¹³C Tracer Reveals Phenolic Acids Biosynthesis in Hairy Root Cultures of Salvia miltiorrhiza

Abstract

Rosmarinic acid (RA) and lithospermic acid B (LAB) are two typical phenolic acids with significant bioactivities that may contribute to the therapeutic effects of Salvia miltiorrhiza. Precise knowledge of the biosynthetic pathway leading to RA and LAB is a necessary prerequisite to optimize the production of important phenolic compounds in S. miltiorrhiza. In vivo isotopic labeling experiments using [ring-(13)C]-phenylalanine, combined with dynamic measurements of metabolite levels by UPLC/Q-TOF, were used to investigate the metabolic origin of phenolic acids in S. miltiorrhiza. These data indicate the in vivo phenolic biosynthetic pathway: two intermediates from the general phenylpropanoid pathway and the tyrosine-derived pathway, 4-coumaroyl-CoA and 3,4-dihydroxyphenyllactic acid (DHPL), are coupled by the ester-forming enzyme rosmarinic acid synthase (SmRAS) to form 4-coumaroyl-3',4'-dihydroxyphenyllactic acid (4C-DHPL). The 3-hydroxyl group is introduced late in the pathway by a cytochrome P450-dependent monooxygenase (SmCYP98A14) to form RA. Subsequently, RA is transformed to a phenoxyl radical by oxidation, and two phenoxyl radicals unite spontaneously to form LAB. The results indicate aspects of the complexity of phenolic acid biosynthesis in S. miltiorrhiza and expand an understanding of phenylpropanoid-derived metabolic pathways. The candidate genes for the key enzymes that were revealed provide a substantial foundation for follow-up research on improving the production of important phenolic acids through metabolic engineering in the future.

Hydroxycinnamoyltransferases in plant metabolism

“…Several putative ''RAS'' sequences from Perilla frutescens or Salvia miltiorrhiza can be found in the databases, but none of them has been proven to have RAS activity on biochemical level. One putative SmRAS gene/enzyme (accession numbers ADA60182, D6BR39), however, was shown to influence RA accumulation in RNAi suppression experiments (Di et al 2013). The RAS protein from Lavandula angustifolia differs from the others by an additional stretch of 29 or 32 amino acid residues compared to the proteins from Coleus blumei and Melissa officinalis (Landmann et al 2011).…”

SmMYB36, a Novel R2R3-MYB Transcription Factor, Enhances Tanshinone Accumulation and Decreases Phenolic Acid Content in Salvia miltiorrhiza Hairy Roots

“…For example, AtMYB123 (TT2) 33 , HvMYB10 34 , DkMYB2 35 , LjTT2c 36 , PpMYBPA1 37 , OgMYB1 39 , PtMYB134 40 and FaMYB11 41 belong to subgroup 5 according to their structures, and all function as activators to promote proanthocyanidin accumulation 26 . AtMYB75 (also called AtPAP1) 11, 42 , AtMYB90 (also called AtPAP2) 43 , PURPLE 44 , AtMYB113 43 , GMYB10 45 , VIMYBA1-3 46 , GmMYB10 47 , CsRUBY 48 , PcMYB10 49 , PyMYB10 50 , AN2 51, 52 , FaMYB10 53 , NtAN2 54 and AtMYB114 43 , belonging to subgroup 6, could act as activators to regulate anthocyanidin biosynthesis 26, 30 . Therefore, it is possible to analyse the structure by discussing the function of MYB.…”

Engineering microbial cell factories for the production of plant natural products: from design principles to industrial-scale production

“…Traditionally, reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE) [10–12], RNA interference (RNAi) [13, 14], virus-induced gene silencing (VIGS) [15] and isotopic tracer methods [16] had been used to uncover novel genes in the biosynthetic pathways of PNPs. However, these technologies are usually time-consuming, laborious, and consequently expensive.…”