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

Dose linearity of inhaled fentanyl (FT) with comparative pharmacokinetics to transmucosal fentanyl (A)

8629 Background: Cancer patients frequently experience breakthrough pain which is a transitory flare of moderate or severe pain occurring on top of otherwise controlled, persistent pain. Fentanyl TAIFUN (FT), a novel breath-actuated dry powder inhaler is being developed for the treatment of breakthrough cancer pain in patients with ongoing opiate therapy. Methods: A randomized, open-label, crossover phase I study with 5 periods derived pharmacokinetics after fentanyl oromucosal (Actiq, A) and pulmonary (FT) administration in 30 healthy volunteers. Each single dose of study medication (200 mcg A; or 100, 200, 400 or 800 mcg FT) was administered following premedication with 50 mg of naltrexone with a minimum of 7 days between doses. Pharmacokinetic parameters were calculated from the plasma concentrations using a non-compartmental model. Results: The plasma concentrations of FT increased proportionally to the increasing dose and t1/2 was independent of the dose. FT had a linear elimination phase. FT had a substantially faster absorption and higher peak fentanyl concentration (Cmax) than A. Median Tmax was 1 and 60 min for FT and A, respectively. Moreover, there was an 8-fold increase in bioavailability of fentanyl during the first 20 min when 200 mcg FT is compared to 200 mcg A. Conclusions: The plasma concentrations from FT increases proportionally to the increasing dose while t1/2 is independent of the dose, and there is a linear elimination phase. Overall, FT is substantially more bioavailable than A during the important first 20–30 minutes after administration. Inhalation of FT allows an immediate and comparable availability of fentanyl suggesting potential for rapid pain relief. [Table: see text] [Table: see text] </jats:p

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