本氏烟草悬浮细胞NBS-1的创建及底盘特征分析

Development and characterization of tobacco suspension cell chassis NBS-1

摘要:

植物合成生物学在植物天然产物的发掘制造方面具有明显的理论优势，但因缺乏高效的底盘系统及相关使能技术，其对生物合成领域的贡献有限。最常用的植物底盘——烟草由于操作周期长、改造难度大、代谢及纯化背景复杂、烟碱毒性和农业生产难精准控制等问题，而烟草悬浮细胞底盘体系可有效解决上述问题。本研究旨在开发出生长快速、生物量高、分散度好、转化效率高、烟碱含量极低并具备较高科研价值及工业化潜力的烟草悬浮细胞底盘。选取分子技术高适用性的本氏烟草(Nicotiana benthamiana)，诱导获得的悬浮细胞NBS-1生长迅速、分散性好，最高生物量可达到476.39 g/L (鲜重)，各项参数均远超常用的烟草BY-2 (Nicotiana tabacum L. cv Bright Yellow 2)细胞系。常用的pEAQ-HT高效瞬时表达系统在NBS-1中的转化表达效率可达81%，远高于BY-2。利用转录组数据对BY-2和NBS-1的代谢特征及偏好性进行分析，发现NBS-1的黄酮类等化合物合成通路基因的表达显著高于BY-2，且NBS-1的生物碱合成通路基因的表达显著低于BY-2，并通过代谢物含量测定实验验证了该分析结果，表明NBS-1极低烟碱含量的同时，为该底盘对应产品的选择提供参考。综上，本研究开发出的生长转化优异、黄酮类含量较高且烟碱含量极低的本氏烟草悬浮细胞底盘NBS-1，对于开发烟草悬浮细胞底盘有重要指导意义。

Abstract:

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.