Study On Biosynthetic Regulation Of Phenolic Acids In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge (Dan-shen in Chinese), is a commonly used traditional Chinese medicine for improving body function (e.g. promoting circulation and improving blood flow), as well as for the treatment of angina pectoris, myocardial infarction and other cardiac symptoms. The phenolic acids including rosmarinic acid (RA) and lithospermic acid B (LAB) aroused scientists'interest in the last twenty years because of their notable pharmacological activities and the conventional use of herbs by decocting with water. LAB is a dimer of RA, and has been suggested to be derived from RA, but the detailed biosynthesis process has not yet been identified. S. miltiorrhiza provides a perfect plant material for elucidation of the unknown LAB biosynthesis pathway, in that compared with other Lamiaceae plants, it has especially high level in accumulating LAB.The biotechnological production of valuable secondary metabolites by elicitor treatment is an attractive alternative to the extraction of whole plant material, which is more economically feasible and environmental friendly. S. miltiorrhiza hairy root cultures provide a stable and efficient platform for active constituent accumulations by using elicitation with suitable biotic and abiotic elicitors. In this study, we found abotic elicitors including methyl jasmonate (MeJA) and silver ion (Ag+) could greatly stimulate the biosynthesis of phenolic acids at various levels in S. miltiorrhiza hairy root cultures.MeJA (0.1 mM) dramatically enhanced both RA and LAB accumulation, from approx. 3.25% to 6.02%, and 2.94% to 19.2% of dry weight, respectively. Several RA biosynthetic genes transcripts were coordinately induced, with phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) transcripts displaying the most rapid and substantial increases. Liquid chromatographic–tandem mass spectrometry (LC-MS/MS) was used to characterize the profile of metabolites involved in RA biosynthesis pathway, in both control and elicited-treated cultures.Further canonical correlation analysis constructed a gene-to-metabolite network, locating possible gene candidates which would directly link to phenolic acids (RA and LAB) production. Based on this relationship, several single-gene over-expression vectors containing C4H, TAT, HPPR respectively, a single-gene suppress-expression vector containing bypass-enzyme gene HPPD, as well as a double-gene co-expression vector containing both TAT and HPPR, were successfully constructed and transformed to Salvia miltiorrhiza hairy root. Results from levels determination of related gene expression and secondary metabolites accumulation showed all used metabolic regulation strategies activated RA biosynthesis pathway at different degrees, and successfully enhanced the target compounds (LAB and RA) content. The strategy that co-expression of TAT and HPPR, obtained the best results, dramatically enhancing LAB and RA accumulation to the extent of 11.5% to 10.5% of dry weight, respectively, which was 17.1 and 14.5 times higher than the control vector transformed hairy roots, respectively, also 3.3 and 4.4 times of non-vector transformed ones. This was the first report indicating phenolic acids biosynthetic pathway could be effectively activated by mean of metabolic engineering, thereby resulting in phenolic acids accumulations. This study provided an excellent example for realization of metabolic engineering breeding.Ag+ (15μM) did not stimulate RA accumulation but dramatically enhanced LAB from approx. 3.58% to 18.8% of dry weight, and their content was found perfectly competitive at each time point after treatment. Profiling analysis of genes and metabolites (intermediates) involved in RA synthesis pathway was performed, the result indicated the activation of this pathway well-corresponded to LAB production. Thus, a potential (putative) biosynthetic route from RA to LAB was presumed, which was suggested to be fantastically activated by Ag+ in S. miltiorrhiza hairy root cultures. Further intermediates monitoring and compound feeding experiments were applied to rank the strength of this hypothesis. Our study, for the first time, provided the evidence for that RA was precursor for leading to LAB synthesis.For the unique pharmacological properties of S. miltiorrhiza, RA formation provides an excellent model to investigate regulatory mechanisms of plant secondary metabolism because two parallel and presumably concertedly regulated pathways are involved in its biosynthesis. This study provided a new responsive model system to profile modulations in biosynthesis of important plant secondary metabolites, and will certainly help us to globally and deeply understand metabolic flux of RA synthesis, both at stressed-elicitation and genetic-regulation levels, also be a great motivation for bioengineering the secondary metabolics in S. miltiorrhiza.