Shujuan Zhao

Enhancing diterpenoid concentration in Salvia miltiorrhiza hairy roots through pathway engineering with maize C1 transcription factor

Tanshinones are valuable natural diterpenoids from danshen (Salvia miltiorrhiza Bunge). Here, it was demonstrated that maize transcription factor C1 improved the accumulation of tanshinones by comprehensively upregulating the pathway genes, especially SmMDC and SmPMK in danshen hairy roots, yielding total tanshinones up to 3.59mg g(-1) of dry weight in line C1-6, a 3.4-fold increase compared with the control. Investigation of 2024bp of the SmMDC promoter fragment revealed that C1-mediated upregulation of terpenoid genes was possibly due to the direct interaction of C1 with its recognition sequences. The increase of tanshinones was accompanied by a decrease of salvianolic acid production, the other bioactive ingredient in danshen, by up to 37% compared with the control. This was the result of the downregulation of SmTAT, the entry-point gene of the tyrosine pathway, which promoted metabolic flow to anthocyanins rather than to salvianolic acids. Based on the findings of the present study, it was concluded that cis-acting elements shared by terpenoid and phenylpropanoid biosynthetic genes are partially responsible for the C1-stimulated variation of tanshinone and salvianolic acid concentrations.

Biosynthesis of rare 20(R)-protopanaxadiol/protopanaxatriol type ginsenosides through Escherichia coli engineered with uridine diphosphate glycosyltransferase genes

Background Ginsenosides are known as the principal pharmacological active constituents in Panax medicinal plants such as Asian ginseng, American ginseng, and Notoginseng. Some ginsenosides, especially the 20(R) isomers, are found in trace amounts in natural sources and are difficult to chemically synthesize. The present study provides an approach to produce such trace ginsenosides applying biotransformation through Escherichia coli modified with relevant genes. Methods Seven uridine diphosphate glycosyltransferase (UGT) genes originating from Panax notoginseng, Medicago sativa, and Bacillus subtilis were synthesized or cloned and constructed into pETM6, an ePathBrick vector, which were then introduced into E. coli BL21star (DE3) separately. 20(R)-Protopanaxadiol (PPD), 20(R)-protopanaxatriol (PPT), and 20(R)-type ginsenosides were used as substrates for biotransformation with recombinant E. coli modified with those UGT genes. Results E. coli engineered with GT95syn selectively transfers a glucose moiety to the C20 hydroxyl of 20(R)-PPD and 20(R)-PPT to produce 20(R)-CK and 20(R)-F1, respectively. GTK1- and GTC1-modified E. coli glycosylated the C3—OH of 20(R)-PPD to form 20(R)-Rh2. Moreover, E. coli containing p2GT95synK1, a recreated two-step glycosylation pathway via the ePathBrich, implemented the successive glycosylation at C20—OH and C3—OH of 20(R)-PPD and yielded 20(R)-F2 in the biotransformation broth. Conclusion This study demonstrates that rare 20(R)-ginsenosides can be produced through E. coli engineered with UTG genes.