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Revision as of 07:32, 3 October 2016
Project
Background
Cardiovascular Diseases: the Protégés of Death
Caridovascular disease (CVD) is the No. 1 killer of human beings. On average, there are about 17.5 million people dying from CVD each year, which accounts for 31% of all deaths of human beings on Earth. Many more people are suffering from CVD or are at risk of developing it (Table 1).
Cardiovascular Diseases---the problem is serious. But how can we deal with it?
We can use tanshinone. One of the priorities of the WHO programme on Cardiovascular Diseases is the development of “cost effective and equitable health care innovations”. The health care for cardiovascular diseases includes various drugs, and tanshinone is one of the most important ingredients in multiple medicines for cardiovascular diseases, especially in China.
Tanshinone is a compound present in the rhizomes of Salvia miltiorrhiza, a plant known as Danshen in China. Salvia miltiorrhiza has positive effects on ischemic diseases, which has been proved by various clinical trials (Sze et al, 2005). This may be the result of its active component, tanshinone, acting as an inhibitor of angiotensin converting enzyme (ACE), hence leading to a decrease in blood pressure and blood clotting, as well as a dilation of arteries (Adams et al., 2006). An isomer of tanshinone, tanshinone II A, has been demonstrated to prevent atherogenesis, cardiac injuries and hypertrophy (Gao et al., 2012). Now, Salvia miltiorrhiza and the tanshinone extracted from it are clinically used as the prevention and treatment of cardiovascular diseases (e.g. coronary heart diseases) (Luo et al, 2015).
Extraction of Tanshinone from Salvia miltiorrhiza: Trials and Tribulations
The quality of Salvia produced through traditional cultivation degrades seriously, and the production costs are very high (Chen and Peng, 2006). Unfortunately, Salvia miltiorrhiza is the only source of tanshinone available in nature. Common extraction methods of tanshinone involve refluxing extraction, soxhlet extraction, ultrasound extraction and supercritical carbon dioxide extraction. But the extraction efficiency of all these methods is not ideal (Table 2) (Zhang et al., 2013; Yang et al., 2006). And since the concentration of tanshinone in Salvia miltiorrhiza is low in the first place, the production of tanshinone is far away behind the rising demand for it. Adding Ag+ and other nutrients may enhance tanshinone production in plants (Zhang et al., 2004), but such method cannot bring about fundamental changes to the situation.
Our solution
As a result, using synthetic biology to produce tanshinone should be a more ideal way for mass production, because comparing to traditional cultivation of Salviamiltiorrhiza, bacteria production would be much faster. Furthermore, using bacteria to produce tanshinone will also avoid the great difficulty in tanshinone extraction from Salviamiltiorrhiza. The product’s purity could be increased, and the production cost would be reduced.
Synthesis of Tanshinone in Salvia miltiorrhiza: A Blind Watchmaker
How did such a compound come into being? Who “designed” it and manufactured it? The credit belongs to Salvia miltiorrhiza…
The biosynthesis of tanshinone in Salvia miltiorrhiza is complicated.
Knowledge on the enzymes involved and techniques for manufacturing these enzymes artificially are crucial for synthesizing tanshinone outside the plant. Tanshinone is an abietanoid diterpene (Yang, 2013; Wang and Wu, 2010). All abietanoid diterpenes are derived from isopentenl diphosphate (IPP) and one of its isomers (Withers and Keasling, 2007). Two pathways, the mevalonate (MVA) pathway and the deoxyxylulose-5-phosphate (DXP) pathway, are involved in the synthesis of IPP (Lange et al., 2000). IPP will be converted to Geranylpyrophosphate (GPP), which marks the start of the downstream biosynthesis of tanshinone. At least five enzymes involved in this part of the pathway have been identified: SmFPS, SmGGPPS, SmCPS, SmKSL and SmCYP76AH1. SmFPS converts GPP to farnesyl pyrophosphate (FPP), and SmGGPPS converts FPP to geranylgeranylpyrophosphate (GGPP) (Liu et al., 2015). SmCPS then converts GGPP into copalyl diphosphate (CPP), which is converted by SmKSL into miltiradiene (Wang and Wu, 2010). Another enzyme, SmCYP76AH1 converts miltiradiene into ferruginol. The rest of the biosynthetic pathway remains unclear, though a number of speculations have been made (Yang, 2013). What is known for certain is that SmCPS and SmKSL are key enzymes involved in synthesis of tanshinone (Figure 1). Without them, the synthesis of tanshinone would not have been possible.
