[Development and characterization of tobacco suspension cell chassis NBS-1]

Plant synthetic biology has significant theoretical advantages in exploration and production of plant natural products. However, its contribution to the field of biosynthesis is currently limited due to the lack of efficient chassis systems and related enabling technologies. Synthetic biologists often avoid tobacco as a chassis system because of its long operation cycle, difficulties in genetic and metabolic modification, complex metabolism and purification background, nicotine toxicity, and challenges in accurately controlling for agricultural production. Nevertheless, the tobacco suspension cell chassis system offers a viable solution to these challenges. The objective of this research was to develop a tobacco suspension cell chassis with high scientific and industrial potential. This chassis should exhibit rapid growth, high biomass, excellent dispersion, high transformation efficiency, and minimal nicotine content. Nicotiana benthamiana, which has high applicability in molecular technology, was used to induce suspension cells. The induced suspension cells, named NBS-1, exhibited rapid growth, excellent dispersion, and high biomass, reaching a maximum biomass of 476.39 g/L (fresh weight), which was significantly higher than that of BY-2. The transformation efficiency of the widely utilized pEAQ-HT transient expression system in NBS-1 reached 81%, which was substantially elevated compared to BY-2. The metabolic characteristics and bias of BY-2 and NBS-1 were analyzed using transcriptome data. It was found that the gene expression of pathways related to biosynthesis of flavonoids and their derivatives in NBS-1 was significantly higher, while the pathways related to alkaloid biosynthesis were significantly lower compared to BY-2. These findings were further validated by the total content of flavonoid and alkaloid. In summary, our research demonstrates NBS-1 possesses minimal nicotine content and provides valuable guidance for selecting appropriate chassis for specific products. In conclusion, this study developed NBS-1, a tobacco suspension cell chassis with excellent growth and transformation, high flavonoid content and minimal nicotine content, which has important guiding significance for the development of tobacco suspension cell chassis.