Getting to the bottom of Taxol biosynthesis by fungi

t Taxol(paclitaxel)is a highly-oxygenated diterpenoid natural product first isolated from the pacific yew tree(Taxus brevifolia).It is one of the most widely used anticancer drugs.Soon after the discovery of its unique mode of action and the resulting high demand,an extensive search was initiated for alternative sources to replace the slow-growing and scarce pacific yew.Thus far,however,Taxol and related compounds have only been found in the genus Taxus,which comprises a small number of slow-growing plants with a broad but generally isolated geographical distribution.In 1993,Stierle and colleagues reported the independent biosynthesis of Taxol in an endophytic fungus isolated from Taxus brevifolia,which resulted in more than 160 subsequent publications and patents addressing the biosynthesis of Taxol and related taxanes by microorganisms.The literature on fungal taxane synthesis contains numerous inconsistencies,prompting us to thoroughly re-examine Taxol biosynthesis in endophytic fungi associated with Taxus spp.Using a combination of phytochemistry,molecular biology and genome sequencing,we were unable to find any evidence for independent taxane biosynthesis in any of the endophytes,including the isolate described in the original publication(Taxomyces andreanae)and several more recent isolates from Taxus trees.Our findings therefore resolve a long-standing mystery concerning the evolution of a complex terpenoid biosynthetic pathway in two distantly-related organisms.

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.

Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves

Taxus leaves provide the raw industrial materials for taxol,a natural antineoplastic drug widely used in the treatment of various cancers.However,the precise distribution,biosynthesis,and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown.Matrix-assisted laser desorption/ionization–mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei,confirming the tissue-specific accumulation of different active metabolites.Single-cell sequencing was used to produce expression profiles of 8846 cells,with a median of 2352 genes per cell.Based on a series of cluster-specific markers,cells were grouped into 15 clusters,suggesting a high degree of cell heterogeneity in T.mairei leaves.Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways.According to the cell-type annotation,most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells;phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells(including the stomatal complex and guard cells);and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells.A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified,including MYB17,WRKY12,WRKY31,ERF13,GT_2,and bHLH46.Our research establishes the transcriptional landscape of major cell types in T.mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.