Description





Introduction

Around the world, the potential health problems caused by UV radiation is one of the most pressing health and social issue that have been highlighted for a long time. Ultraviolet radiation can seriously injure human skin. The two types of UV radiation which will do harm to human health are UVB and UVA. UVB has a shorter wavelength that will induce sunburn, inflammation, skin aging, and skin cancer. UVA, with a longer wavelength, could also cause skin cancers in the long term. Noticing the harm brought by UV radiation, people would often use sunscreens as a precaution. However, sunscreens on the current market will damage human skin by blocking pores and being harmful to human tissues, also poisonous to the species in the sea, and would pollute the environment.

So we, LINKS_China, decide to use gene editing to make the family of Mycosporine-like Amino Acids (MAAs) in Saccharomyces cerevisiae. MAAs are widely found natural in marine creatures and are able to prevent UV radiation without causing harm to nature. We will also use Catalase and SOD to reduce oxidative stress already done by UV-radiation in human skins.






What is UV radiation? What harm does it induce?

UV radiation, which exists in three main forms of energy classified as UVA, UVB, and UVC energy radiation, is one type of general energy radiation measured on the electromagnetic (EM) spectrum. In general, UVA rays have a wavelength of 315-400nm that can penetrate the dermis of human skins, and UVB rays are about 280-315nm, which makes them only capable of penetrating the epidermis of human skin (Almudena et al., 2018). Only UVA and UVB rays are able to penetrate through the atmosphere, as most UVC and some UVB rays are commonly absorbed by the Earth’s ozone layer when emitted by the sun. Thus, the types of UV rays that come in contact with human skin are UVA (95%) and UVB (5%) (Center for Devices and Radiological Health, 2020).

UVA rays have a wavelength of 315-400nm that can penetrate the dermis of human skin, and UVB rays are about 280-315nm, which makes them only capable of penetrating the epidermis of human skin. (Almudena et al., 2018)

Once UV radiation passes through human skin, the naturally emitted energy waves can distort normal metabolic functions and cause a variety of health problems such as inflammation, premature skin aging, and skin cancer. Acute exposure to common types of UV radiation for an extended period of time causes skin burns and inflammation. Overexposure to UV light causes skin cells to pass on and weakens the immune system, reducing the skin's ability to defend against foreign invaders such as viruses in the air (Health Effects of UV Radiation, 2022).n addition to severe sunburn as physical damage, UV radiation, under natural conditions, would also trigger actinic keratosis and chronic skin-aging within regions of the epidermis that were overexposed, and without proper medical treatment at this stage, these pre-malignant symptoms of skin-aging and overgrowth would eventually evolve into skin cancers (A Singh et al., 2021).

Skin cancers are some of the most common forms of cancers that account for almost 40% of all cancer cases globally. Over the year, approximately 2-3 million people would be diagnosed with skin cancers, making it a large health issue that should not be underestimated (Cakir et al., 2012). Amongst the different types of skin cancer diagnosed, melanoma remains to be the most serious form as it contributes to almost 75% of all skin cancer deaths. Caused mainly by UVR exposure during childhood periods, the such disease would quickly spread throughout the human body, leading to consequences that are irreversible. Other forms of non-melanoma skin cancers such as basal and squamous cell carcinomas are also dangerous, but under proper treatment, the induced tumors caused by these cancers could be eradicated more efficiently (A Singh et al., 2021). Thus, Sunscreen is essential for resisting UV rays that can cause skin cancer via DNA damage.






Sunscreens

The current market is composed of two kinds of sunscreen products, physical and chemical sunscreen. Physical sunscreen is made of titanium dioxide and zinc oxide, reflecting mainly UVA (315 to 400nm). This kind of sunscreen contains an advantage in its long-lasting properties and is insensitive to the skin. However, it still lacks essential protection against UVB (280 to 315nm), has low solubility in water, and is unable to be absorbed by the human skin. Chemical sunscreen, on the other hand, commonly consists of oxybenzone, which absorbs and transforms the energy of UV rays through chemical reactions to reduce the damage to the skin. Moreover, it has a certain absorption effect on both UVA and UVB light waves. However, the chemicals it consists of cause a certain degree of irritation and damage to the skin and thus must be cautiously selected for consumption to avoid symptoms of skin allergies (Bedosky, 2021).

