Biology subjects

Dang, T.-T. T.

Publications and source records attributed to Dang, T.-T. T..

3 recordsLinked to original sources

bioRxiv2025

Production of the anticancer drug intermediate strictosidinic acid in engineered yeast

Strictosidinic acid is a key intermediate in the biosynthetic pathway of camptothecin, a plant alkaloid that serves as a precursor for semisynthetic anticancer drugs. At the moment, camptothecin is mainly sourced from trees, causing limited supply and high costs. Improving access to strictosidinic acid would help to elucidate yet unknown biosynthetic steps and in the long term enable sustainable production of camptothecin in heterologous hosts. While structurally similar to the common monoterpene indole alkaloid precursor strictosidine, strictosidinic acid has not been the target of metabolic engineering efforts before. Here, we present a strategy to produce strictosidinic acid from glucose and tryptophan in engineered yeast. First, we create a basic strain that generates 75 mg/L strictosidine. We further optimise this strain by introducing a membrane steroid binding protein and a second copy of the farnesyl pyrophosphate synthase mutant gene ERG20WW, boosting strictosidine levels by 5.5-fold to 398 mg/L. At these higher titres, a previously overlooked shunt product, (2E,6E)-2,6-dimethylocta-2,6-dienedioic acid (DOA), was identified that diverts flux from the pathway. Lastly, we reprogrammed our strictosidine strain to strictosidinic acid production by four genomic modifications. Final fed-batch cultivation in shake flasks resulted in 843 mg/L strictosidine or 548 mg/L strictosidinic acid, respectively, after 168 hours. Taken together, our work now grants access to strictosidinic acid by metabolic engineering, while revealing strategies to further enhance the production of strictosidine and related monoterpene indole alkaloids. These findings will help to produce plant alkaloids in microbial cell factories in the future at scale.

bioengineering↗

bioRxiv2025

Single cell mass spectrometry reveals intercellular compartmentalization of camptothecin biosynthesis in the tree Camptotheca acuminata

The medicinal tree Camptotheca acuminata produces camptothecin, a monoterpenoid indole al- kaloid (MIA) precursor for several leading chemotherapeutic agents (Lorence and Nessler 2004). Alt- hough the biosynthesis of camptothecin remains poorly understood, a putative route has been hypothe- sized based on in planta metabolite profiling and feeding studies (Fig. 1a) (Sheriha and Rapoport 1976; Sadre et al. 2016). However, pathways proposed on whole-tissue or organ-level metabolomic and tran- scriptomic data lack resolution on the intricate, cell-specific compartmentalization of natural products biosynthesis. Such gaps could be addressed by single cell technologies, which have recently shown tremendous potential to transform gene discovery in herbaceous plants (Li et al. 2023; Zhan et al. 2023; Vu et al. 2024; Wu et al. 2024; McClune et al. 2025). Nevertheless, single cell mass spectrometry (scMS) has not been adapted for woody species, largely due to the challenges associated with their highly lignified tissue and complex cellular architecture. In this study, we developed an scMS pipeline for the woody tree C. acuminata to investigate the intercellular organization of camptothecin biosynthesis. O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/661346v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17f7710org.highwire.dtl.DTLVardef@161c469org.highwire.dtl.DTLVardef@1a5ee1borg.highwire.dtl.DTLVardef@b361b9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Single cell metabolomics of Camptotheca acuminata. (a) Proposed camptothecin biosynthetic pathway. (b) Schematic experimental pipeline. (c) Dot plots showing concentrations of key metabolites in single cells across organs. Each data point rep- resents a protoplast. For each metabolite, dots were offset from the center based on the distribution of concentrations among sin- gle cells. (N.D.: not detected). (d) Stacked bars showing concentrations in individual cells of loganic acid, secologanic acid, strictosidinic acid, strictosamide, pumiloside, and camptothecin). Cells are organized into groups with loganic acid only (A), secologanic acid only (B), loganic and secologanic (C), strictosidinic acid only (D), and other alkaloids (strictosamide, pumiloside, and camptothecin) (E). In each group, cells are ordered by total metabolite concentration with highest concentration in the center and lower producers closer to the edges. Panel widths scale with the number of cells in each group, and colours cor- respond to individual metabolites. C_FIG

biochemistry↗

bioRxiv2025

Single cell omics extends metabolic regulon via orthologous transcription factors from a pair of medicinal plant species

Camptotheca acuminata Decne is a woody medicinal tree that produces over a hundred bioactive compounds, including camptothecin, which has been used as the starting material to semi-synthesize many leading anticancer drugs (Lorence and Nessler 2004). Camptothecin and its derivatives are potent inhibitors of DNA topoisomerase I and are widely used for the treatment of lung, cervical, ovarian, and colon cancers. Camptothecin biosynthesis in C. acuminata involves complex catalytic steps, most of which remain undeciphered. In this pathway, tryptamine and secologanic acid are coupled, leading to strictosidinic acid. The formation of strictosidinic acid is catalyzed by strictosidine/strictosidine acid syn-thase enzymes (STR) (Fig. 1A). While a biosynthetic route for the conversion of the indole ring to the quinoline ring has been proposed, most of the underlying biosynthetic genes have yet to be identified (Fig. 1A) (Sadre et al. 2016). In addition, the cell type specificity of this pathway also remains undescribed. Here, we generated a single cell multiome (RNA-seq and Assay for Transposase Accessible Chromatin by sequencing [ATAC-seq] from the same nuclei) to probe the cell type specificity of camptothecin biosyn-thetic genes. O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/650021v2_fig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@81dcf0org.highwire.dtl.DTLVardef@1d03ae1org.highwire.dtl.DTLVardef@1c9b0a9org.highwire.dtl.DTLVardef@6d5238_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Single cell multi-ome of Camptotheca acuminata leaf. (A) The proposed biosynthetic pathway for camptotheci . Solid arrows indicate previously characterized enzymatic steps. Dashed arrows indicate proposed enzymatic steps (see Table S7 for gene name abbreviations). (B) Uniform Manifold Approximation and Projection (UMAP) of nuclei of the single nuclei RNA-seq dataset (n = 4,012), color coded by cell clusters. (C) Gene expression heatmap of MIA biosynthetic genes across cell clusters. Rows are expressed biosynthetic genes, which are ordered from upstream to downstream. Color scale shows the average scaled expression of each gene at each cell cluster. Cell clusters are sorted by cell types. Dot size indicates the percentage of cells where a given gene is detected. The predicted cell type for each cell cluster is annotated by the color strip below the x-axis. Box highlights expression of STR genes. (D) Heat map showing accessibility of cell type marker peaks across cell clusters. Each row is an ATAC-seq peak. Each column is a cell cluster. Color scale is maxed out at 90th percentile of normalized ATAC-seq signal. The predicted cell type for each cell cluster is annotated by the color strip below the x-axis, with the same color palette as (B). (E) Heatmap showing gene expression across cell clusters. Each row is a gene within 2-kb of a STR+ marker peak. Each column is a cell cluster. The predicted cell type for each cell cluster is annotated by the color strip below the x-axis, with the same color palette as (B). (F) DNA motif enriched in STR+ marker peaks, as well as a reference MYB motif. C_FIG

plant biology↗