Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the B. subtilis genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway ( dxs , ispD , ispF , ispH , ispC , ispE , ispG ) together with ispA (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of Pantoea ananatis ) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of B. subtilis . The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in B. subtilis . We determined that the expression of GGPPS through the crtE gene is essential for the formation of sufficient precursor, GGPP, in B. subtilis as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.

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Objective: The research aimed to provide new information regarding the secondary metabolites content of purple and white-purple Orthosiphon aristatus (Blume) Miq. callus, which can then be used as a basis for developing towards cell suspension and ultimately producing secondary metabolites using bioreactors. Methods: Callus induction of two varieties of O. aristatus were performed by inoculating sterile leaf explants grown on Murashige and Skoog basal media supplemented with 2,4-dichlorophenoxyacetis acid 0.4 ppm. The secondary metabolites were analysed and quantified using high-performance liquid chromatography with gradient elution. Results: The results showed the growth of callus two varieties of O. aristatus in growth media MS with 2,4-D 0.4 ppm. Rosmarinic acid content in the acetone extract of the purple variety callus was 1.28% w/w, and the white-purple variety was 2.22% w/w. Conclusion: This study could form the basis for the development of rosmarinic acid production by In vitro culture modification.

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