Background

Japan, where members of iGEM Tsukuba live, is an island country. Therefore, we inevitably rely on marine transportation more than 99% of Japan's trade is carried by ocean transport. The amount of cargo transported by sea is expanding worldwide, which makes it essential as a means of transportation. (Reference1)

However, "marine sessile organisms" have brought an issue in such situations. Barnacles and Mediterraneanmussels are typical examples. In addition, ascidians, moss worms, borers, and seaweed can be included. These organisms are known to adhere to man-made structures such as ships, not only natural rock walls, which are their original habitat. They cause an increase in ship weight when adhering to ships` bodies and water flow resistance on ship surfaces. Consequently, it is estimated that fuel consumption will go up by approximately 40%. (Reference2) This is a serious problem.

In addition, excessive energy consumption leads to larger emissions of carbon dioxide, sulfur oxides, and nitrogen oxides. This is directly connected to global warming and air pollution. (Reference3)Furthermore, the fishermen we interviewed at the Hiraikata fishing port said “due to the physical damage to the bottom of ships they cause, the repair cost is also an economic problem.”

Moreover, damage caused by marine biofouling has also been reported at power plants and in fishing areas. Many power plants such as thermal power plants use ocean water for refrigeration. Marine organisms seem to attach and obstruct the water flow in the waterways, reducing cooling efficiency. In the fishing industry, they adhere to the nets used for cultivating marine products, causing deterioration. There was actually damage at the Hiraikata fishing port. In this way, marine sessile organisms are causing environmental and economic problems in a variety of industries related to the sea, and its solution is an urgent issue.

Conventional Solutions and Their Problems

We humans have been fighting these problems caused by marine fouling organisms for a long time, especially in the shipping industry. The following is a summary of its history.

In the 1960s, people used toxic substances such as arsenic and mercury to coat the bottoms of ships. Although these substances did work by killing organisms, they were reportedly harmful to other marine creatures and even to humans. As a matter of course, they stopped using those substances.

In the 1970s, the mainstream countermeasure was to apply organotin compounds to the bottom of ships. These compounds protect ship surfaces by gradually hydrolyzing, thereby stripping off sessile organisms. However, it is reported that organotin compounds also harm marine ecosystems, and they were banned in Japan in 1990 and worldwide in 2008. (Reference5)

Currently, there are a total of 17 types of adhesion inhibitors in use. However, many of them have also been reported to have adverse effects on marine ecosystems. Even if the substance itself is not, some of their degradation products are highly poisonous. Therefore, substances currently in use still harm the marine environment. Along with the recent increase in environmental awareness, these substances are expected to be regulated. Another way to solve the problem is to physically modify the structure of ship surfaces, which is also under research. However, different marine organisms have different attachment mechanisms. Therefore, a surface structure that is effective for a particular organism is likely to be ineffective for others. In summary, the factor that makes it difficult to develop

attachment inhibitors for marine organisms lies in that all the following four properties must be satisfied.

1. They must be effective for whole marine organisms.
2. Safety must be ensured for the entire ocean ecosystem.
3. The substance must be highly biodegradable or effective in minute amounts so that it does not remain in high concentrations in seawater.
4. No toxic can be found on its degradations.

Thus, although various studies have been conducted to date, research on the prevention of marine organisms from adhering to the structures continue to face difficulties to this day and remains a serious social problem worldwide.(Reference5)

Research on Natural Organic Compounds that Inhibit Adhesion

Natural organic compounds produced by marine organisms are attracting attention as a solution to this problem. Marine organisms such as sea slugs, seaweed, and sponges produce substances that prevent organisms from attaching to their own body. These substances act on the larvae before they attach. Unlike the attachment inhibitors that had been used in the past, they do not affect the survival of the sessile organisms themselves. Those organisms indeed harm human activities, but they are also members of the marine ecosystem. Therefore, it is crucial to protect the marine ecosystem by not affecting sessile organisms. This indicates that natural organic compounds are much better for the environment than conventional adhesion inhibitors. Also, because they derive from nature, they are thought to have little impact on marine organisms other than the organisms they are attached to. Furthermore, a single type can act on multiple types of sessile organisms.

These substances act at very low concentrations, which is another advantage of using natural organic compounds. However, since the amount of substance obtained from each organism is very small, it is not practical to recover and utilize natural organic compounds directly from marine organisms. (Reference6)

Our Solution

We believe that synthetic biology is the key to solving this problem. We have examined 15 natural organic compounds with adhesion-inhibitory properties. Among them, we decided to synthesize Cyclo-L-Trp-L-Ala in this project because we believe that its biosynthesis in synthetic biology has great advantages. It is a cyclic dipeptide composed of two amino acids, tryptophan and alanine. This substance inhibits mussel attachment at very low concentrations. Analogues of this substance are also effective for other sessile organisms such as barnacles and mussels. We have three advantages in synthesizing this substance using synthetic biology below.

