<center>Home | Warwick

The Problem

Limonene and perillyl alcohol (PA) are naturally occurring chemical compounds that have vast application in many industries: food (permitted by the USA Food and Drug Administration (FDA)), cosmetics, cleaning products and as a solvents (Bhatia et al., 2008)(Chen et al., 2015). With the largest limonene market being in North America, the limonene industry is expected to increase in size by 4.5% from 2023-2028 (Limonene Market Insights, n.d.) and 6% from 2023-2030 (Ltd, n.d.). Despite this, the main interest in limonene and its less prevalent metabolite perillyl alcohol, is their application as anticancer therapeutic drug (Chen et al., 2021).Perillyl alcohol in particular has been shown to regress both small and advanced carcinomas over a 10 week period, halt any tumour growth and have chemoprotective effects on the skin (Haag & Gould, 1994)(Yuri et al., 2004)(Barthelman et al., 1998). With the promising application of intranasal PA administration undergoing increased clinical testing, the demand for PA will surely increase.

This is where the problem arises, limonene and perillyl alcohol are found at the highest concentration in the peels of citrus fruits and undergo a lengthy extraction process (Jung & Row, 1998). Conventional extraction methods include dissolving in a solvent, cold pressing or distillation to get the essential oil of the peel and then a final purification (typically chromatography, that shows the highest purities but is the most energy expensive step)(Siddiqui et al., 2022). The overall peels to essential oil to limonene yield is ~1% of the total mass of peel used - which is undoubtedly even lower for PA (Zema et al., 2018)(Siddiqui et al., 2022)(NileRed, 2015). With products like PA showing great potential as an extremely useful therapeutic drug, will we be able to keep up with the demand for it using these inefficient and energy expensive extraction techniques?

Solution

When looking into previous biomanufacturing attempts to synthesise limonene or PA, they had achieved production of the chemicals but they didn't have high enough yields to implement industrially. One of the main bottlenecks preventing its application is the accumulation of PA/limonene that is antimicrobial and toxic to the cells that produce it (Alonso-Gutiérrez et al., 2013)(Jongedijk et al., 2016). Therefore, we are aiming to produce perillyl alcohol (and its precursor limonene) in E. coli using some unique strategies to mitigate the toxicity of our products.

When limonene and PA build up in the cell, they cause a lot of membrane stress which will eventually kill the cell. Our design will utilise this, specifically the Cpx membrane stress response that is native to E. coli. By adding a Cpx promoter to one of our inserted plasmids (pJBEI-6411), activation of the native stress responses that make CpxR stress proteins when toxicity and stress is too high will induce the promoter - activating the next steps of our product. The first addition made is a pump sequence downstream of the Cpx promoter, that, when expressed, will help pump out more of the toxic products from the cell. Secondly, adding a Tet protein sequence on pJBEI-6411 downstream of the Cpx promoter and a Tet promoter upstream of key production enzymes on pJBEI-6410 will mean that Tet protein indirectly made in response to stress will shut down production of enzymes, preventing any further production of enzymes. Found out more detail on the specifics of how our system works on the parts section of the contributions page and on the project description page.

Alonso-Gutiérrez, J., Chan, R., Batth, T. S., Adams, P. D., Keasling, J. D., Kim, Y., & Lee, T. S. (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, 19, 33–41. https://doi.org/10.1016/j.ymben.2013.05.004

Barthelman, M., Chen, W., Gensler, H. L., Huang, C., Dong, Z., & Bowden, G. T. (1998). Inhibitory effects of perillyl alcohol on UVB-induced murine skin cancer and AP-1 transactivation. PubMed, 58(4), 711–716. https://pubmed.ncbi.nlm.nih.gov/9485025

Bhatia, S., McGinty, D., Letizia, C., & Api, A. (2008). Fragrance material review on p-mentha-1,8-dien-7-ol. Food and Chemical Toxicology, 46(11), S197–S200. https://doi.org/10.1016/j.fct.2008.06.071

Chen, T. C., Da Fonseca, C. O., Levin, D., & Schönthal, A. H. (2021). The monoterpenoid perillyl alcohol: anticancer agent and medium to overcome biological barriers. Pharmaceutics, 13(12), 2167. https://doi.org/10.3390/pharmaceutics13122167

Chen, T. C., Da Fonseca, C. O., & Schönthal, A. H. (2015). Preclinical development and clinical use of perillyl alcohol for chemoprevention and cancer therapy. PubMed, 5(5), 1580–1593. https://pubmed.ncbi.nlm.nih.gov/26175929

Da Fonseca, C. O., Khandelia, H., Salazar, M. D., Schönthal, A. H., Meireles, O. C., & Quírico‐Santos, T. (2016). Perillyl alcohol: Dynamic interactions with the lipid bilayer and implications for long-term inhalational chemotherapy for gliomas. Surgical Neurology International, 7(1), 1. https://doi.org/10.4103/2152-7806.173301

Figure 1 created in https://www.biorender.com/

Haag, J. D., & Gould, M. N. (1994). Mammary carcinoma regression induced by perillyl alcohol, a hydroxylated analog of limonene. Cancer Chemotherapy and Pharmacology, 34(6), 477–483. https://doi.org/10.1007/bf00685658

Jongedijk, E., Cankar, K., Buchhaupt, M., Schrader, J., Bouwmeester, H. J., & Beekwilder, J. (2016). Biotechnological production of limonene in microorganisms. Applied Microbiology and Biotechnology, 100(7), 2927–2938. https://doi.org/10.1007/s00253-016-7337-7

Jung, Y. A., & Row, K. H. (1998). Extraction and purification of perillyl alcohol from Korean orange peel by reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 828(1–2), 445–449. https://doi.org/10.1016/s0021-9673(98)00785-7

Limonene Market Insights. (n.d.). https://www.mordorintelligence.com/industry-reports/limonene-market

Ltd, R. a. M. (n.d.). Limonene Market Size, Share & Trends Analysis Report By Source (Orange, Mandarin, Grapefruit), By End-use (Personal Care Products, Food Products), By Region, And Segment Forecasts, 2023 - 2030. Research and Markets Ltd 2023. https://www.researchandmarkets.com/report/limonene

NileRed. (2015, March 12). How to extract Limonene from Orange Peels [Video]. YouTube. https://www.youtube.com/watch?v=o4CBXkfVHDc

Siddiqui, S. A., Pahmeyer, M. J., Assadpour, E., & Jafari, S. M. (2022). Extraction and purification of d-limonene from orange peel wastes: Recent advances. Industrial Crops and Products, 177, 114484. https://doi.org/10.1016/j.indcrop.2021.114484

Yuri, T., Danbara, N., Tsujita-Kyutoku, M., Kiyozuka, Y., Senzaki, H., Shikata, N., Kanzaki, H., & Tsubura, A. (2004). Perillyl Alcohol Inhibits Human Breast Cancer Cell Growth in vitro and in vivo. Breast Cancer Research and Treatment, 84(3), 251–260. https://doi.org/10.1023/b:brea.0000019966.97011.4d

Zema, D. A., Calabrò, P. S., Fòlino, A., Tamburino, V., Zappia, G., & Zimbone, S. M. (2018). Valorisation of citrus processing waste: A review. Waste Management, 80, 252–273. https://doi.org/10.1016/j.wasman.2018.09.024