Controlled Production of Carnosic Acid and Carnosol in Cell Suspensions of Lepechinia meyenii Treated with Different Elicitors and Biosynthetic Precursors

Lepechinia meyenii is a medicinal plant specialized in the biosynthesis of different types of antioxidants including the diterpenes carnosic (CA) acid and carnosol (CS). Herein we present the results of plant tissue culture approaches performed in this medicinal plant with particular emphasis on the generation and evaluation of a cell suspension system for CA and CS production. The effect of sucrose concentration, temperature, pH, and UV-light exposure was explored. In addition, diverse concentrations of microbial elicitors (salicylic acid, pyocyanin, Glucanex, and chitin), simulators of abiotic elicitors (polyethylene glycol and NaCl), and biosynthetic precursors (mevalonolactone, geranylgeraniol, and miltiradiene/abietatriene) were evaluated on batch cultures for 20 days. Miltiradiene/abietatriene obtainment was achieved through a metabolic engineering approach using a recombinant strain of Saccharomyces cerevisiae. Our results suggested that the maximum accumulation (Acc(max)) of CA and CS was mainly conferred to stimuli associated with oxidative stress such as UV-light exposure (Acc(max), 6.2 mg L-1) polyethylene glycol (Acc(max), 6.5 mg L-1) NaCl (Acc(max), 5.9 mg L-1) which simulated drought and saline stress, respectively. Nevertheless the bacterial elicitor pyocyanin was also effective to increase the production of both diterpenes (Acc(max), 6.4 mg L-1). Outstandingly, the incorporation of upstream biosynthetic precursors such as geranylgeraniol and miltiradiene/abietatriene, generated the best results with Acc(max) of 8.6 and 16.7 mg L-1, respectively. Optimized batch cultures containing 100 mg L-1 geranylgeraniol, 50 mg L-1 miltiradiene/abietatriene (95 : 5 %) and 5 g L-1 polyethylene glycol treated with 6 min UV light pulse during 30 days resulted in Acc(max) of 26.7 mg L-1 for CA and 17.3 mg L-1 for CS on days 18-24. This strategy allowed to increase seven folds the amounts of CA and CS in comparison with batch cultures without elicitation (Acc(max), 4.3 mg L-1).

Automated summary

In this study, a cell suspension system for the production of antioxidants carnosic acid (CA) and carnosol (CS) in Lepechinia meyenii was generated and evaluated through tissue culture approaches. External stimuli such as UV light exposure, polyethylene glycol, and NaCl were found to promote the maximum accumulation of CA and CS. Additionally, the bacterial elicitor pyocyanin was effective in increasing the production of both diterpenes. Metabolic engineering using Saccharomyces cerevisiae successfully led to the synthesis of miltiradiene/abietatriene. Optimized batch cultures with specific conditions resulted in a significant increase in the production of CA and CS.