Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.

Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents.

T-DNA activation tagging as a tool to isolate Salvia miltiorrhiza transgenic lines for higher yields of tanshinones.

The study of functional genomics has paved the way for directed approaches to the generation of genetically modified plants that produce novel and/or improved yields of pharmaceuticals. In the present study, an activation tagging mutagenesis (ATM) population of Salvia miltiorrhiza Bunge, a medicinal plant, was established by Agrobacterium-mediated transformation. The optimum conditions for Agrobacterium transformation were determined by the expression of green fluorescent protein. Under these optimized conditions, we isolated 1435 ATM cell lines with our initial antibiotic selection. Of these 1435 ATM cell lines, six lines (T1-T6) showed a red color on a selective medium containing 4.5 microM 2,4-dichlorophenoxyacetic acid (2,4-D), which is used as a phenotypic model system to identify the accumulation of tanshinones. 700 out of 1435 ATM cell lines were tested with a beta-glucuronidase (GUS) assay, 35 showed GUS activity. Southern blotting analysis revealed that the T1-T7 ATM cell lines have a single copy of the T-DNA insertion. Comparative analysis by high-performance liquid chromatography of the tanshinones expressed by non-transformed and ATM-transformed calli revealed varying quantities of tanshinones. There were negligible tanshinones in non-transformed white calli induced with 2,4-D. ATM lines T1-T6 showed significant increases in the yields of tanshinone-I (up to 43-fold), tanshinone-IIA (up to 26-fold) and cryptotanshinone (up to 104-fold) compared with those of the non-transgenic lines on 2,4-D medium. Interestingly, the yield of cryptotanshinone from line T4 on 2,4-D medium was two times higher than that of the non-transgenic lines on Trans-zeatin riboside medium. To the best of our knowledge, this is the first report of a quantitative and qualitative improvement in quinoid diterpene production achieved in a medicinally important plant species by activation tagging.