Production of taxol from Lasiodiplodia theobromae and biochemical characterization of its 10-deacetylbaccatin III-10-O-acetyltransferase enzyme and evaluation of anticancer activity of enzymatically synthesized baccatin III
Taxol (paclitaxel) originally identified from the inner bark of Taxus brevifolia, is a clinically approved anticancer drug for the treatment of various kinds of cancer, particularly ovarian cancer, breast cancer and Kaposi's sarcoma. The mode of action of taxol is unique against cancer cells. It inhibits cell proliferation by preventing the depolymerization of tubulin during cell division. Taxol induces apoptosis by increasing the microtubule stability in the cell and finally leading to the apoptotic death of cancer cells. The increase in the number of cancer patients every year is leading to a higher demand for taxol, which can barely be met by the extremely low amounts of taxol produced by Taxus species plants, resulting in crisis of taxol. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the total dry weight. At commercial scale to purify 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000-3000 mature trees. Therefore, to meet the demand of taxol, alternative sources are continuously being searched and have been reported from multiple sources such as plant cell suspension methods, semi-synthesis, and chemical synthesis. However, these sources have their own limitations, forcing researchers to search for other sustainable alternative sources. In the last two and half decades, research suggests that endophytic fungi can be the potential alternative source for Taxol production. Totally, 25 Taxus plant-associated endophytic fungi and 24 non-Taxus plant-associated endophytic fungi producing Taxol have been reported (Gond et al., 2014). The taxol biosynthesis pathway contains approximately 19 enzymatic steps and has been studied extensively in the Taxus species. However, at the molecular level, the taxol biosynthesis pathway is poorly understood in endophytic fungi (Zhang et al., 2009). Several endophytic fungi isolated and characterized from Taxus and non-Taxus spp. plants are known to produce taxol in axenic cultures. The earlier report suggests that the taxol-producing strain Lasiodiplodia theobromae has been isolated from both Taxus and non-Taxus host plants. In search of Lasiodiplodia theobromae, taxol-producing strains from non-Taxus medicinal plants such as Piper nigrum, Zizypus jujube and Mesura ferrae were selected. In this study, totally eight strains of L. theobromea were obtained; among them one strain L. theobromea-1251 was found to produce 10-deacetylbaccatin III and taxol while two strains namely L. theobromea-SY and L. theobromea-1250 were found to produce 10-deacetylabaccatin III. All three strains identification was confirmed by the morphological characteristics and ITS rDNA sequence analyses. Further, L. theobromea-1251 isolated from non-Taxus Piper nigrum was selected in this study. Taxanes (Taxol, 10-deacetylbacctin III and Baccatin III) produced by L. theobromea were found to be identical to the authentic 10-deacetylbaccatin III and taxol as analyzed by TLC, HPLC, APCI-MS and MS/MS analyses. The quantity of taxol produced by the strain L. theobromea-1251 was estimated to be 247μg/L, and fungal taxol showed potent cytotoxic activity towards cancer cell line (Sah et al., 2017). The dbat (10-deacetylbaccatin III-10-O-acetyltransferase) is a key gene in the taxol biosynthetic pathway encoding the DBAT eznyme, which catalyzes the conversion of 10-deacetylbaccatin III (10-DAB) into baccatin III (Walker and Croteau, 2000). As an evidence to support the independent production of taxol by L. theobromea, the dbat gene was isolated from L. theobromae-1251 using nested PCR with primers designed from conserved amino acid and nucleotide sequences. Later, the dbat gene was cloned and sequenced for the first time. The obtained amino acids and nucleotide sequences of the L. theobromea and the plant DBAT show high homology to DBATs from the taxol-producing T. cuspidata plant as well as the endophytic fungus Aspergillus species. This study suggests that the dbat gene in L. theobromae might have evolved independently, and sheds light on the molecular basis of the taxol biosynthetic pathway in taxol-producing endophytic fungi. Further, the heterologous expression and purification of the LtDBAT enzyme were performed in E.coli which was cloned from L. theobromea-1251. The recombinant LtDBAT enzyme purified by affinity and gel filtration chromatography methods was shown to be capable of catalyzing 10-deacetylbaccatin III in the presence of acetyl CoA (radiolabeled) into baccatin III which was analyzed by densitometry methods. The biochemical properties such as optimum concentration, time, temperature and kinetic parameters (Km, Vmax, and Kcat) were evaluated for LtDBAT. Far UV CD (Circular dichroism) spectra of LtDBAT were analyzed in the absence and presence of both substrates (10-DAB and Acetyl CoA) and results indicate that both substrates bring substantial changes in the secondary structure of the recombinant LtDBAT protein. The localization of LtDBAT was performed using S. cerevisiae and microscopic observation suggests that LtDBAT is localized with lipid droplets. In addition, large-scale baccatin III was enzymatically synthesized using 10-DAB and cold acetyl CoA which was purified and characterized by mass spectroscopy. Further, the in vitro anticancer activity of ESB III was evaluated. Baccatin III was found to be cytotoxic and the cause of apoptotic cell death in different human cancer cells (Chakravarthi et al., 2013). ESB III showed cytotoxic and apoptosis-inducing activity in different human cancer cells. Cytotoxic activity was confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and propidium iodide (PI) staining. Further, ESB III induced cell cycle arrest leading to apoptosis in a ROS dependent manner through mitochondrial membrane dysfunction.
- Biochemistry (BC)