https://static.igem.org/mediawiki/igem.org/0/0c/T--CIEI-BJ--backgroundfigure1.jpg
Our objectives: SmCPS1 and the Artificial Synthesis of Tanshinone
Our project aims to synthesize SmCPS1 and SmKSL1, two of the key enzymes involved in synthesis of tanshinone. By combining various BioBricks and the gene SmCPS1/SmKSL1, we designed two gene circuits to synthesize SmCPS1 and SmKSL1 in E. coli. If the synthesis is successful, it will provide a better understanding of the tanshinone synthesis process in Salvia miltiorrhiza, and the enzymes we produced would contribute to the artificial production of tanshinone. Implications for the drug industry would include an increased purity, a reduced production time and a lower cost. With the help of this cheap and purified product of tanshinone, more lives would be saved.
Adams J D, Wang R, Yang J, Lien E J. Preclinical and clinical examinations of Salvia miltiorrhiza and its tanshinones in ischemic conditions. Chinese Medicine. 2006. 1:3.
Chen W, Peng L. Applications of the tissue culture of herbs. Shanxi Agricultural Science. 2006. 5: 62-65 (In Chinese)
Gao S, Liu Z, Li H, Liu P. Cardiovascular actions and therapeutic potential of tanshinone IIA. Atherosclerosis. 2012. 220(1): 3-10.
Kim SY, Moon TC, Chang HW, Son KH, Kang SS, Kim HP. Effects of tanshinone I isolated from Salvia miltiorrhiza Bunge on arachidonic acid metabolism and in vivo inflammatory responses. Phytotherapy Research. 2002. 16(7): 616-620.
Lange B M, Rujan T, Martin W, Croteau, R. Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes. Proceedings of the National Academy of Sciences of the United States of America. 2000. 97(24): 13172-13177.
Lee CY, Sher HF, Chen HW, Liu CC, Chen CH, Lin CS, Yang PC, Tsay HS, Chen J. Anticancer effects of tanshinone I in human non-small cell lung cancer. Molecular Cancer Therapeutics. 2008. 7(11): 3527-3538.
Liu LJ, Yang X, Peng YS, Zhao C, Liu C, Yu SQ, Hu XH, Wang RF. Research progress on key enzymes involved in biosynthesis of tanshinone. Chinese Traditional and Herbal Drugs. 2015. 46(1): 140-147. (in Chinese)
Luo J, Song W, Xu H, Chen K. Compound Danshen (Salvia miltiorrhiza) Dripping Pill for Coronary Heart Disease: An Overview of Systematic Reviews. The American Journal of Chinese Medicine. 2015. 43(1): 25-43.
Shan H, Li X, Pan Z, Li Z, Cai B, Zhang Y, Xu C, Chu W, Qiao G, Li B, Lu Y, Yang B. Tanshinone IIA protects against sudden cardiac death induced by lethal arrhythmias via repression of microRNA1. British Journal of Pharmacology.2009. 158(5): 1227-1235.
Zhang J, Quan Z, Fu J, Zhu Y, Liu F. Study of extraction of tanshinone IIA from Salvia miltiorrhiza via supercritical CO2. Chinese Traditional Patent Medicine. 2013. 35(6): 1329-1332.
Sze FK, Yeung F, Wong EM, Lau J. Does Danshen improve disability after acute ischaemic stroke. Acta Neurologica Scandinavica. 2005. 111(2): 118-125.
Wang JW, Wu JY. Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures. Appl Microbiol Biotechnol. 2010. 88: 437–449.
Withers ST, Keasling JD. Biosynthesis and engineering of isoprenoid small molecules. Applied Microbiology and Biotechnology. 2006. 73(5): 980-990.
Yang L. Biosynthesis of Tanshinone and Its Regulation in Salvia miltiorrhiza. Botanical Research. 2013. 2: 73-78. (in Chinese)
Yang G, Zhang J, Zhang L, Liu Y. Studying extraction methods of tanshinone IIA and crypototashinone from Radix salviae miltiorrhizae. Chinese Journal of Pharmaceutical Analysis. 2006. 12: 1807-1810.
Zhang C, Yan Q, Cheuk J. Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding. Planta Medica. 2004: 70(2): 147-151.