Physical sunscreen reflects UV rays while chemical sunscreen absorbs and transforms the energy of UV rays (Tolan et al., 2018).

Besides the drawbacks of both market sunscreens on tactile experience, physical and chemical sunscreens are also hostile to the marine ecosystem. Approximately 14,000 tons of sunscreen are washed off and absorbed into the ocean annually (Wang et al., 2022). About 10% is hydroxybenzophenone, far exceeding the minimum concentration that causes coral bleaching (Wang et al., 2022). Consequently, the disposal of hydroxybenzophenone leaves about 15% of the world's coral reefs and 40% of coastal waters under deadly threat from chemical sunscreens (Wang et al., 2022). The components of physical and chemical sunscreens, mainly oxybenzone, hydroxybenzone, and octyl methoxycinnamate, lead to the bleaching of coral reefs as well as the extinction of large numbers of fish and other marine life (Wang et al., 2022). These sunscreens also cause a direct impact on the viability of parrotfish, wrasse, and eels, with Oxybenzone and Octinoxate being capable of impairing the neurological and reproductive abilities of marine life and acting as hormone disruptors (Beachapedia, 2020).

To address this issue, biological sunscreens should be manufactured. The natural biological sunscreens on the market indirectly absorb UV rays through antioxidants from plant extracts, making them inefficient. This includes vitamin C, carotenoids, and flavonoids, which are used to reduce the reactive oxygen species (ROS) produced by UV exposure. However, biological sunscreens do not provide instant sun protection and will face the problem of insufficient repair (Solano, 2020). Therefore, in order to solve the problem, we decided to introduce Mycosporines and Mycosporine-like amino acids.






What are MAAs? Why did we choose MAAs?

In nature, there is a special compound synthesized by Cyanobacteria that functions mainly as natural protection against ultraviolet radiation (UVR), known as Mycosporine-Like Amino Acids (MAAs) (Rahman, 2018). They are naturally present in many organisms, including red algae, sea urchins, corals, and sea stars.

Figure 3: Different types of MAAs.

MAAs generally have a core molecular structure of a cyclohexanone or cyclohexenimine ring, and they are colorless, water-soluble secondary metabolites that are able to disperse surplus energy as heat after absorbing harmful UVR through their conjugated double bonds (Pandika, 2018). Research has also shown secondary properties of MAAs, involving antioxidation, antiaging, wound healing, and anti-inflammatory (Singh et al., 2021).

Moreover, it is proved that the accumulation of these MAA compounds in larger organisms such as fish is attributed to being acquired from diet or partnering with an MAA-producing microorganism (Osborn et al., 2015). Thus, it will not cause any damage to the environment but rather benefits.






MAAs Production

As molecules with strong UVR absorption ability, MAAs can be produced naturally by algae such as Cyanobacteria and red algae (and other marine organisms that are exposed to strong UVR). However, their slow growth has limited the wide use of MAAs molecules as an essential component in the cosmetic industry. In the biosynthetic production of MAAs molecules, S7P is the crucial precursor which is made through the reaction of several sugar molecules in the Pentose Phosphate Pathway. This precursor will have a considerable accumulation in organisms with high utilization efficiency of sugar. The brewer’s yeast (Saccharomyces cerevisiae) with a fast-growing rate (60-75minutes to complete a growth cycle) and effective sugar metabolic pathway, has already been widely used in the cosmetic industry, making it the best choice for chassis cells.

Figure 4: Metabolic pathway of MAAs.

S7P is converted to MAAs molecules by a successive series of catalytic reactions of enzymes. First, S7P converts into 4DG by the reaction caused by DDGS and OMT. The molecule 4DG has the basic structure of MAAs---Mycosporine-like structure. Then different ligases will add amino acids to the structure to form various MAAs molecules. For example, ATP-grasp ligase can fuse 4DG with L-glycine to form the M-glycine molecule. Building upon the previous step, the ligase D-ALA-D-ALA can blend M-glycine and L-serine to produce shinorine. D-ALA-D-ALA can also fuse M-glycine with L-Alanine when this amino acid becomes the substrate. This fusion will form porphyra-334. Finally, adding the MysE enzyme will remove the carboxyl group on shinorine and porphyra-334 to produce Palythine.

