Implementation

Overview

Aquatic products, highly valued for their abundant protein content and essential nutritional benefits, are a popular food source. According to official statistics, in 2024 China's total aquatic product output reached 73.5759 million tons, generating a fishery output value of 167.5480 billion yuan, with a national per capita consumption of 52.25 kg (1). However, their high protein and moisture content render them particularly susceptible to spoilage and deterioration from microbial contamination after harvesting. This spoilage not only shortens shelf life and leads to resource losses during capture, transportation, and storage, but also poses significant health risks to consumers. Spoiled aquatic products may accumulate histamine toxins and harbor pathogenic bacteria, potentially leading to foodborne illnesses such as diarrhea, vomiting, and even acute poisoning (2, 3).

In our project CRUSTA, we identified a variant of chitosanase SaCsn46A (BBa_25C3NO7W) after several rounds of random mutation whose degradation products exhibit exceptionally strong antibacterial activity (see Design for details). These compounds can effectively inhibit spoilage-causing microorganisms on the surface of food. Through this approach, our project aims to enhance food safety, lower the carbon footprint of food preservation, and contribute to the realization of a sustainable ocean circular economy.

1. Potential Users

Our project has the potential to greatly benefit chitooligosaccharides (COS) producers, fishing and fish processing enterprises, and researchers.

1.1 COS Producers

The production of COS predominantly utilizes chemical and physical methods. Chemically, COS is prepared through degradation by acids or oxidizing agents, but these processes pose control challenges and often result in products with poorly defined structures. They additionally create pollution and generate significant waste due to excessive hydrolysis from acid-alkali interaction. Physical methods, such as microwave radiation, can degrade COS but usually require advanced equipment and make precise control of degradation difficult. In our project CRUSTA, we successfully screened a variant of chitosanase SaCsn46A (BBa_25C3NO7W) that represents a promising candidate for enzymatic industrial production of COS, enabling their rapid and large-scale synthesis. COS producers can integrate this enzyme variant into industrial pipelines to scale up high-value COS production while lowering operational costs.

In the future, they can produce COS based on the route developed in our CRUSTA project through synthetic biology, which has the potential to reduce chemical waste, carbon emissions, and production costs. Manufacturers can also benefit from our technological solutions, as the polymerization of COS in production is more controllable, the process is simplified, and the costs are lower.

In the aspect of production of chitosan oligosaccharides, the enzymatic approach decreases about 99.1% of CO₂ emissions and 96.4% of cost compared to the chemical one. Additionally, our method saves about 61.0% of CO₂ emissions and 71.7% of cost compared to the chemical production of traditional preservative, potassium sorbate, in the same functional performance (see LCA for more details).

1.2 Food processing enterprises

We applied COS to food preservation and observed significant inhibitory effects on microbial growth (see Results for details), demonstrating the strong potential of COS as a novel biological preservative. If applied to the preservation of aquatic products as well as fruits and vegetables, COS could reduce the need for strict refrigeration during transportation. This would lower water consumption associated with refrigeration, decrease equipment costs for enterprises, and enhance food safety, while simultaneously contributing to carbon emission reduction.

1.3 Researchers

This year, we enhanced the signal peptide LMT, originally discovered by our team in 2021 (XMU-China 2021, XMU-China 2023, XMU-China 2024), by introducing targeted mutations, resulting in a significant improvement in excretion of protein. For proteins required in industrial-scale quantities, this signal peptide enables extracellular secretion through engineered strains, thereby eliminating the need for complex purification steps during production. This provides research groups and iGEM teams worldwide with an effective method and platform for obtaining large amounts of target proteins without relying on time-consuming purification procedures.

2. Usage

Co-incubation of the chitosanase mutant with chitosan under optimal conditions produces substances with significant antibacterial properties. The application of this solution to aquatic products, either by soaking or spraying, effectively inhibits microbial growth, thereby slowing deterioration and extending their shelf life.

3. Safety

3.1 Suicide Switch

To prevent engineered bacteria in the chitosanase production chain from contaminating other processes and posing biosafety risks, we designed two independent suicide switches. The first is a ccdB/ccdA toxin-antitoxin system regulated by cuminic acid, and the second is an IPTG-inducible system that triggers overexpression of the tyrS gene. Together, we integrated these two systems into a same plasmid, they can operate independently and provide mutual backup for each other, thus guarantee rapid cell death, keeping the engineered bacteria in a controllable state (see Design for details).

3.2 Risk Assessment

The use of engineered bacteria in our program may pose potential risks to public health and the environment. Therefore, before deploying these strains beyond the laboratory, comprehensive testing must be conducted to confirm their safety for both humans and ecosystems.

China implemented the Biosafety Law on April 15, 2021, and amended it in April 2024. This legislation establishes a comprehensive biosafety risk prevention and control system, covering risk monitoring and early warning, risk investigation and assessment, and information-sharing mechanisms. It also includes provisions for the prevention, control, and response to specific biosafety risks, such as major emerging infectious diseases, epidemic outbreaks, and the research, development, and application of biotechnology. Accordingly, if we advance the application of engineered bacteria, we will apply for the necessary permits and approvals in compliance with this law.

3.3 Food Safety

In China, COS is classified as a novel food ingredient. The National Health Commission (NHC) has approved COS following the guidelines established by the Food Safety Law of the People's Republic of China and the Administrative Measures for the Safety Review of New Food Ingredients. This designation requires safety assessment and approval before their use in both general and health foods. Product data from the National Medical Products Administration (NMPA) reveals that edible COS is predominantly sold in capsule form (4).

