Molecular cloning and functional characterization of multiple ApOSCs from Andrographis paniculata.

Triterpenoids have been described in Andrographis paniculata. Oleanolic acid exhibits high biological activity and is widely used in the clinic, and ß-sitosterol not only has good biological activity but also plays an important physiological role in plants. However, analysis of the biosynthetic pathway of triterpenoids in Andrographis paniculata has not been reported. Here, we provide the first report of the isolation and identification of nine 2, 3-oxidosqualene cyclases (ApOSC3 to ApOSC11) from A. paniculata. The results showed that ApOSC4 represented a monofunctional synthase that could convert 2, 3-oxidosqualene to ß-amyrin. ApOSC5 as a bifunctional 2, 3-oxidosqualene cyclases, could transfer 2, 3-oxidosqualene to ß-amyrin and α-amyrin. ApOSC6 to ApOSC8 composed the multifunctional 2, 3-oxidosqualene cyclases that could convert 2, 3-oxidosqualene to ß-amyrin, α-amyrin and one or two undetermined triterpenoids. This study provides a better understanding of the biosynthetic pathway of triterpenoids in A. paniculata, and the discovery of multifunctional 2, 3-oxidosqualene cyclases ApOSC5 to ApOSC8 of the facilitates knowledge of the compounds diversity in A. paniculata.

Molecular cloning and functional characterization of multiple geranylgeranyl pyrophosphate synthases (ApGGPPS) from Andrographis paniculata.

KEY MESSAGE: We found that ApGGPPS1, ApGGPPS2, and ApGGPPS3 can convert IPP and DMAPP to GGPP. ApGGPPS2 is probably involved in andrographolide biosynthesis. ApGGPPS3 may be responsible for the synthesis of the cytosolic GGPP. Andrographis paniculata is a traditional herb for the treatment of sore throat, flu, upper respiratory tract infections and other disorders. In A. paniculata, GGPP is not only the precursor of andrographolide and its primary bioactive compounds, but also the precursor of chlorophylls, carotenoids, gibberellins, and abscisic acid, which are the biomolecules of photosynthesis, growth regulation and other physiological and ecological processes. In this study, four cDNAs (named ApGGPPS1, ApGGPPS2, ApGGPPS3 and ApGGPPS4) encoding geranylgeranyl pyrophosphate synthases from A. paniculata were putatively isolated. Bioinformatic and phylogenetic analyses suggested that these ApGGPPS are highly similar to the geranylgeranyl pyrophosphate synthases in other plants. Prokaryotic expression showed that ApGGPPS1, ApGGPPS2 and ApGGPPS3 could convert IPP and DMAPP to GGPP, although ApGGPPS4 lacks a similar function. The expression of ApGGPPS2 was similar as ApCPS2 under MeJA treatment, ApCPS2 involved in the biosynthesis pathway of andrographolide (Shen et al., Biotechnol Lett 38:131-137, 2016a), has been proven through Virus-induced Gene Siliencing (VIGS) (Shen et al., Acta Bot Boreal 36:17-22, 2016b), and the subcellular localization of ApGGPPS2 was shown to localize in the plastid, suggested that ApGGPPS2 could be the key synthase in the biosynthesis pathway of andrographolide. In addition, ApGGPPS3 was shown to localize in the cytoplasm, suggested that ApGGPPS3 may be responsible for the synthesis of cytosolic GGPP, which may participate in the synthesis of cytosolic oligoprenols as side chains to produce ubiquinone, dolichols or other isoprenoids, in the synthesis of polyisoprenoids, and in protein prenylation.

The experimental study of shunt-decompression arterialized vein flap.

BACKGROUND: Arterialized vein flap is a kind of unphysiological flap. Unphysiological reconstruction of blood circulation leads to higher load than that supported by physiological flap and is the culprit of flap swelling, blood stasis, skin blistering, and necrosis after flap grafting. To resolve the multiple disadvantages of traditional flap grafting, by introducing the principles of fluid mechanics, shunt-decompression surgery is prepared to decline the circulation preload and improve the prognosis of arterialized vein flap grafting. METHODS: By introducing the principles of fluid mechanics, we established the model of shunt-decompression arterialized vein flap, which satisfied the common properties of general fluid that the interface pressure between object and fluid is reduced when the velocity of fluid is increased and vice versa-the effect of Bernoulli. Under this rule, we anastomose the arterialized vein to the branch of main artery of recipient region or make end-to-side anastomosis, which can maintain the blood flow of main artery, decrease the perfusion of flap, and preserve the decompressive effect of main artery to branches. From March, 2016 to September, 2016, we performed animal experiments on ten male bama mini pigs with average weight of 28 ± 2.35 kg. Superior epigastric artery of pig was used for feeding artery to arterialize the superficial epigastric veins. The total area of flap is 8 cm × 6 cm. End-to-side anastomosis and end-to-end anastomosis were established in experimental group and control group, respectively. Doppler speckle perfusion imaging apparatus was used to monitor the alterations of flap perfusion, blood flow of flap, tissue swelling and survival of flaps. RESULTS: The average flap perfusion (PU) at 1 week after surgery is 83.62 ± 3.14 in experimental group and 98.14 ± 6.54 in control group, respectively (P < 0.05), indicating the significant reduction of flap blood perfusion in experimental group as compared with control group. As to the survival of flaps, 7 flaps completely survived, 3 showed partial necrosis, and no one was found as complete necrosis in experimental group, while only 3 flaps survived, and 4 flaps and 3 flaps showed partial necrosis and complete necrosis in control group, respectively (P < 0.05). CONCLUSION: Based on the physiological features of arterialized vein flap and its problems in clinical application, we improved the anastomosis strategy of flap grafting and obtained excellent experimental outcomes, which provides an insight for the clinical application of arterialized vein flaps.

