Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild‐type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial‐scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.
Methyl jasmonate (MJ) enhances the production of a range of secondary metabolites including triterpenoid saponins in a variety of plant species. Here, it enhanced production of bacoside A, a valuable triterpenoid saponin having nootropic therapeutic activity in in vitro shoot cultures of Bacopa monnieri, the only known source of bacoside A. The highest yield was with 50 μM MJ giving 4.4 mg bacoside A/g dry wt; an 1.8-fold increase (compared to control) after 1 week.
Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The Km values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.
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