Ginsenosides, the predominant bioactive components of Panax species, are biosynthesized by glycosylation at C3–OH and/or C20–OH of protopanaxadiol (PPD), and C6–OH and/or C20–OH of protopanaxatriol (PPT). Dammarenediol-II (DM), the direct precursor of PPD, has two hydroxyls at C3 and C20 positions, but DM glucosides have scarcely been identified from Panax species. Herein, we used two crude recombinant UDP-glycosyltransferases (UGTs), PgUGT74AE2 and UGTPg1 from Panax ginseng, to catalyze the glycosylation of DM with UDP-glucose (UDPG) as the sugar donor to produce DM glucosides 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (3β-O-Glc-DM) and 20-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (20S-O-Glc-DM), respectively. The in vitro and in vivo assays demonstrated that both 3β-O-Glc-DM and 20S-O-Glc-DM exhibited higher anti-colon cancer activities than natural ginsenosides. In order to produce DM glucosides in an economical, efficient and convenient way, we refactored the complete biosynthetic pathways of 3β-O-Glc-DM and 20S-O-Glc-DM by introducing the codon-optimized genes encoding DM synthase (DS) together with PgUGT74AE2 or UGTPg1 into Saccharomyces cerevisiae, respectively. Furthermore, multistep metabolic engineering strategies were applied, including optimization of chassis cell, multi-copy integration of heterologous genes via the CRISPR/Cas9 system, increase of precursor supply by overexpressing rate-limiting enzymes, down-regulation of the competitive pathway to redirect the metabolic flux towards the target products, and overexpression of the transcriptional activator. Finally, the titers of 2.4 g L⁻¹ 3β-O-Glc-DM and 5.6 g L⁻¹ 20S-O-Glc-DM were achieved through fed-batch fermentation in a 3 L bioreactor. This is the first study to demonstrate the anti-colon cancer activities of DM glucosides and to achieve the de novo biosynthesis of DM glucosides with high titers in microbial cell factories. This study has established a green and sustainable approach for the industrial production of DM glucosides, which provides promising candidates for new drug research and development.

Quinomycin G (1), a new analogue of echinomycin, together with a new cyclic dipeptide, cyclo-(l-Pro-4-OH-l-Leu) (2), as well as three known antibiotic compounds tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated from Streptomyces sp. LS298 obtained from a marine sponge Gelliodes carnosa. The planar and absolute configurations of compounds 1 and 2 were established by MS, NMR spectral data analysis and Marfey’s method. Furthermore, the differences in NMR data of keto-enol tautomers in tirandamycins were discussed for the first time. Antibacterial and anti-tumor activities of compound 1 were measured against 15 drug-sensitive/resistant strains and 12 tumor cell lines. Compound 1 exhibited moderate antibacterial activities against Staphylococcuse pidermidis, S. aureus, Enterococcus faecium, and E. faecalis with the minimum inhibitory concentration (MIC) values ranged from 16 to 64 μg/mL. Moreover, it displayed remarkable anti-tumor activities; the highest activity was observed against the Jurkat cell line (human T-cell leukemia) with an IC₅₀ value of 0.414 μM.

Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy. However, metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity. Herein, choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types, and a choline-modified strategy for small molecule-drug conjugates (SMDCs) design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy, instead of directly inhibiting choline metabolism. As a proof-of-concept, choline-modified SMDCs were designed, screened, and investigated for their druggability in vitro and in vivo. This strategy improved tumor targeting, preserved tumor inhibition and reduced toxicity of paclitaxel, through targeted drug delivery to tumor by highly expressed choline transporters, and site-specific release by carboxylesterase. This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy.

Construction of engineered yeast for de novo synthesis of unnatural ginsenoside 12β- O -Glc-PPD.