1. (1) Only one gene is involved in the synthesis of the substance.
2. (2) We can increase the amount of tryptophan and alanine, the precursors of synthesis, in the cell, and this is expected to increase the amount of synthesis.
3. (3) Japan has many companies and researchers excelling in amino acid fermentation, and their knowledge can be utilized.

(Reference7)

Why Synthetic Biology?

Currently, there is a lot of research in the field of chemistry on natural organic compounds with adhesion-inhibitory properties. However, the approach of chemically synthesizing and utilizing these substances has not yet been successful. For example, a chemical synthesis called isocyanocadinene, a natural organic compound derived from sea slugs, has been achieved, but the synthesis requires 28 steps, and the yield is only a few percent. Similar problems remain for other compounds. In addition, toxic substances used in the process may contaminate chemical synthesis. (Reference8)

Synthetic biology solves these problems. It allows us to use the elaborate synthetic systems of living organisms. Moreover, because the organisms themselves synthesize the substance, there is little chance of contamination.

Specific Strategies

So far, we have broadly described the advantages of synthesizing Cyclo-L-Trp-L-Ala (hereafter, cWA). From this point on, we will describe the detailed policy of this project in the following three phases.

Phase 1: Introduction of the synthase into E. coli and devising culture media conditions

Introduce cWA synthase into E. coli and culture it in various media with different amino acid contents. cWA synthase is known to react with other amino acids and synthesize cyclic time peptides that are not cWA. To avoid this, it is essential that other aminoacyl tRNAs (proline aminoacyl tRNAs, for example) not be incorporated. Therefore, the synthesis of cWA is increased by limiting the amount of other amino acids and culturing the cells in a medium with large amounts of alanine and tryptophan.

Phase 2: Increase alanine synthesis in E. coli

Modify the primary metabolic system of E. coli to directly raise the amount of alanine in the body of E. coli. Three types of enzymes involved in alanine synthesis are selected, including exogenous ones, and each is expressed in small amounts. By increasing the number of alanine synthesis pathways and expressing them in small amounts, we intend to avoid depletion of the raw material for alanine and the resulting deterioration of development and to stably raise the amount of alanine synthesis.

Phase 3: cWA synthesis in a cell-free system

Synthesize cWA without relying on E. coli. We believe that if cWA is synthesized in vivo, such as in E. coli, the synthesis of the raw material may be limited due to the feedback mechanisms and other factors. To avoid this, we will synthesize and isolate cWA synthase, aminoacyl tRNA synthetase, and RNA polymerase itself in E. coli so that cWA synthesis can be performed from RNA nucleotides and amino acids. We are sure that biosynthesis by constructing a cell-free system may break through the limitation of biosynthesis in E. coli.

Implementation in society

We need to consider how to implement the synthesized substance in society. In fishing ports and power plants, in which biofouling is causing damage, we can prevent it by simply distributing the substance in waterways with effective concentration. Therefore it is relatively easy in this case. On the other hand, the biosynthesized compounds must be turned into paint to solve the problem on the ship's surface. Our final goal in social implementation is to create material that can prevent microorganisms from adhering even when the ship is moving.

Regarding these problems along with social implementation, we kept seeking countermeasures in the process of Human Practice and Modeling and devised approaches about how to use it practically and to make it paint.

Prospects for this project

Until now, there have been few approaches to the problem of sessile organisms from the perspective of synthetic biology. Through iGEM, we will announce a synthetic biology approach to the problem. We hope that the success of this project will lead to further research on the genes involved in the synthesis of natural organic compounds with adhesion-inhibiting ability, which has rarely been explored. We'd also like to assert the importance of biodiversity, including marine organisms, and their conservation, by publicizing this project both domestically and internationally.

Reference

Reference1. Japan maritime center. SHIPPING NOW 2023-2024. p20, p26

Reference2. International paint. 2003. Propeller16, 6.

Reference3. IMO MEPC. 2010. 60/4/21 (International Maritime Organization Marine Environment Protection Committee.)

Reference4. The sessile organisms society of Japan. 2006. Fujitsubogaku no saishingaku, 59. P209-223

Reference5. Kazunobu Takahashi. 2010. Journal of the JIME, 45, 4

Reference6. Tatsuhumi Okino. 2021. Kagaku to seibutsu, 59, 1

Reference7. Elena Bovio, et al. 2019. Marine Biotechnology, 21, p743-752

Reference8. Keisuke Nishikawa, et al. 2009. Japan Chemistry Organisms Proceedings, 89, p2