Background
Danshen, derived from the root of Salvia miltiorrhiza, is a widely utilized class of traditional Chinese herbal medicines. Its primary applications lie in the treatment of cardiovascular and cerebrovascular diseases, earning it the accolade of "possessing the efficacy of four herbs in a single flavor of danshen." Pharmacological studies have elucidated its active components, comprising primarily fat-soluble diterpene quinone compounds, exemplified by tanshinones, and water-soluble phenolic acid compounds, led by salvianolic acids [1]. Amongst these, salvianolic acids play a pivotal role in promoting blood circulation and eradicating blood stagnation. They are predominantly found in plants belonging to the Labiatae and Ziziphus families. Researchers have isolated approximately 40 monomer compounds, such as caffeic acid, danshensu, rosmarinic acid, and salvianolic acid B, each exhibiting a diverse array of pharmacological effects, including anti-platelet aggregation, antithrombotic properties, enhancement of microcirculation, tissue repair promotion, free radical scavenging, and anti-lipid peroxidation [2].
In S. miltiorrhiza, the salvianolic acid biosynthesis pathway (Figure 1) primarily utilizes 4-coumaroyl coenzyme A, derived from the phenylalanine pathway, as the acyl donor, and 4-hydroxyphenyllactic acid or danshensu, stemming from the tyrosine pathway, as the acyl acceptor. This process is catalyzed by rosmarinic acid synthetase (RAS) and cytochrome P450 subfamily 98A monooxygenase (CYP98A), ultimately leading to the formation of rosmarinic acid and further complex phenolic acids [3]. By delving into the metabolic engineering and synthetic biology of these active ingredients, we can significantly mitigate the reliance on cultivation and collection pressures for danshen resources, while also enhancing production efficiency and utilization rates. This approach, which involves reconfiguring and optimizing the biosynthetic pathways of phenolic acids in microbes or plant cells, offers an innovative strategy for the sustainable development and utilization of medicinal plants like S. miltiorrhiza. Additionally, it aids in reducing ecological disturbances, safeguarding biodiversity, and fostering growth in the pharmaceutical and healthcare industries, thereby exhibiting immense scientific value and application potential.
Figure 1 The biosynthesis pathway of salvianolic acid in S. miltiorrhiza [3].
Our Strategy
In synthetic biology, host cells serve as vital biological platforms for the design, construction, and expression of artificial biological components, circuits, and even comprehensive biological systems. Common host cells, including Escherichia coli, yeasts, mammalian cells, plant cells, and specific microorganisms, are carefully selected based on factors like the complexity of the desired product, the necessity for post-translational modifications, product toxicity, and the intended production scale. This project aims to reconstruct and optimize the biosynthetic pathway of salvianolic acids, utilizing tobacco (N. benthamiana) leaves as the host (Figure 2). Tobacco offers several distinct advantages in synthetic biology studies involving plant active ingredients [4-5]. Firstly, it inherently contains a diverse array of biologically active components and possesses a rich metabolic network for synthesizing multiple secondary metabolites, laying a solid foundation for enhancing or creating new metabolic pathways through metabolic engineering to produce specific active ingredients. Secondly, tobacco demonstrates a robust capacity for heterologous protein expression, enabling high-level expression of complex plant secondary metabolites like phenolic acids, flavonoids, alkaloids, and other active components in leaves, seeds, or other tissues, making it highly suitable for industrial production. Lastly, tobacco cells are large and receptive to exogenous microorganisms, simplifying and streamlining the introduction of exogenous genes via Agrobacterium-mediated genetic transformation and other methods. Additionally, tobacco is among the first plants to have undergone a successful genetic transformation, boasting a well-established genetic manipulation technology system.
Figure 2 The morphology of tobacco (N. benthamiana).
In this project, tobacco was selected as the host system, leveraging the 4-coumaroyl-coenzyme A generated through its phenylalanine pathway as the acyl donor. We introduced exogenously danshensu + SmRAS + SmCYP98A14 or 4-hydroxyphenyllactate acid + SmRAS + SmCYP98A14 + SmCYP98A75 to generate rosmarinic acid, aiming to reconfigure the salvianolic acid pathway. Through comparing the yields of rosmarinic acid under these two approaches, we determined the optimal exogenous recombination conditions, laying a solid foundation for the sustainable development and utilization of danshen.
References
[1]Li ZM, Xu SW, Liu PQ. Salvia miltiorrhiza Burge (Danshen): a golden herbal medicine in cardiovascular therapeutics. Acta Pharmacol Sin. 2018, 39(5):802-824. doi: 10.1038/aps.2017.193.
[2]Xiao Z, Liu W, Mu YP, Zhang H, Wang XN, Zhao CQ, Chen JM, Liu P. Pharmacological Effects of Salvianolic Acid B Against Oxidative Damage. Front Pharmacol. 2020, 11:572373. doi: 10.3389/fphar.2020.572373.
[3]Zhou Z, Feng J, Huo J, Qiu S, Zhang P, Wang Y, Li Q, Li Y, Han C, Feng X, Duan Y, Chen R, Xiao Y, He Y, Zhang L, Chen W. Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis. Plant Biotechnol J. 2024, 22(6):1536-1548. doi: 10.1111/pbi.14284.
[4]Zhu X, Liu X, Liu T, Wang Y, Ahmed N, Li Z, Jiang H. Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells. Plant Commun. 2021, 2(5):100229. doi: 10.1016/j.xplc.2021.100229.
[5]Wang HT, Wang ZL, Chen K, Yao MJ, Zhang M, Wang RS, Zhang JH, Ågren H, Li FD, Li J, Qiao X, Ye M. Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants. Nat Commun. 2023, 14(1):6658. doi: 10.1038/s41467-023-42393-1.