In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.
The availability and quality of wild berry material is strongly affected by seasonal variations, human resources, and both chemical and biological pollutants. Here we describe for the first time the industrial scale biotechnological production of cloudberry (Rubus chamaemorus) cells of consistent quality and defined chemical composition. Callus was initiated from sterile cuts of the wild cloudberry leaves. Bright yellow, soft callus of uniform quality was selected by regular subculturing, and a stable line successfully used for suspension culture was obtained within 18 months. The cultivation process of the cell culture was initiated in 250 mL shake flasks and stepwise expanded to a stirred tank bioreactor of 300 L working volume. Each growth period in flasks and fed-batch cultures in bioreactors was 10 ± 3 days. Viability of the cultures was approximately 90% throughout the cultivation processes. The 300 L culture was harvested by pressure filtration and the biomass was freeze-dried. The 300 L yields of fresh cells and freeze-dried material were 19 and 2 kg, respectively. The unusual phenolic profile of cloudberry cells including flavanols, as well as their fatty acid composition with a high proportion of α-linolenic acid and high protein content, make them a unique and interesting alternative for industrial applications for various industrial fields.
While this Article was undergoing peer review, Salim et al. (2013), Asada et al. (2013) and Salim et al. (2014) published individual steps of the (seco)iridoid biosynthesis pathway in Catharanthus roseus, including the molecular and biochemical characterization of 7-deoxyloganic acid hydroxylase, 7-deoxyloganetic acid glucosyl transferase and 7-deoxyloganetic acid synthase, which are in agreement with our results. These papers should have been cited at the end of the Discussion section.
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