Pan Liao

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.

Molecular mechanism of elicitor-induced tanshinone accumulation in Salvia miltiorrhiza hairy root cultures

To develop an optimal bioprocess for the production of tanshinone which is mainly used for the treatment of cardiocerebral vascular disease, the tanshinone biosynthetic pathway regulation must be better understood. In this paper, expression of tanshinone biosynthetic pathway related genes as well as tanshinone accumulation in Salvia miltiorrhiza hairy root cultures were investigated, in response to biotic and abiotic elicitors, respectively. Our results showed tanshinone accumulation in S. miltiorrhiza hairy roots was highly regulated by the coordination of the expression of several genes involved in tanshinone biosynthesis pathway. Our results showed a positive correlation between gene expression and tanshinone accumulation, suggesting that tanshinone accumulation may be the result of the coexpression up-regulation of several genes involved in tanshinone biosynthesis under the treatment of various elicitors. Meantime, SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS were identified as the potential key enzymes in the pathway for targeted metabolic engineering to increase accumulation of tanshinone in S. miltiorrhiza hairy roots. This is the first report integrating comprehensively the transcript and metabolite biosynthesis of tanshinone in S. miltiorrhiza hairy roots.

Molecular characterization and expression of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from Salvia miltiorrhiza

Abstract1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.

Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI-transformed hairy roots

Tropane alkaloids are used medicinally as anticholinergic agents with increasing market demand, so the improvement and production of active components from medicinal plants using molecular biotechnology show great potential for applications that should benefit human healthcare. Two tropinone reductases constitute a branching point in the biosynthesis of tropane alkaloids. In the present paper, we report for the first time the cloning and characterization of two fulllength cDNAs encoding TRI (tropinone reductase I) (GenBank® accession number EU424321) and TRII (tropinone reductase II) (GenBank® accession number EU424322) from the solanaceous plant Anisodus acutangulus by rapid amplification of cDNA ends. Sequence comparison indicated that AaTRI (A. acutangulus TRI) and AaTRII (A. acutangulus TRII) had high homology with other tropinone reductases from Hyoscyamus niger, Datura stramonium etc., but AaTRI and AaTRII showed identity of only 60.8%. Phylogenetic‐tree analysis showed that AaTRI and AaTRII belong to different clusters and have the closest relationship with H. niger TRI and TRII respectively. Expression‐pattern analysis showed that AaTRI and AaTRII were expressed in all tissues tested, including root, stem and leaf, but the transcript level of AaTRI was much lower than AaTRII. Expression of AaTRI and AaTRII could be enhanced by methyl jasmonate, with a weak effect for AaTRI and a strong effect for AaTRII. AaTRI‐transformed hairy‐root lines were accompanied by a mean 1.87‐fold higher level of hyoscyamine and a mean 8‐fold higher level of scopolamine compared with control roots, indicating that AaTRI is a promising target for genetic engineering to increase tropane alkaloid in A. acutangulus.

Molecular characterization and expression analysis of two distinct putrescine N‐methyltransferases from roots of Anisodus acutangulus

Putrescine N-methyltransferase (PMT; EC. 2.5.1.53) catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine to form N-methylputrescine, which was the first committed step in tropane alkaloid biosynthetic pathway. Two PMT cDNA clones [Anisodus acutangulus putrescine N-methyltransferase 1 (AaPMT1), GenBank Accession No. EU670745; AaPMT2, GenBank Accession No. EU670746] were obtained and characterized together from Anisodus acutangulus for the first time. The full-length cDNA of AaPMT1 was 1322 bp containing a 1014-bp open reading frame (ORF) encoding a polypeptide of 338 amino acids and AaPMT2 was 1219 bp containing a 1041-bp ORF encoding a polypeptide of 347 amino acids. Comparison of the deduced amino acid sequences of AaPMTs with those from tropane alkaloid-producing plants revealed that AaPMTs had high similarity with other plants PMT. Phylogenetic tree analysis displayed that AaPMT1 showed extensive homology with PMT from Anisodus tanguticus, and AaPMT2 had closer relationship with PMT2 from Atropa belladonna, which indicated PMTs belonged to PMT superfamily. Southern hybridization analysis of the genomic DNA revealed the occurrence of two PMT copies in A. acutangulus genome. Tissue expression pattern analysis revealed that AaPMT1 expressed strongly in roots, weakly in steams and leaves, besides, AaPMT2 presented a similar weaker trend. It indicated that AaPMTs were constitutive expression genes, which were the first reported tissue-independent PMT genes compared with other known PMT genes. AaPMT1 expression was upregulated by methyl jasmonate (MeJA) in all tissues, reaching the highest level after 24 h of the treatment. AaPMT2 also exhibited a very similar trend, whereas the expression was much weaker than that in AaPMT1. So, AaPMTs were considered to be MeJA elicitor-responsive genes and could be effectively elicited at least at the transcriptional level. The work would provide useful knowledge for tropane alkaloids biosynthesis and metabolic engineering to increase the production.

Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Camptotheca acuminata

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank accession no. EU677841) was successfully isolated from young leaves of C. acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp long and contained a 1413-bp open reading frame encoding a polypeptide of 471 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Southern hybridization analysis showed that there were at least two HMGS gene members in the C. acuminata genome. CPT content was found to be much higher in cotyledons and hypocotyls as compared to roots. RT-PCR analysis revealed strong expression in hypocotyls and cotyledons, but no expression in roots, indicating good correlation between CaHMGS expression and CPT content in the tested tissues. The expression of CaHMGS could be regulated by exogenous elicitors, including salicylic acid and methyl jasmonate, suggesting that CaHMGS was elicitor-responsive. This work is a first step to acquire a better understanding on the role of HMGS in CPT biosynthesis.