16) and the engineered yeast strains were cultivated in SC-His, SC-His-Ura, YPD medium (17), or YPD medium supplemented with 20 g/L glycerol and different concentrations of G418 at 30°C and 220 rpm.

Purification of ST-1, ST-2, and ST-3

Purification of ST-1, ST-2, and ST-3 was performed as previously reported (12).

Cell lines and cell viability assay

Tumor cell lines (Table S1) were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) or RPMI1640 (Invitrogen) with 10%

ST-3 significantly inhibited the proliferation of NSCLC and glioma cells

We first compared the inhibition rates of ST-1, ST-2, and ST-3 together with squalene and (3S)-2,3-oxidosqualene ((3S)-2,3-OS) on the proliferation of NSCLC HCC827 cells (Fig. 1A). ST-1, ST-2, and ST-3 all inhibited the proliferation of these cells in vitro, among which ST-3 had the strongest effect. At a concentration of 10 μM, the inhibition rate of ST-3 on HCC827 cells was approximately 80%, which was nearly three times higher than that of ST-1 and ST-2. In contrast, squalene, (3S)-2,3-OS,

Acknowledgments

This work was supported by the National Key R&D Program of China (2018YFA0900600), the National Natural Science Foundation of China (nos. 31971344 and 31600071), Shanghai Municipal Natural Science Foundation (nos. 17ZR1448900 and 18ZR1420300), Interdisciplinary Program of Shanghai Jiao Tong University (YG2017MS82) and Construction Project of High Level Local Universities in Shanghai and Pharmacy (XD18011). We thank Prof. Jian-Jiang Zhong (Shanghai Jiao Tong University) for his helpful

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    The first two authors contributed equally to the work.

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