Team:TU Munich/Project/Limonene


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Responsible: Lara Kuntz and Andrea Richter

Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. Limonene has been shown to inhibit rat mammary and other tumor development (Tsuda et al. 2004). Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [Sun, 2007].

Producing the flavoring substance limonene in our beer might result in a fresh, lemon-like taste on the one hand. On the other hand, we might have beneficial effects on health such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.

Background and principles

Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [Fietzek, 2001]. Therefore D-Limonene ((+)-Limonene, (R)-enantiomer) is used as a component of flavorings and fragrances.


Limonene is produced by limonene synthase which uses geranyl pyrophosphate (GPP) as educt which is the universal precursor of monoterpenoids. (+)-limonene synthase from Citrus limon consists of 606 aminoacids (EC= and catalyzes the following reaction: Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate.

Fig. 1: Reaction catalyzed by limonene synthase.Source?

Saccharomyces cerevisiae produces geranyl pyrophosphate via the mevalonate pathway where it occurs exclusively as an intermediate of farnesyl pyrophosphate (FPP) synthesis [Oswald et al., 2007]. It has been established that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [Herrero et al., 2008, Oswald et al., 2007].

Fig. 2: Simplified isoprenoid pathway in S. cerevisiae, including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [Rico et al., 2010]

The molecular and physiological effects of limonene

Limonene is listed in the Code of Federal Regulations as generally recognized as safe (GRAS) [FDA] for a flavoring agent and does not pose a mutagenic, carcinogenic, or nephrotoxic risk to humans. Today it can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding [Sun, 2007]. As natural compound of Plants Limonene has practical advantages with regard to availability, suitability for oral apllication, regulatory approval and mechanisms of action. It has been shown to inhibit rat mammary and other tumor development by Apoptosis induction and modulation of oncogene signal transduction [Tsuda, 2004]. Furthermore It is used as excellent solvent of cholesterol, therefore d-limonene has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it also has been used for relief of heartburn [Sun, 2007].




Gel Picture of finished construct

SDS Page of Limonene Synthase

InVitro detection of limonene

To test functionality of purified Limonenesynthase in vitro we used an optimized protocol of an Enzyme Assay with previous Extraction of Landmann et al, 2007. The Enzyme Assay was carried out in Tris-HCl buffer with Glycerol, DTT and Cofactors. An identified amount of substrate (geranyl pyrophosphate) and purified recombinant limonene synthase. The mixture was extracted with pentane, dried with Sodiumsulfate and reduced under a stream of nitrogen. The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.

InVivo detection of limonene


Toxicity Assay

To establish whether limonene has an effect on yeast cells , we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium and the used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket.

Limonene at high concentrations affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed.

The in vivo GCMS detection of limonene [B] displayed a concentration of 50 µM. Hence the amount of limonene we will produce with the modified yeast will not reach a toxic concentration at all.

Evaluation of the Toxicity Assay for Limonene.


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  • [Tsuda et al., 2004] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. Drug Metab Pharmacokinet. 19(4):245-63.
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  • [FDA]