In the United States, chitosan has undergone multiple submissions for Generally Recognized as Safe (GRAS) notifications, with the FDA consistently raising no objections and affirming its safety. However, COS has not yet been granted GRAS status. The FDA expert panel further concluded that veterinarians and caregivers have minimal concerns when administering SYNOPLEX (an antimicrobial product containing COS), noting that COS-based products have already been applied as medical devices in humans. To date, no allergic reactions or adverse events have been reported (5, 6).

Within the European Union, the European Commission's Food Safety Panel permits the use of chitosan as a nutraceutical or food supplement. Studies show that chitosan powders, incorporated into pasta and bakery products are safe, well-tolerated, and non-toxic, and can be integrated into a balanced diet as a substitute for conventional pasta. However, the application of COS in food products has not been specifically addressed (7).

4. Future plan

4.1 Patent Protection

Our CRUSTA program features two major innovations. First, we have identified a variant of chitosanase SaCsn46A (BBa_25C3NO7W) characterized by its remarkably potent bacteriostatic activity. Second, we developed a variant of the signal peptide (BBa_25LUSMUT) with significantly higher protein excretion than the original. These innovations will be safeguarded through patents, securing a competitive edge. Patent protection will enhance constructive collaborations with industry stakeholders and support sustainable development.

4.2 Industrial Production

Because we have obtained an efficient signal peptide and a chitosanase variant, it is possible to rapidly produce large quantities of both the enzyme and COS products. This demonstrates the feasibility of applying our design to industrial-scale production. By bringing these innovative technologies into practice, we aim to advance the development of the COS industry as well as other sectors that utilize engineered bacteria for protein production.

4.3 Food Preservation

The success of our laboratory research marks only the first step for CRUSTA. We plan to scale up production and commercialize our products. As the next stage, we plan to establish a company dedicated to scaling up and pilot testing the production process. This will include optimizing enzyme production and chitosan degradation, as well as implementing a rigorous quality control system to ensure that every batch consistently delivers stable and effective bacteriostatic performance. In parallel, we will strengthen our ties with potential partners by participating in industry exhibitions, while actively building a global sales network to enhance brand awareness and accelerate market entry. To realize this vision, we will seek strategic investments and partnerships, directing funds toward advancing technological research, improving enzyme and protein performance and productivity, and developing a robust new product pipeline to maintain our position at the forefront of industry innovation.

From a competitive perspective, the CRUSTA project is uniquely positioned in the global market for antibacterial products, underpinned by our patented high-performance variant of chitosanase SaCsn46A (BBa_25C3NO7W) and our highly efficient signal peptide variant (BBa_25LUSMUT). Our core advantage lies in the superior antibacterial efficacy of our enzymatic products, which not only provide a natural and safe alternative to traditional chemical preservatives (e.g., sodium benzoate) but also deliver performance and cost advantages over other natural preservatives (e.g., bio-based or botanical extracts) thanks to faster onset of action and strong patent protection. By licensing our technology to industry partners with established distribution channels and brand recognition, we can rapidly penetrate high-demand markets such as food preservation, cosmetics, and pharmaceuticals, promoting the adoption of natural, highly effective preservative solutions and reshaping the traditional preservative market.

In terms of cost structure, our initial investments will focus on R&D (Research & Development) optimization, international patent protection, and product compliance certifications—critical prerequisites for securing technological leadership and market exclusivity. Once scaled, our high-efficiency signal peptide technology will reduce downstream separation and purification costs. Additionally, the strong antimicrobial potency of our products enables efficacy at lower dosages, further lowering the total cost of ownership for end users. This combination of high performance and cost efficiency positions CRUSTA as not only a technologically advanced solution but also an economically attractive choice, creating long-term sustainable value (see LCA for details).

The industrialization strategy of the CRUSTA project is grounded in the principles of sustainable development. By using chitin derived from crustacean waste as a raw material, we significantly reduce the environmental burden of waste disposal. Moreover, our enzymatic hydrolysis process is environmentally friendly and energy-efficient, minimizing industrial pollution and resource consumption while contributing to global carbon neutrality targets. Importantly, chitosan-derived end products are fully biodegradable, allowing them to naturally reintegrate into ecosystems after fulfilling their antimicrobial function. This approach maximizes resource utilization and conservation, aligning with circular economy policies and global market demands. By embedding environmental responsibility at the heart of our design, CRUSTA delivers not only economic benefits but also distinctive green value—realizing the vision of “sourced from aquatic products, used for aquatic products”.

References

1. 2024 national fisheries economic statistics bulletin (in chinese). https://yyj.moa.gov.cn/gzdt/202507/t20250707_6475475.htm.
2. O.A. Odeyemi, C.M. Burke, C.C.J. Bolch, R. Stanley, Seafood spoilage microbiota and associated volatile organic compounds at different storage temperatures and packaging conditions. Int. J. Food Microbiol. 280, 87-99 (2018).
3. D.L. Nevado et al., Detection, identification, and inactivation of histamine-forming bacteria in seafood: A mini-review. J. Food Prot. 86, 100049 (2023).
4. Announcement on the approval of six new food ingredients including chitosan oligosaccharide (no. 6 of 2014) (in chinese).
   https://www.nhc.gov.cn/wjw/c100175/201405/8cd4e5d85d6b412cbb00edb2652bfb2b.shtml.
5. Original request for addition to the index of legally marketed unapproved new animal drugs for minor species, U.S.F.a.D. Administration, Editor, (2012).
6. Gras notice (grn) no. 997 with amendments, U.S.F.a.D. Administration (2021).
7. Summary of the application: Powder of chitosan, I.M.o.H.i.c.w.R. (EC) (2015)