Plant secondary metabolites have been increasingly used in the pharmaceutical industry. Camptothecin, a monoterpene indole alkaloid, is an important anti-cancer drug extracted from the plants - Camptotheca acuminata and Nothapodytes nimmoniana (native to south-east Asia), which have been excessively harvested to meet the ever-increasing market demand. As a promising alternative, a high camptothecin-yielding cell line of N. nimmoniana has been developed using a rational metabolic engineering approach. We reconstructed a genome-scale metabolic model for camptothecin production (NothaGEM iSM1809) and experimentally validated it. Biomass precursors determined experimentally were used to formulate the
N. nimmoniana - specific biomass objective function. We further used in silico tools to determine and rank suitable enzyme targets for over-expression and knock-out. Among the over-expression targets predicted, some of them include the camptothecin biosynthesis pathway enzymes such as strictosidine synthase and geraniol 10-hydroxylase reported earlier, and some novel targets in the plant’s metabolism, including reactions from the amino acid biosynthesis and tricarboxylic acid cycle. Among the top-ranked targets for knock-outs, reactions involving the formation of folates, serine, and conversion of acetyl CoA and oxaloacetate to malate and citrate have been predicted. One of the top-ranked over-expression targets, strictosidine synthase, was chosen to generate metabolically engineered cell lines of N. nimmoniana using Agrobacterium tumefaciens-mediated transformation. The transformed cell line showed a 5-fold increase in camptothecin production. We thus demonstrate maximizing camptothecin productivity by multiple folds through a rational metabolic engineering strategy, which can be used with minimal time and resources spent over hit-and-trial experimental approaches.

Publications:
1. Murali, S., Ibrahim, M., Rajendran, H., Shagun, S., Masakapalli, S. K., Raman, K., & Srivastava, S. (2023). Genome-scale metabolic model led engineering of Nothapodytes nimmoniana plant cells for high camptothecin production. Frontiers in Plant Science, 14, 1207218.
https://doi.org/10.3389/fpls.2023.1207218

2. Murali, S., Rajendran, H., Srivastava, S. (2021). Plant Cell Biofactories as In Vitro Production Platforms of the Anticancer Drug Camptothecin. In: Malik, S. (eds) Exploring Plant Cells for the Production of Compounds of Interest. Springer, Cham. https://doi.org/10.1007/978-3-030-58271-5_2