Periodic Reporting for period 1 - Taxol biosynthesis (De nova of transcription regulators from taxol®-producing endophytic fungi)
Okres sprawozdawczy: 2021-09-01 do 2023-08-31
Globally, Paclitaxel/Taxol® is an essential anticancer drug and there is a high demand for Taxol but there is a low yield from Taxus species. Two hundreds of reports have been shown for the production of taxol in endophytic fungi. Finding strategies for producing cost-effective microbial taxol is imperative to the demand. The endophytic fungi offer several advantages and the ability to produce Taxol easily with very less prices by fermentation technology, but a lack of information about the Taxol biosynthetic pathway, involved transcription factors and their industrial utilization has remained elusive. We have made objectives cloning of TB genes and TFs involved in fungal Taxol biosynthesis, In the this project, WRKY, AP2 ERF (ortholog to Taxus plant) bHLH and Zn(dt) (putative) TFs were obtained from two different endophytic fungal cDNA for the first time. Finding the elicitor-induced expression of fungal TFs and production of fungal taxol, our study finding noted that elicitors such as salicylic acid, methyl jasmonate (MeJA), sodium benzoate etc., have effectively stimulated taxol biosynthetic gene (HMGR, GGPPS, TDS, Tα5OH and PAL) expression and the enhanced production of fungal taxol, which was analyzed using qRT-PCR and various chromatography techniques, respectively. We have successfully overexpressed fungal TFs DgbHLH and PmERF into E. coli BL21 cells and purification of recombinant TFs from E. coli extracts, separately, the fungal TB gene promoters and determination of their DNA-binding sites to the TF proteins is underway. The study demonstrates that the TFs play significant roles in elicitor induced taxol biosynthesis from fungi. In addition, this project highlights that promising targets Transcription factors and Taxol biosynthetic genes for genetic and metabolic engineering approaches to further enhance fungal Taxol and precursors in future.
We took advantage finding three Transcription factors (WRKY, AP2, ERF), two Taxol biosynthetic gene (TDS and GGPPS) sequences similar to Taxus plants, two putative taxol biosynthetic TFs (bHLH & Zn (df) ) and three putative TB (HMGA, Tα5OH and PAL) genes sequences were identified for the first time in taxol producing endophytic fungi using the diverse of modern analytical techniques to characterize the elicitor induced gene expression changes and their relationships with Taxol production. This project has successfully performed a molecular gene cloning of full-length Transcription factors (PmERF sequences 98% similar with bacteria, Taxus plant, Non Taxus (Glycine max) plant and this reports first time in P. microspora fungus. In addition, achieved the putative full length fungal TF DgbHLH gene involved in the expression of Taxol production. It was actively involved in finding the putative taxol biosynthetic gene promoter through the Illumina NGS platforms and different PCR methods. We have shown advanced UPLC/MS and HPLC techniques for the quantification of fungal taxol. This research achieved for the first time the putative transcription factors induced expression of taxol biosynthetic genes by qRT-PCR analysis in the presence of elicitors. Further, purification of two recombinant TF proteins (PmERF and DgbHLH) to explore cis and trans-acting elements on two putative taxol biosynthetic gene promoters are underway. It may involve state-of-the-art protein and synthetic DNA promoter binding measurements by electrophoretic mobility shift assay (EMSA). We require fungal transformation by over-expressing Taxol biosynthetic genes due to the absence of suitable transformation systems for these fungi. Taxol biosynthetic genes through whole genome sequencing; number of putative genes were achieved by genome annotation.
We have achieved through whole genome sequencing and PCR based approaches fungal Taxol biosynthetic transcription factors PmbHLH, DgbHLH and a series of putative taxol biosynthetic genes, gene promoters HMGR, GGPPS , TDS, Tα5OH and PAL. These genes are responsible for elicitor induced enhanced production of Taxol in the fungal liquid culture. It may lead to achieving potential (anti-cancer drug) fungal Taxol production in industrial applications. In addition, the metabolic engineering approaches offer a possible way to counteract fungal Taxol production and provide a remarkable benefit to society in future.