Chunxian Yang

Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market.

An analytical pipeline to compare and characterise the anthocyanin antioxidant activities of purple sweet potato cultivars

Purple sweet potato (Ipomoea batatas L.) is rich in anthocyanin pigments, which are valuable constituents of the human diet. Techniques to identify and quantify anthocyanins and their antioxidant potential are desirable for cultivar selection and breeding. In this study, we performed a quantitative and qualitative chemical analysis of 30 purple sweet potato (PSP) cultivars, using various assays to measure reducing power radical-scavenging activities, and linoleic acid autoxidation inhibition activity. Grey relational analysis (GRA) was applied to establish relationships between the antioxidant activities and the chemical fingerprints, in order to identify key bioactive compounds. The results indicated that four peonidin-based anthocyanins and three cyanidin-based anthocyanins make significant contributions to antioxidant activity. We conclude that the analytical pipeline described here represents an effective method to evaluate the antioxidant potential of, and the contributing compounds present in, PSP cultivars. This approach may be used to guide future breeding strategies.

Engineering the MEP pathway enhanced ajmalicine biosynthesis

The 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR‐overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co‐overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes.

A new isopentenyl diphosphate isomerase gene from sweet potato: cloning, characterization and color complementation

Isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate and its isomer dimethylallyl diphosphate, which are the essential precursors for isoprenoids, including carotenoids. Here we report on the cloning and characterization of a novel cDNA encoding IDI from sweet potato. The full-length cDNA is 1155 bp with an ORF of 892 bp encoding a polypeptide of 296 amino acids, which was designated as IbIDI (GenBank Acc. No: DQ150100). The computational molecular weight is 33.8 kDa and the theoretical isoelectric point is 5.76. The deduced amino acid sequence of IbIDI is similar to the known plant IDIs. The tissue expression analysis revealed that IbIDI expressed at higher level in sweet-potato’s mature leaves and tender leaves than that in tubers, meanwhile, no expression signal could be detected in veins. Recombinant IbIDI was heterologously expressed in engineered Escherichia coli which led to the reconstruction of the carotenoid pathway. In the engineered E. coli, IbIDI could take the role of Arabidopsis IDI gene to produce the orange β-carotene. In summary, cloning and characterization of the novel IDI gene from sweet potato will facilitate our understanding of the molecular genetical mechanism of carotenoid biosynthesis and promote the metabolic engineering studies of carotenoid in sweet potato.

A new 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene encoding the committed-step enzyme in the MEP pathway from Rauvolfia verticillata.

Abstract 1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank® to be assigned with an accession number (DQ779286). The fulllength cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPHbinding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.

Molecular cloning and characterization of the Strictosidine synthase Gene from Rauwolfia verticillata

The strictosidine synthase (STR, EC 4.3.3.2) catalyzes the condensation of tryptamine and secologanin to form strictosidine, which is the universal precursor for a wide range of pharmaceutical terpenoid indole alkaloids (TIAs). The full-length cDNA encoding STR was cloned and characterized from Rauwolfia verticillata a Chinese native plant producing TIAs, such as reserpine and ajmalicine. The new cDNA was designed as RvSTR and submitted to GenBank to get an accession number DQ017054. The full-length cDNA of RvSTR was 1211 bp containing a 1035-bp open reading frame encoding a deduced 344-amino-acid polypeptide with a calculated mol wt of 38.2 kD and an isoelectric point of 5.19. Comparative and bioinformatic analysis revealed that RvSTR showed a higher similarity to STRs from Apocynaceae species, including Catharanthus roseus and Ophiorrhiza pumila, but a relatively lower similarity to other plant STRs. The unique essential catalytic residue Glu-309 was conserved in all alignment plant species. The phylogenetic analysis revealed that STRs were divided into two groups, including plant and bacterial enzymes. The tissue expression pattern analysis indicated that RvSTR expression could be detected in all tested organs of R. verticillata, including roots, stems, leaves, fruits, and flowers. The lowest transcription level was observed in flowers and the highest was found in fruits; subsequently, the order of transcription level decrease was stems > roots > leaves. The cloning and characterization of RvSTR give a new STR sequence involved in TIA biosynthesis of R. verticillata, and provide a candidate gene for metabolic engineering of the TIA pathway in R. verticillata.

Variation Laws of Anthocyanin Content in Roots and Their Relationships with Major Economic Traits in Purple-Fleshed Sweetpotato [Ipomoea batatas (L.) Lam]

Abstract Variation laws of anthocyanin content in root during the development and among the varieties, and their relationships with major economic traits in purple-fleshed sweetpotato [Ipomoea batatas (L.) Lam] were studied in the present article. The dynamics of 20 economic traits in 13 purple-fleshed sweetpotato varieties at 20, 40, 60, 80, 100, 120, and 140 d after their transplanting were investigated, and these traits included anthocyanin content in root, length of the longest vine, number of base branches, root number, dry matter contents in stem, foliage and root, fresh/dry weight of root, fresh/dry weight of stem, fresh/dry weight of foliage, fresh/dry weight of stem and foliage, fresh/dry weight of whole plant, and rations of photosynthate to root, stem, and foliage. The correlations between the variations of anthocyanin content and the other 19 economic traits among varieties and during the whole developing stages, and the correlations of daily increase of anthocyanin content with other 10 kinds of yields were analyzed. The results showed that: (1) During the whole development, the anthocyanin content had three variation types, i.e. a slow-increase type, a fluctuating-change type, and a deviousrising type, and had different responses to the growth of length of the longest vine, number of base branches, fresh/dry yield of root, and photosynthate allotments. (2) The anthocyanin contents among 13 varieties began to have significant difference after 20 d, and showed completed differentiation during 40–100 d, which had significantly negative correlationships with the number of base branches, fresh/dry yield of root, photosynthate allotment ratio to root, and had significant positive correlationships with dry matter content of root, length of the longest vine, fresh/dry yield of stem, dry yield of whole plant and photosynthate allotment ratio to foliage. (3) Because of the significantly negative correlation between daily increase of anthocyanin content and dry matter weight of root, the anthocyanin accumulation competed with dry matter accumulation for photosynthate in root, and the competitive relation was resolved in different ways in different purple-fleshed sweetpotato (PFSP) varieties. So, there had three variation types of anthocyanin content among PFSP varieties during their development, and had different correlations between these variations of anthocyanin content and the major economic traits.

Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant

Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of tropane alkaloids. With the purified recombinant SlTRI from Escherichia coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing tropane alkaloid biosynthesis in S. lurida via metabolic engineering.

A Root-Preferential DFR-Like Gene Encoding Dihydrokaempferol Reductase Involved in Anthocyanin Biosynthesis of Purple-Fleshed Sweet Potato

Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase (IbDHKR) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway.