Sleeping is a necessary period of rest for our body and mind.[1] Generally, when sleeping, people can restore their energy and refresh their minds to prepare for the next day. Adults aged 18 through 64 should acquire at least 7 hours [2] of sleep to avoid diseases such as heart attack and obesity[3]. Teenagers should sleep 8-10 hours to prevent such diseases [4]. However, as people get overwhelmed by different work, such as projects and assignments, their typical sleeping routines are also disrupted. They start having less amount of sleep, which exposes them to the risk of having other health issues. According to Sleep Foundation, 35.2% of all adults in the US do not meet the essential requirement of sleeping for 7 hours[2]. Also, many people suffer from insomnia or sleeping disorders for various reasons, including depression, work time, and other mental health issues. The number itself is astonishing enough. In addition, to further understand the significance of problems sleeping in our life, we conducted a survey to study the sleeping quality in China, in which over 600 participants from all across China and of different ages filled it. The result indicates that many of our participants did not get the required amount of sleep.

Since an increasing number of people are getting disrupted in their sleeping routines, the outside manipulation of sleep would be essential to ensure high-quality sleep. However, in the market, sleeping pills have been proven to have several side effects, including impaired memory, headache, and the development of dependence[5]. Therefore, we turn to another popular alternative of sleeping supplements, melatonin.

Melatonin is a type of organic molecule found in animals, plants, and bacteria. For vertebrates, it contributes to the organization of biological functions through circadian rhythms[5] and anti-inflammatory molecules due to its antioxidant properties. However, it seems to function differently in plants, as a signaling molecule to plant defense response and growth and development. [6]

Melatonin in the human body functions similarly to natural sleeping pills secreted by the pineal gland with fewer side effects. However, even with melatonin, many people still have sleeping disorders. Since the production of melatonin may not be enough to manipulate the sleeping routine, the demand for related products. Hence, our team tries to find an effective, economical, and environment-friendly way to produce melatonin artificially.

Melatonin, N-acetyl-5-methoxytryptamine, is an indolamine compound with the 3-amide group and a 5-alkoxy group that is largely applied to regulate circadian rhythms, sleep, body temperature, protect lymphocytes against DNA damage, etc. (Mannino et al., 2021) Apart from medical uses, melatonin is also demanded crop improvement. Research has shown that approximately 60 million Americans suffer from circadian disorders and over 300 million Chinese also suffer from sleep disorders. Humans cannot synthesize L-tryptophan by themselves; therefore, it must be externally ingested (Xie et al., 2022). More and more uses of melatonin are being discovered through recent research and being applied to clinical treatments. With the increasing demand for melatonin in the market, economists predict that the global market for melatonin will reach 3.4 billion dollars by 2026 (Xie et al., 2022). The market needs new approaches and improved procedures for synthesizing melatonin as it is in shortage.

Chemical synthesis is the dominant way of the production of commercial melatonin.

Table 1. New methods of chemical synthesis of melatonin (Hügel & Nurlawis, 2003)

Scientists have been experimenting with new approaches to improve existing methods of synthesis of melatonin. The above table shows some examples of the new methods in the fact that the synthetic steps are reduced. The chemical synthesis pathway does solve the problem of needing industrialized melatonin to reach the demand, but problems also surfaced, as some pathways can be polluting, long, and costly.

Over the past few years, scientists began to seek new approaches, and they put their attention to biosynthesis. The natural biosynthesis of melatonin in plants happens mostly in the mitochondria and chloroplast. In healthy plants, L-tryptophan decarboxylase carboxylates L-tryptophan into tryptamine in the cytoplasm. The tryptamine is then catalyzed by the enzyme tryptamine-5-hydroxylase and acetyl-coenzyme A on the endoplasmic reticulum into serotonin. Serotonin N-acetyltransferase found in the chloroplast acetylates the serotonin into N-acetyl serotonin. Melatonin is produced through the methylation reaction of N-acetyl serotonin through the use of Acetyl serotonin methyltransferase or COMT. The co-conversion of S-adenosyl-l-methionine to S-adenosyl-l-homocysteine is present during the final reaction of the synthesis of melatonin. If the plant has abiotic stress senescence, then AMST/COMT will be likely used to catalyze serotonin into 5-methoxytryptamine. (Xie et al., 2022)

