## 5 Biotechnological Applications  
Natural plant products are often produced in relatively low amounts and are difficult to chemically synthesize. Biotechnology is one of the most important ways to improve the active compounds of plants. Elucidating the secondary metabolites biosynthetic pathway is helpful for further revealing the mechanisms that produce bioactive components. Based on the biosynthetic pathway analyses, synthetic biology and metabolic engineering have been used to produce or improve the production of tanshinone and phenolic acid in both microorganisms and _S. miltiorrhiza_ or its hairy roots (Figure 3). The advances in recombinant DNA technology offered the opportunity of using synthetic biology to produce tanshinones and phenolic acid-related compounds in microorganisms. [PERSON] _et al._ applied a modular pathway engineering (MOPE) strategy for the rapid assembly of synthetic miltiradiene pathways in _S. cerevisiae_, using a fusion of the diterpene synthases _SmCPS1_ and _SmKSL1_ as well as the fusion of _BTS1_ (GGPPS) and _ERG20_ (farnesyl diphosphate synthase, FPPS). This approach led to a significant improvement in the production of miltiradiene and a reduction of byproduct accumulation. The miltiradiene yield of the most prolific strain reached 365 mg/L in a 15-L bioreactor culture [82]. Further study revealed that simultaneous overexpression of _tHMGR-upc2.1_ and _ERG20-BTS1-SaGGPS_ genes had a synergetic effect on the miltiradiene production in yeast strains, and 488 mg/L miltiradiene was produced in fed-batch fermentation [83]. Based on the establishment of miltiradiene production in _S. cerevisiae_, [PERSON] _et al._ engineered the yeast by incorporating modules expressing _CYP76 AH1_ as well as a P450 reductase gene  
Figure 3: Biotechnological applications in _Salvia miltiorrhiza_. (_SmCPRI_) into this efficient pathway, leading to the heterologous production of ferruginol at 10.5 mg/L under flask shaking conditions without process optimization [27]. As the biosynthetic pathway of tanshinones remains unsolved and most of the metabolic intermediates could rarely accumulate in plants, the engineered yeast strain provided a basis strain to identify additional enzymes. The production of RA in microorganisms has also been realized. [PERSON] _et al_. [84] designed a biosynthetic pathway tailored for the production of RA and isorinic acid (IA) in _Escherichia coli_ through the overexpression of a six-enzyme chimeric bacterial and plant pathway. They compared three plant RAS orthologues from _Coleus blumei_ (_CbRAS_), _Lavandula angustifolia_ (_LaRAS_) and _Melissa officinalis_ (_MoRAS_). The strains with different plant RAS genes produced different levels of RA and IA but in the same ratios, and the amount of RA and IA produced could also be increased by the optimization of another pathway. In _S. miltiorrhiza_, the analysis of phenolic acid biosynthesis could provide more candidate modules for pathway optimization. This work provided a microbial platform for the production of RA and IA that uses a modular biosynthetic approach to produce these compounds. The metabolic engineering of _S. miltiorrhiza_ or its hairy roots provides an alternative method for accelerating the production of tanshinones and phenolic acids by overexpression the key enzymes or suppression the competition pathway. _SmGGPPS_, _SmHMGR_, and _SmDXS_ were key enzymes involved in the biosynthesis of tanshinone precursors [17; 38; 48; 49; 50]. To increase the supply of precursors in _S. miltiorrhiza_, _SmGGPPS1_, _SmHMGR1_, and _SmDXS2_ were overexpressed in transgenic hairy root lines. It was revealed that the co-expression of _SmHMGR1_ and _SmGGPPS1_ resulted in the highest production of tanshinones (approximately 2.727 mg/g dw), which was approximately 4.74-fold higher than the production in the control (0.475 mg/g dw) [17]. The simultaneous overexpression of _SmHMGR_ and _SmDXR_ in _S. miltiorrhiza_ hairy roots also significantly increased the production of tanshinones, especially with elicitor-mediated induction [62].