Glucosyltransferase Capable of Catalyzing the Last Step in Neoandrographolide Biosynthesis.

ApUGT, a diterpene glycosyltransferase from Andrographis paniculata, could transfer a glucose to the C-19 hydroxyl moiety of andrograpanin to form neoandrographolide. This glycosyltransferase has a broad substrate scope, and it can glycosylate 26 natural and unnatural compounds of different structural types. This study provides a basis for exploring the glycosylation mechanism of ent-labdane-type diterpenes and plays an important role in diversifying the structures used in drug discovery.

[Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata].

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified in vitro. The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its in vivo function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

[Overexpression and isolation of CYP76AH1 in Salvia miltiorrhiza hairy roots].

Protein complexes are involved in the synthesis of multiple secondary metabolites in plants, and their separation is essential to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0.5% Triton X-100 was selected as the best extraction buffer, and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.

The Biosynthetic Pathways of Tanshinones and Phenolic Acids in Salvia miltiorrhiza.

Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza.

RNA-Seq reveals transcriptomic interactions of Bacillus subtilis natto and Bifidobacterium animalis subsp. lactis in whole soybean solid-state co-fermentation.

Bifidobacteria are anaerobes and are difficult to culture in conventional fermentation system. It was observed that Bacillus subtilis natto enhanced growth of Bifidobacterium animalis subsp. lactis v9 by about 3-fold in a whole soybean solid-state co-fermentation, in a non-anaerobic condition. For the purpose of understanding the metabolic interactions between Bif. animalis subsp. lactis v9 and Ba. subtilis natto, the transcriptome of Bif. animalis subsp. lactis v9 and Ba. subtilis natto was analyzed in single and mixed cultures using RNA-Seq. Compared with the single culture, 459 genes of Bif. animalis subsp. lactis v9 were up regulated and 21 were down regulated in the mixed culture with Ba. subtilis natto, with more than 2-fold difference. Predictive metagenomic analyses suggested that Ba. subtilis natto up regulated transport functions, complex carbohydrates and amino acid metabolism, DNA repair, oxydative stress-related functions, and cell growth of Bif. animalis subsp. lactis v9. In the mixed culture with Bif. animalis subsp. lactis v9, only 3 transcripts of Ba. subtilis natto were over-expressed and 3115 were under-expressed with more than 2-fold difference. The highest down-regulated genes were those involved in carbohydrate and amino acid metabolism. The data presented here demonstrated a parasitic-like interaction regulated at the transcription level, between Ba. subtilis natto and Bif. animalis subsp. lactis in the mixed culture. The over-expression of genes involved in substrate uptake and metabolism in Bif. animalis subsp. lactis in the mixed culture nevertheless, led to its higher cell concentration in the nutrient rich whole soybean medium.

Expression of the Chlamydomonas reinhardtii sedoheptulose-1,7-bisphosphatase in Dunaliella bardawil leads to enhanced photosynthesis and increased glycerol production.

Bioengineering of photoautotrophic microalgae into CO(2) scrubbers and producers of value-added metabolites is an appealing approach in low-carbon economy. A strategy for microalgal bioengineering is to enhance the photosynthetic carbon assimilation through genetically modifying the photosynthetic pathways. The halotolerant microalgae Dunaliella possess a unique osmoregulatory mechanism, which accumulates intracellular glycerol in response to extracellular hyperosmotic stresses. In our study, the Calvin cycle enzyme sedoheptulose 1,7-bisphosphatase from Chlamydomonas reinhardtii (CrSBPase) was transformed into Dunaliella bardawil, and the transformant CrSBP showed improved photosynthetic performance along with increased total organic carbon content and the osmoticum glycerol production. The results demonstrate that the potential of photosynthetic microalgae as CO(2) removers could be enhanced through modifying the photosynthetic carbon reduction cycle, with glycerol as the carbon sink.

Molecular characterization of poly(A)-binding protein from Daucus carota.

Poly(A)-binding proteins (PABs) bind to the poly(A) tails of most eukaryotic mRNAs and thereby influence the translational efficiency as well as the stability of the mRNAs thus bound. Compared with the data on yeast PAB, relatively little is known about the functions of PABs in higher plants. The cDNA encoding PAB was cloned by the method of "virtual subtraction" from carrot somatic embryos cultured with different sucrose concentrations. Sequence alignment reveals a significant homology between the deduced amino acid sequence of DcPAB and those of other PABs. The deduced sequence consists of 658 amino acids with a calculated molecular weight of 71.9 kDa. The cDNA has been expressed as a recombinant protein in Escherichia coli. The differential expression of DcPAB under different conditions suggests a potently unique role in the development of carrot somatic embryo.