Valeriana officinalis is a perennial medicinal plant native to Europe and Asia. The medicinal properties of V. officinalis were known to the ancient Greeks and Romans. The roots of the V. officinalis plant synthesize a suite of terpenoid and alkaloid compounds with important anti-anxiety and anti-insomnia properties. Valerian root was employed in many ethnobotanical settings for hundreds of years for treatment of sleep disorders, nervousness, and attention deficit hyperactivity disorder. For example, valerian root was used in World War I for the treatment of shell-shock, and it is currently marketed as a dietary supplement. The sesquiterpene valerenic acid, and to a lesser extent its intermediate valerena-1,10-diene, exhibit much of the pharmacologically significant GABA-A activity. Furthermore, the roots of V. officinalis produce scant quantities (0.7-0.9% DW) of valerenic acid and valerena-1,10-diene. This present work is motivated by the necessity of creating a more reliable production platform for elaboration of Valeriana sesquiterpenes. These molecules are synthesized from the substrate farnesyl pyrophosphate (FPP) by valerenadiene synthase (VDS). Production of these molecules might be hindered by low levels of FPP substrate or insufficient VDS enzyme catalyst in the roots of V. officinalis. To remedy this problem, we have created a metabolic engineering platform for elevated titers of FPP and valerena-1,10-diene in E. coli. Previously, we synthesized a codon optimized version of the valerena-1,10-diene synthase gene (vds) and demonstrated functional production of valerena-1,10-diene in E. coli. When this construct was co-expressed along with the eight gene mevalonic acid pathway from Saccharomyces cerevisiae, valerena-1,10-diene was produced at 41 mg/L. Despite this success, other groups have reported sesquiterpene yields in excess of 200 mg L-1 in similar shake flask experiments. In this work, we hypothesize that optimization of VDS enzyme levels and expression of the mevalonic acid pathway will further boost productivity. First, we cloned the vds gene under the strong T7 promoter. This should enhance VDS catalyst levels to couple FPP to valerena-1,10-diene. Secondly, we synthesized several different combinations of the mevalonic acid pathway using enzymes from Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis to balance carbon flux to FPP. This work will provide the foundation for development of new terpene anxiolytic drugs.

Combinatorial engineering of the mevalonic acid pathway for optimization of valerena-1,10-diene production

253rd National Meeting (2017)

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