Different algae such as cyanobacteria, actinomycetes, and red algae all have different enzymes for MAAs production- and we want to find the best combination to achieve the highest yield. In this research, we choose to combine the enzymes in Nostoc punctiforme, Nostoc linckia, and Actinosynnema mirum and insert them into S. cerevisiae for expression and fermentation.

A high concentration of the S7P precursor is also crucial to improve MAAs molecules. S7P is made through the reactions of glucose in the Pentose Phosphate Pathway. However, most glucose are guided to glycolysis to maintain the growth of microorganisms. So we insert the metabolic enzymes of Xylose (Xyl1, Xyl2, Xyl3) from Scheffersomyces stipitis. As a result, S. cerevisiae is able to assimilate Xylose and produce X-5-P which can directly convert into S7P. Meanwhile, we knocked out the downstream gene TAL 1 to avoid the possibility that S7P may convert into other substances.

Figure 5: The designed metabolic pathways in S cerevisiae in order to produce MAAs.

There are other mycosporine-like molecules, such as gadusol, that can also absorb UV-radiation. It has an absorption range that covers UVB radiation and shows an absorption maximum at 290nm. Gadusol can also be produced from precursor S7P using ligase EEVS and MTOX, with no need for other amino acids. With gadusol, we provide a wider absorption spectrum for UVR.

Figure 6: UV absorption range of four MAAs. Porphyra-334 and Shinorine mainly protect against UVA-2; Palythine is effective against both UVA-2 and UVB (Singh et al., 2021); Gadusol primarily protect against UVB.






Sunburn Repair Introduction and its Relative Importance

Despite the numerous aforementioned benefits and advantages of MAAs, nothing is perfect – MAAs cannot absorb the entirety of UVR from the sun. The radiation that escapes MAAs is usually too little in quantity to damage our skin, but after a long period of direct exposure to the sun, UV radiation damages could still accumulate, increasing the infamous oxidative stress.

Reactive oxygen species (ROS) in the form of superoxide O2- play a significant role in cellular signaling; however, high concentrations of ROS possess the ability to “non-specific[ally] damage … proteins, lipids, and nucleic acids” (Briegar et al., 2012). Oxidative stress is more or less related to multiple different serious health harms such as photoaging, Alzheimer’s disease, insulin resistance, etc. Normally, cells have a system to limit and remove ROS, which, in unstressed conditions, is balanced with the formation of ROS. However, under UVR, the ROS defense system is overwhelmed with a dramatic increase in ROS levels. To solve this problem, we incorporated enzymes that participate in ROS defense found naturally in cells to maintain ROS levels in equilibrium.

Figure 7 : The mechanism of sunburn repair process in human skin.

To solve these problems, we chosed several enzymes, such as human cytosolic superoxide dismutase 1 (hSOD1) and human catalase (hCatalase), that break down reactive oxygen species (ROS) in the form of superoxide into water and hydrogen. Hydrogen peroxide, as a member of the ROS family, is toxic to human cells. Catalase has a higher efficiency in degrading hydrogen peroxide but is most effective under lower hydrogen peroxide concentrations. Thus, the usage of this specific enzyme would best help to eradicate the hydrogen peroxide with the most effective usage.

Figure 8: The genetic circuit design of sunburn-repair enzymes.

We intend to fuse hCAT and hCatalase with a linker so that the two-enzyme system has the highest efficiency. We predict that SOD1 linked with catalase (SHC) has more enhancements than the pathway that consists of simple, separate enzymes. First, the linker “bundles up” the two enzymes so that any ROS processed by SHC can be directly broken down into non-toxic oxygen and water. What’s more, paying consideration to catalase’s susceptibility to become photoinactivated, linker enzyme designs will also reduce inter-process exposure to light caused by gaps between the two enzymes’ processing and in turn maintain the functioning stability of catalase. This could ensure that the pathway is fixed and stable, and has processing efficiency maximized. Finally, we will express the linked SHC gene inside of E.coli BL21, purify the proteins, and thereby assess their functionality and activity.

To look for further information about the engineering and experimental design of our Project, please click the here (Engineering section) for more relative content.






Citations

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