2021 Annual Meeting
Metabolic Pathway Engineering of paclitaxel Biosynthesis in Taxus Chinensis Plant Cell Culture
Paclitaxel, a chemotherapeutic produced by the plant Taxus chinensis, is one of the most commonly used FDA-approved drugs for treatment of lung, breast, and ovarian cancer and one of few compounds industrially produced using plant cell culture. Commonly used approaches for increasing yield of paclitaxel in plant cell culture, such as methyl jasmonate elicitation, result in decreased cell growth and viability and production of undesired side products, such as phenolics and flavonoids. Thus, the objective of this research is to specifically increase production of paclitaxel via metabolic pathway engineering of the taxane biosynthetic pathway. First, a stable Agrobacterium infiltration-based transformation method for Taxus chinensis cell lines was established using a pCambia1300 binary vector expressing the GUS+ reporter gene. After optimizing transformation methods, over a 120-fold increase in GUS expression was observed for the Taxus chinensis cell line 48.82A.3s one week after Agrobacterium infiltration. Stable genomic integration of the selectable marker (hygromycin resistance) 3 months after transformation was also confirmed using a PCR screen on extracted genomic DNA. Next, four taxane biosynthetic pathway genes (BAPT, DBAT, DBTNBT, and TASY) were each overexpressed using the same pCambia1300 binary vector. After transformation, cells were treated with different concentrations of hygromycin (0, 2.5, 5, and 10 mg/L) to determine the minimum inhibitory concentration (MIC). Following two weeks of incubation, cell viability was measured using an Alamar blue assay, expression of taxane biosynthetic pathway genes was measured using RT-qPCR, and paclitaxel content was analyzed using ultra performance liquid chromatography (UPLC). Hygromycin was determined to be a suitable selection agent, with a 97% decrease in cell viability for wild type cells at 10 mg/L. Preliminary results from these experiments indicate that overexpression of DBTNBT resulted in a log2-fold increase in DBTNBT expression of over 16 and a 5.7-fold increase in paclitaxel production when normalized by viable cell count. In order to more successfully identify the transformed cells in these heterogeneous cell populations, we developed a non-destructive method for fluorescence screening of positive clones. A C-terminal mCherry tag was added to the aforementioned taxane biosynthetic pathway genes (along with a 10 amino acid linker) using Gibson assembly and these constructs were then transformed into Agrobacterium tumefaciens EH105 using electroporation. This novel non-destructive method will enable us to more easily develop stable homogeneous cell lines and improve our study of taxane biosynthetic pathway engineering.