During our team's brainstorming session, we came across the work done by previous iGEM teams on addressing the issue of eutrophication in rivers and oceans. One of the problems caused by eutrophication is the excessive growth of algae, which depletes the dissolved oxygen in the water through photosynthesis and has a significant impact on the environment and ecology. After conducting in-depth research, we found that in many treatments for eutrophication, excess algae is simply removed and discarded. This led us to wonder if there could be a way to utilize these algae. After further investigation, we discovered that algae commonly contain alginates, which can be broken down into alginate oligosaccharides—a valuable form of biofertilizer. Among the existing degradation methods, chemical degradation generates harmful substances and causes environmental pollution, while physical degradation consumes a significant amount of energy and may impact the structure of alginates.

Our team is conducting an experimental research project that employs genetic engineering to express alginate lyase for the production of alginate oligosaccharides. We have developed an integrated system that combines biodegradation and enzymatic degradation methods to enhance the efficiency and quality of alginate degradation.

Alginate, a natural polysaccharide, is abundantly present in various species of brown algae found in the vast ocean. It is a water-soluble acidic polysaccharide derived from the cell walls of brown algae such as kelp, giant kelp, and Sargassum. Alginate consists of two sugar acid monomers, 1,4-β-D-mannuronic acid (M) and its C5 epimer 1,4-α-L-guluronic acid (G). As a versatile biopolymer, alginate exhibits wide-ranging biological activities and plays a vital role as a structural component in organic life forms. Alginate has extensive applications in food, medicine, cosmetics, and agriculture.

However, its inherent resistance to degradation poses challenges, resulting in significant waste generation and environmental pollution during production and utilization.

Alginate degradation methods can be classified into four main categories: chemical, physical, enzymatic, and biodegradation. Chemical degradation involves using reagents like sodium hydroxide and hydrochloric acid to break alginate into smaller molecules but can be environmentally harmful. Physical degradation employs extreme conditions like high temperature and pressure but may require substantial energy and affect alginate's structure. Enzymatic degradation is efficient and eco-friendly but requires suitable enzymes and conditions. Biodegradation utilizes microorganisms for sustainable and harmless degradation, considering microbial growth and selective degradation abilities.

Application of Alginate Lyase in Agriculture:

Alginate lyase's utilization in the realm of agriculture primarily revolves around its capacity to generate alginate oligosaccharides, functioning as a novel form of "plant vaccine." These oligosaccharides offer a multitude of advantages, including the promotion of plant growth, enhancement of crop yield, and reinforcement of plant resilience against environmental stressors and viral diseases.

To address these challenges, we employ an innovative approach to enhance alginate degradation efficiency by developing three systems in genetically engineered E. coli

Our project features a meticulously designed gene circuit with a focus on precision control. Utilizing the T7 promoter, we initiate transcription and integrate our alginate lyase gene. A significant innovation lies in our engineered arabinose-inducible promoter. In the absence of arabinose, the latter part of the lyase gene remains dormant, resulting in no expression. However, with the introduction of arabinose, this promoter is activated, leading to lyase protein production. This key protein is responsible for cleaving the engineered bacteria, thereby releasing both alginate lyase and cellulase enzymes.

The second gene circuit mirrors the first, differing only in the incorporation of bacterial cellulase genes. This meticulous gene circuitry enhances enzyme production with precise control mechanisms.

Escherichia coli, as a chassis microorganism in synthetic biology, offers significant advantages. It boasts rich genetic tools, making it easily amenable to genetic engineering and rapid production of target products. E. coli's fast growth rate and robust adaptability make it an ideal choice for synthetic metabolic engineering and gene regulation studies. Furthermore, its well-studied metabolic and biosynthetic pathways facilitate the design and optimization of bioproduction processes.

We inserted the Alg6B gene from Pseudoalteromonas distincta to E.coli Rosetta to express alginate lyase, an enzyme capable of breaking the ketosidic linkages between uronate and glycosyl residues in sulfated alginates. This enzymatic action hydrolyzes alginate into smaller oligosaccharides and monosaccharides.

Additionally, we incorporate Bgls, a cellulase enzyme produced by Bacillus subtilis, into our system. Bgls efficiently breaks down cellulose into smaller monomers, which can be utilized as a carbon source. Combining these enzymatic and biodegradation systems enhances the efficiency of alginate degradation. Furthermore, Bgls improves the solubility of hydrolysis products, making their extraction and utilization more effective.

Our project capitalizes on the synergy between alginate lyase and cellulase systems, significantly boosting efficiency compared to individual enzymes. Together, these enzymes work harmoniously to rapidly and thoroughly degrade biomass, ensuring maximum conversion into biofertilizers. This cooperative action not only enhances effectiveness but also simplifies operations, minimizes energy consumption, and broadens the range of biofertilizers, all while promoting sustainability.

As the bacterial cellulase and alginate lyase are expressed through genetic engineering in Escherichia coli, and our substrates are located outside the bacterial cells, we need to lyse the bacteria to allow the expressed enzymes to come into contact with the substrates and facilitate the reaction. Additionally, as we are incorporating Escherichia coli in our fertilizer production process, ensuring biosafety is of utmost importance.

We therefore introduced a lysis gene to break down the Escherichia coli cells. SRRz cleavage gene was utilized, which is a chain of genes consisting of the periplasmic penetration gene S, the phage lysin (transglycosylase) gene R, and the gene RZ.

S gene's product is to modify the permeability of the cell membrane, resulting in the formation of a porous structure on the membrane, which allows the enzymes expressed by the R and RZ genes to penetrate the cell wall and exert their action on it.

Consequently, the cell wall undergoes disruption, leading to the release of the substances contained within the cells. In our case, the enzymes released are the alginate lyase and cellulase expressed by Escherichia coli.

Safety Considerations:

To ensure Biosafety, We inserted the SRRz cleavage gene in every E.coli, trying to make them all lyse and release the enzymes. However, considering arabinose might not be able to reach every single E.coli, we also used an Ultrasonic microbial lysis instrument and high temperature to ensure that all the E.coli were dead before we used the alginate oligosaccharides as fertilizers.

Our final product would be our two genetically engineered freeze-dried bacteria powders. In addition, we would provide how our hardware works and how to make new hardware capable of generating alginate oligosaccharides with seaweed, freeze-dried bacteria, and potentially kitchen wastes.

Our targeted clients are fertilizer companies, farmers, and those who wish to make fertilizers themselves.

To use our product, one can use our hardware scheme to make hardware capable of crushing seaweed into small pieces and going through fermentation with our genetically engineered bacteria. After a certain time based on the environment temperature, we can then.

(See the hardware section for details)

Our project marks a transformative leap towards sustainable agriculture through the development of genetically engineered freeze-dried bacterial powders. This pioneering approach, complemented by user-friendly hardware, enables the efficient production of alginate oligosaccharides from seaweed and potentially kitchen waste. Designed for fertilizer companies, farmers, and DIY enthusiasts, our eco-friendly solutions prioritize biosafety, ensuring responsible biotechnology practices. In summary, we aspire to revolutionize agriculture by providing practical, efficient, and environmentally conscious alternatives, contributing to a greener and more sustainable future in farming.

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[5]Qian Li, Ling Zheng, Zilong Guo, Tiancheng Tang & Benwei Zhu (2021): Alginate degrading enzymes: an updated comprehensive review of the structure, catalytic mechanism, modification method and applications of alginate lyases, Critical Reviews in Biotechnology, DOI: 10.1080/07388551.2021.1898330

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[8]Endoglucanase Exoglucanase β-glucosidase producing glucose

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