The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors

The efficacy of a new salbutamol metered-dose powder inhaler in comparison with two other inhaler devices

AbstractAn open cross-over and randomized study was carried out in order to compare the efficacy and safety of inhaled salbutamol delivered from a new 50 μ g dose−1metered-dose dry powder inhaler Taifun®, and a commercially available 50 μ g dose−1dry powder inhaler Turbuhaler®, and a conventional 100 μ g dose−1pressurized metered-dose inhaler with a spacer (pMDI+S). Twenty-one patients, aged 21–70 years, with stable asthma and with demonstrated reversibility upon inhalation of salbutamol were included in the study. On three separate study days, the patients received a total dose of 400μ g of salbutamol from the dry powder inhalers and a dose of 800 μ g from the pMDI+S in a cumulative fashion: 1, 1, 2 and 4 doses at 30 min intervals. The per cent change in forced expiratory volume in 1 sec (FEV1), was used as the primary efficacy variable. Salbutamol inhaled via the Taifun®produced greater bronchodilation than the other devices. The difference in percent change in FEV1between the Taifun®and the other devices was statistically significant at the two first dose levels, but diminished towards the higher doses when the plateau of the dose–response curve was reached. The estimated relative dose potency of the Taifun®was approximately 1·9- and 2·8-fold compared to the Turbuhaler®and the pMDI+S, respectively. The Taifun®caused a slight, but clinically insignificant, decrease in serum potassium concentration. There were no significant changes in the other safety parameters (blood pressure, heart rate and electrocardiogram recordings) with any of the used devices. In conclusion, this study indicates that salbutamol inhaled via the Taifun®is more potent than salbutamol inhaled from the other devices tested. In practise, a smaller total dose of salbutamol from the Taifun®is needed to produce a similar bronchodilatory response. All treatments were equally well tolerated

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids

Key Message: Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. Abstract: The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.Peer reviewed: YesNRC publication: Ye

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30 months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87 mg l−1) and hyoscyamine (107.7 mg 1−1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60 g/l sucrose or Gamborg’s B5 with 40 g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine