On October 24, 2019, Wang Yong's research group of the Center for Excellence in Molecular Plant Science of the Chinese Academy of Sciences published a research paper titled "Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana" in the international academic journal Nature Communications. In this study, heterogeneous biosynthesis of paclitaxel's key intermediate, 5a-hydroxytaxadiene, was achieved for the first time in a plant chassis.

　　Paclitaxel is a rare diterpenoid compound with anticancer activity isolated from the yew bark. It is a clinical first-line drug widely used in the treatment of various cancers. The content of paclitaxel in the yew bark is only about one ten-thousandth of the dry weight. At present, it is mainly obtained by semi-synthetic methods that consume yew resources, which is far from meeting clinical needs. The use of synthetic biotechnology to construct an artificial biosynthesis system of paclitaxel is expected to solve this problem. Some progress has been made in the synthesis of paclitaxel intermediates with microbial chassis, but in recent years, this type of work has faced difficulties: the biosynthetic pathway of paclitaxel is extremely complicated, including 19 steps of biochemical reactions, and nearly half involve the participation of cytochrome P450 enzymes. Functional expression P450 enzymesis a huge challenge for microbial chassis. Compared with microbial chassis, plant chassis has obvious advantages in terms of membrane protein expression, precursor supply, product tolerance, and partitioned synthesis. Nevertheless, to date，only the first step of taxol synthesis has been successfully reported in plant chassis, and the paclitaxel intermediates involved in the cytochrome P450 enzyme have no successful precedent in plant chassis.

　　In this study, the researchers introduced taxadiene synthase (TS), taxadiene 5α-hydroxylase (T5H) and its reductase (CPR) into the Tobacco system. It was found by fluorescence confocal microscopy that these enzymes are located in different regions in the cell. This different partitioning is the key to the failure of the P450 enzyme to participate in the successful synthesis of paclitaxel intermediates in plant systems. Through the chloroplast partition engineering strategy, the authors carried out chloroplast localization and transformation of taxadiene-5α-hydroxylase and cytochrome P450 reductase to ensure the spatial consistency of the GGPP-TS-T5H / CPR metabolic pathway and successfully achieved 5α- Synthesis of hydroxytaxadiene with a yield of 0.9 μg / g fresh weight leaves. Further experiments to selectively block the MEP and MVA pathways have confirmed that the mother core of taxanes in the tobacco system is mainly derived from the MEP pathway. It is clear that DXS is an important engineering target for the synthesis of taxol in tobacco. GGPPS can increase the yield of taxadiene by a factor of 10 to 56 μg / g fresh weight, and the yield of 5α-hydroxytaxadiene to 1.3 μg / g fresh weight. This study provides a successful case based on plant chassis for the heterogeneous synthesis of complex natural products. The engineered tobacco system established provides the possibility to further analyze the unknown synthetic pathway of paclitaxel.

　　Professor Yong Wang，from the Center for Excellence in Molecular Plant Science of the Chinese Academy of Sciences，is the corresponding author of the paper. Dr. LiJianhua is the first author of the research paper. Foreign postdoc Ishmael Mutanda and Wang Kaibo, a graduate student from Henan University, participated in the research work. Special thanks to Researcher Wang Jiawei of the Center for Excellence in Molecular Plant Science of the Chinese Academy of Sciences and Researcher Yang Lei of the Shanghai Chenshan Botanical Garden Research Center for their help. The research was funded by the National Key R & D Program, Pioneer B of the Chinese Academy of Sciences, and the Shanghai Natural Science Foundation.

　　Link: https://www.nature.com/articles/s41467-019-12879-y 

　　Contact:

　　Dr. Yong Wang, Professor

　　Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences/Shanghai Institute of Plant Physiology and Ecology (SIPPE), Chinese Academic of Sciences

　　Tel: 86-21-54924295

　　Email: yongwang@sibs.ac.cn