Animals can’t synthesize L-tryptophan themselves, meaning that animals have lower melatonin metabolism than plants. For animals to synthesize melatonin, cofactor BH4 and oxygen are required to produce 5-hydroxytryptophan from tryptophan hydroxylase. Serotonin, the dominant precursor, is produced from the conversion of 5-hydroxytryptophan with tryptophan carboxylase. Thereafter, “alkylamine N-acetyltransferase produces N-acetyl serotonin at the expense of acetyl-CoA.” (Xie et al., 2022) Melatonin is produced through N-acetyl-serotonin methyltransferase with the co-conversion of SAM to SAH.

Apart from plants and animals, microbe was found available to carry out melatonin synthesis. The synthesis of melatonin can be improved in microbe pathways including raising the supply of precursors, maximizing the expression of pathway enzymes, refine the use of cofactors and coenzymes.

Tryptophan is the precursor used in melatonin synthesis, and it can be increased by weakening the repressor protein as it activates the enzyme in L-tryptophan.

Serotonin, the essential intermediate in the process, can be improved by adding heterologous enzymes as it helps with the 5HT production, by controlling the expression level of related enzymes, and by optimizing the supply of cofactors and oxygen.

Scientists realized the need to move from chemical synthesis to biosynthesis, however, some failed to generate high yield due to the insolubility and low enzyme activity in heterologous hosts.

The cofactor is an essential part of the synthesis of melatonin, therefore boosting BH4 and NADPH (examples of cofactors) will also contribute to the optimization of the process. Pterin-dependent L-tryptophan hydroxylation was made through directed metabolic pathway evolution with the use of a minimum set of heterologous enzymes and a host. Then, the BH4 biosynthetic pathway was deleted and pterin turned into an alternative cofactor by the host strain. This could then be used to increase the production of BH4 and GTP.

A group of scientists, Yanfeng Zhang, Yongzhi He, et al, successfully carried out a biosynthesis pathway of melatonin using Escherichia coli. They combined the physostigmine biosynthetic genes and gene encoding phenylalanine 4-hydroxylase and caffeic acid COMT. The group found that by adding 3% glycerol and by putting the expression of heterologous protein in equality, melatonin production increased threefold. The production was raised another fivefold by the creation of the COMT variant with C303F and V321T mutations. The supply of S-adenosylmethionine, a cofactor of COMT was boosted through the cancelation of the speD gene, which essentially leads to the boost in overall yield. The group conducted cofactor engineering in an attempt to raise the supply of acetyl-CoA and SAM. COMT was extracted from S. Albulus and O. Sativa. Then, both were cloned to pBAD-SaCOMT and pBAD-OsCOMT plasmids, which were then transformed to EcSaCOMT and EcOsCOMT, and that only soluble protein was obtained. EcOsCOMT yielded more melatonin than the other, but both are available to be used in the process. (Zhang et al., 2021)

To minimize the side effects during the production while maintaining high-yielding melatonin, we try to improve the biosynthetic method--which is environmental-friendly--by increasing the yield of melatonin in poplar. Our main goal is to make poplar trees over-express COMT, the gene that manages the production of melatonin, to increase the efficiency of the production of melatonin. Poplar tree is one of the most abundant trees planted in the world, so that it can provide a solid production foundation. The over-expression doesn’t harm the plant since it increases its resilience by reducing oxidative stress. [7]

In the lab, we extract the target gene: COMT, and link the target gene to different vectors: storage and expression vectors. Finally, we connect the COMT gene on Agrobacterium tumefaciens GV3101, then GV3101, which carries COMT, would infect the plant. The result shows that we did improve the yielding with no by-products. This means that compared to the chemical synthesis, we have a similar yield, but it doesn’t bring any side effects.

