In aromatic plants species, biosynthesis of essential oils oocurs through two complex natural biochemical pathways involving different enzymatic reactions. Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the universal precursors of essential oils biosynthesis and are produced by the cytosolic enzymatic MVA (mevalonic acid) pathway or by plastidic and enzymatic 1-deoxy-D-xylolose-5-phosphate (DXP), also called the 2-Cmethylerythritol 4-phosphate (MEP) pathway. In the particular plant cell part, prenyl diphosphate synthases condense isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) further to form prenyl diphosphates, which are used as substrates for geranyl diphosphate, C 10 (GPP) or for fernesyl diphosphate, C 15 (FPP). Essential oils are final terpenoid products and are formed by a huge group of enzymes known as terpene synthases (TPS). Essential oils are important secondary metabolites of plants and have been used not only in different industries but also in ethnobotanical medicines for centuries. Hence, considerable research has been undertaken to understand the essential oils biosynthetic pathways. This review will be a valuable source of information in the field of natural products as we give detailed insights about biosynthesis of essential oils in plants, and thus indicate also new unexplored horizons for further research.

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1 to 5×104. This demonstrates the potential for using yeast to perform phenotypic screening of genetically-encoded cyclotide-based libraries in eukaryotic cells.

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We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with ahighly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human a-synuclein in live yeast cells.C yclotide MCoCP4 was selected by phenotypic screening from cells transformed with am ixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in ar atio of 1:510 4 .T his demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

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