Background and Our solution
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 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, and the skin of its root is the only tissue to contain tanshinone. Furthermore, extraction efficiency of tanshinone is very low (1.23%-10.4%) (Table 2) (Zhang et al., 2013; Yang et al., 2006). Based on all these reasons, 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 & Design
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 is one of the most important key enzymes involved in synthesis of tanshinone (Figure 1). Without them, the synthesis of tanshinone would not have been possible.
Figure 1 The synthesis pathway of tanshinone (Wang and Wu, 2010)
Our objectives: SmCPS1 and the Artificial Synthesis of Tanshinone
Our project aims to synthesize the enzyme SmCPS1, the first and most important enzyme involved in synthesis of tanshinone. By combining various BioBricks and the gene SmCPS1, we designed the following gene circuit to synthesize SmCPS1 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.
The gene circuit includes a Tac promotor, which is a strong hybrid promotor produced by combination of promotors from the trp and lac operons and is used to control and increase the expression levels of the target gene.
Thrombin protease and TEV are the two type of restriction enzyme cleavage sites which can let SmCPS1 gene insert in between and later help the SmCPS1 enzyme to be easily purified. GFP was used to test the expression of our target gene SmCPS1. The target gene (SmCPS1) was obtained from Institute of Botany, Chinese academy of Science.
In our research, pGEX-KG was used as the vector of our Biobrick device, because of its unique advantages and characteristics that plays a vital role in transition of tanshinone expression system. pGEX-kG encompasses expression of Lacl inducible promoter, Ribosome binding sites (RBS), and Glutathione transferase GST tag. With the exploitation of Lacl inducible promoter, the E. coli are able to generate the target protein in short term without affecting the growth of the bacteria. In order to connect the SmCPS1 into the pGEX-kG vector, acquire the final protein to be without GST or GFP (green fluorescent protein) at same time, and furthermore, to purify the fusion protein and disconnect the upstream GST and downstream GFP, it is necessary that Protease Thrombin was added to the Enzyme cleavage site at front of the target gene as well as TEV protease adding at end of target gene.
Because SmCPS1 contains the same restriction enzyme cutting site as the biobricks provided by iGEM, we performed site-directed mutagenesis to avoid our target gene be digested by the restriction enzyme after we combine it with the plasmid backbone iGEM provided.
Here is the complete process of our work. Firstly, we obtained our target gene from Institute of Botany, Chinese Academy of Science. Then, we used PCR to amplify our target gene (SmCPS1). After we got enough of our target gene, we did three site-directed mutagenesis. After the mutagenesis was succeeded, the target gene was transformed into PGEX-KG vector for further propagation and production of the enzyme SmCPS1.
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