SmHPPR And SmRAS Yeast And The Construction Of Semi - Synthetic Rosmarinus

Salvia miltiorrhiza Bunge, a perennial herb of Labiatae, is one of most important traditional Chinese medicine (TCM) in China. Water-soluble phenolic compounds are one of the main active ingredients in S. miltiorrhiza. Due to the root water decoction is the traditional medications in TCM, researches on water-soluble components have drawn considerable attention in recent years, Rosmarinic acid (RA), and its downstream products, salvianolic acid B (Sal B), exhibited remarkable biological activities, such as anti-tumor, anti-liver damage and cardiovascular protection. Therefore, studies on RA synthesis pathway help us make rational utilization of its medicinal resources and clarify metabolic pathway of downstream complex phenolic acids. It is reported that S. miltiorrhiza shares similar RA synthesis pathway with Coleus blumei Benth, however, the detail evidences are lack. The fuction of some genes in this pathway has been studied, such as SmPAL, SmC4H, Sm4CL, SmTAT and so on. However, protein activites of SmHPPR and SmRAS are still unclear.It is well studied that the recombinant microorganisms heterologously ferment and produce plant secondary metabolic products (PSMPs) by integrating related genes on plant secondary metabolic pathways, which work as bioreactors. This method makes up the shortcomings of traditional plant extracts and chemical synthesis, which does not require a lot of plant material and tedious synthetic steps. It can produce a plenty of native conformation PSMPs with low cost and high efficiency. Here we heterologously produced SmHPPR and SmRAS proteins in Pichia pastoris, and detected their functions in RA synthesis pathway in vitro. In addition, we constructed engineering P. pastoris strains which contain SmTAT, SmHPPR and SmRAS by genetic engineering techniques for heterologously producing RA in the future.Main results and conclusions are as follows.1) The open reading frame (ORF) of SmTAT, SmHPPR and SmRAS were cloned (1236 bp,942 bp and 1284 bp, respectively). Intracellular expression vector pPICZB-SmHPPR and extracellular expression vector pPICZaA-SmHPPR were successfully constructed, in which SmHPPR fused with 6×HIS to construct. We also successfully constructed the intracellular expression vector pPICZB-SmRAS and extracellular expression vector pPICZaA-SmRAS, in which SmRAS fused with 6XHIS.2) Optimizing electroporation conditions with the highest conversion efficiency of P. pastoris X-33, the optimizing electroporating pulse was 750 V,0.4 ms×25 pulse with 1 mm-gap cuvette. The highest transformation rate was 1.57×102. We obtained 4 no-load strains,3 recombinant strains containing SmHPPR, and 4 recombinant strains containing SmRAS by transforming X-33. In this study, we optimized protein inducing conditions. The optimum pH of SmHPPR protein is 6.5. Best methanol dosage to express SmHPPR protein is 0.5%. SmHPPR protein band from S. miltiorrhiza was around 70 kDa, which existed as dimers. SmRAS protein expressed only at pH7.0. Best methanol dosage to express SmRAS protein is 0.5%. Purification product of SmRAS protein showed two bands. One is about 70 kDa, and the other is about 80 kDa. It may be glycosylated in different degrees when secreted.3) Detecting the product of SmHPPR enzyme by HPLC. The added precursor, 4-hydroxyphenylpyruvic acid, was almost exhausted and undetectable. However, the expected product,4-hydroxyphenyllactic acid, was not accumulated. We speculated that an unknown product might be synthesized, or the protein was not folded properly when secreted, which resulted in failing to form product and release substrate.4) Constructing three-gene co-expression vectors by two ways, three expression cassettes strategy and self-cleaving 2A strategy, which named pPICZB-SmRAS-SmTAT-SmHPPR-3K and pPICZB-SmHPPR-SmRAS-SmTAT-2A vectors, respectively. P. pastoris X-33 transformed with of the above vectors, could express SmTAT, SmHPPR and SmRAS, which encodes the key enzyme of tyrosinase branch. A new product with a retention time of 18.3 min was detected by HPLC. Results indicated that the RA synthesis pathway in S. miltiorrhiza is not exactly the same as that of Coleus blumei Benth. Unknown steps may exsit between SmHPPR and SmRAS.In this study, SmHPPR and SmRAS proteins were successfully heterologously secreted by P. pastoris. Engineering strains were acquired in two ways, which expresses gene SmTAT, SmHPPR and SmRAS of tyrosinase pathway. An unknown product was detected in recombinant strains, which provides important information for future research for engineering RA synthesis pathway and lays a foundation for subsequent exploration of RA heterologous synthesis. In conclusion, a new strategy for studying water-soluble phenolic acid biosynthesis was developed. Our study provides the basis for heterologous production and possibility for large-scale industrial production of medicinal phenolic acids.