[1] sleep. (n.d.) Miller-Keane Encyclopedia and Dictionary of Medicine, Nursing, and Allied Health, Seventh Edition. (2003). Retrieved August 18 2022 from  https://medical-dictionary.thefreedictionary.com/sleep

[2] Suni, Eric. Truong, Kimberly. (2022) Sleep Statistics. Retrieved August 18, 2022 from https://www.sleepfoundation.org/how-sleep-works/sleep-facts-statistics

[3] Sleep in Middle and High School Students | Healthy Schools | CDC. (2020, September 10). Centers for Disease Control and Prevention. Retrieved August 18, 2022, from https://www.cdc.gov/healthyschools/features/students-sleep.htm

[4] National Heart, Lung, and Blood Institute. (2022, March 24). Sleep Deprivation and Deficiency - What Are Sleep Deprivation and Deficiency? | NHLBI, NIH. Retrieved August 18, 2022, from https://www.nhlbi.nih.gov/health/sleep-deprivation

[5] Pacheco, D. (2022, April 13). Side Effects of Sleep Medication. Sleep Foundation. Retrieved August 18, 2022, from https://www.sleepfoundation.org/sleep-aids/side-effects-of-sleeping-pills

[6] Mannino, G., Pernici, C., Serio, G., Gentile, C., & Bertea, C. M. (2021). Melatonin and Phytomelatonin: Chemistry, Biosynthesis, Metabolism, Distribution and Bioactivity in Plants and Animals—An Overview. International Journal of Molecular Sciences, 22(18), 1–2. https://doi.org/10.3390/ijms22189996

[7]Mannino, G., Pernici, C., Serio, G., Gentile, C., & Bertea, C. M. (2021). Melatonin and Phytomelatonin: Chemistry, Biosynthesis, Metabolism, Distribution and Bioactivity in Plants and Animals-An Overview. International journal of molecular sciences, 22(18), 9996. https://doi.org/10.3390/ijms22189996

Reference 2

Chemistry and Biosynthesis of Melatonin. (n.d.). Encyclopedia.pub. Retrieved August 18, 2022, from https://encyclopedia.pub/entry/16502

Harper, K. (2014). The Science of Sleep - American Chemical Society. American Chemical Society. https://www.acs.org/content/acs/en/education/resources/highschool/chemmatters/past-issues/archive-2014-2015/the-science-of-sleep.html

Hügel, H., & Nurlawis, F. (2003). SHORT SYNTHESES OF MELATONIN. HETEROCYCLES, 60(10), 2349–2354. https://chemistry.mdma.ch/hiveboard/rhodium/pdf/novel.melatonin.syntheses.pdf

Mannino, G., Pernici, C., Serio, G., Gentile, C., & Bertea, C. M. (2021). Melatonin and Phytomelatonin: Chemistry, Biosynthesis, Metabolism, Distribution and Bioactivity in Plants and Animals—An Overview. International Journal of Molecular Sciences, 22(18), 9996. https://doi.org/10.3390/ijms22189996

Melatonin. (n.d.). Www.ch.ic.ac.uk. https://www.ch.ic.ac.uk/local/projects/s_thipayang/synth.html

National Center for Complementary and Integrative Health. (2021, January). Melatonin: What You Need To Know. NCCIH. https://www.nccih.nih.gov/health/melatonin-what-you-need-to-know

Xie, X., Ding, D., Bai, D., Zhu, Y., Sun, W., Sun, Y., & Zhang, D. (2022). Melatonin biosynthesis pathways in nature and its production in engineered microorganisms. Synthetic and Systems Biotechnology, 7(1), 544–553. https://doi.org/10.1016/j.synbio.2021.12.011

Zhang, Y., He, Y., Zhang, N., Gan, J., Zhang, S., & Dong, Z. (2021). Combining protein and metabolic engineering strategies for biosynthesis of melatonin in Escherichia coli. Microbial Cell Factories, 20(1). https://doi.org/10.1186/s12934-021-01662-8