1. Module I focuses on converting L-tyrosine to Zosteric Acid. The reference introduced two key enzymes: TAL and SULT1A1. These enzymes convert L-tyrosine to pHCA and then to Zosteric Acid, forming the core of our biosynthesis process.
Abstract
Marine biofouling—initiated by microbial biofilms and macro-organism adhesion imposes billions of dollars in extra fuel, maintenance and emissions costs to global shipping and aquaculture each year (Schultz et al., 2010) [1]. Conventional copper- or silicone-based coatings face regulatory bans and short service lives due to toxicity or poor durability (IMO：Anti-fouling Systems, n.d., 2023) [2].
Naturally derived Zosteric Acid (ZA), a metabolite of seagrass (Zostera marina), possesses intense anti-biofilm activity by inhibiting microbial adhesion and quorum sensing, while remaining non-toxic and environmentally benign (Cattò et al., 2015) [3]. However, chemical synthesis is costly and unsustainable, limiting its commercial adoption. To overcome this, we engineered E. coli strains capable of high-yield, low-cost ZA biosynthesis through metabolic pathway optimization and sulfotransferase engineering.
HullGuard integrates wetlab, drylab, human practices, and entrepreneurship to deliver a sustainable antifouling solution:
Wetlab: Constructed engineered E. coli strains, optimized key enzymes such as TAL and SULT1A1, and designed cysH knockouts to enhance ZA production.
Drylab: Guided by ProtSSN residue scoring, ΔΔG stability prediction, and ConSurf conservation analysis, we designed and screened multiple site-directed mutations of SULT1A1. We also engineered a synthetic linker between TAL and SULT1A1 to enhance substrate channeling and catalytic efficiency.
Human Practices: Collaborated with shipyards, coating companies, and policymakers to ensure feasibility and regulatory alignment, and proposed policy recommendations on toxic paint regulation.
Education & Outreach: Designed large-scale public engagement activities to promote awareness of synthetic biology.
Entrepreneurship: Established two commercial models—Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA)—to evaluate cost-effectiveness and environmental sustainability of HullGuard as a scalable antifouling platform.
HullGuard is not only a technical innovation in antifouling but also a practical step toward greener shipping and sustainability, aligning with IMO goals and the United Nations SDGs.
Background
In recent years, the maritime transportation industry has faced persistently high fuel costs. However, the more pressing issue lies beyond economics: increased fuel consumption directly translates into higher CO₂ emissions, accelerating global climate change and global warming. For example, biofouling alone can raise a ship's fuel consumption by 5% to 86%. A medium-sized cargo ship may burn an additional 2 tons of fuel per day due to biofouling, incurring around $1,000 in extra cost, while a large tanker may consume an extra 5 tons of fuel daily, costing about $2,500 (Schultz et al., 2011)[1]. This heightened consumption not only imposes economic burdens but also significantly worsens the environmental footprint of global shipping, contributing to greenhouse gas emissions and marine ecosystem degradation.
Figure 1. Different positions within the hull where biofouling gathers(left); Fishing Net and Barnacles: A Marine Biofouling Scene(right) Source: pinterest.com
Biofouling, driven by microbial biofilm formation and the attachment of macroorganisms, has thus been recognized as a critical challenge in the maritime industry. It is the accumulation of microorganisms, algae, or small animals on surfaces such as ship hulls, fishing nets, pipes, and water inlets. Beyond shipping, biofouling also imposes substantial losses on aquaculture. In 2016, world aquaculture production reached 110.2 million tonnes (US$243.5 billion), yet biofouling was estimated to account for 5–10% of production costs (Bannister et al., 2019b)[12]. Although the precise global economic impact remains difficult to quantify, it is likely underestimated given the indirect costs across different sites, species, and management practices (Fitridge et al., 2012)[13].
Figure 3. Barnacles attached to ships
Most existing antifouling solutions remain inadequate. Physical methods are often inefficient and costly to maintain, while chemical methods, although widely applied, can be environmentally harmful or toxic. Thus, rather than mitigating the problem, many of the current solutions inadvertently create new ecological risks.
Simply put, if sustainable, advanced, and cost-effective antifouling strategies are not developed, the issue will only intensify with rising global shipping traffic and the expansion of marine infrastructure. Addressing biofouling in a more effective and greener way is therefore not only about reducing fuel costs for the shipping industry but also about mitigating climate change, protecting marine biodiversity, and ensuring the long-term health of oceans.
Current Solution
Current biofouling mitigation strategies include physical methods, such as altering the attachment environment to prevent adhesion (Swain & Schultz, 1996; Brady & Singer, 2000) [25][26] and manual or mechanical removal, while effective, tend to be labor-intensive or energy-consuming. chemical methods, such as superoleophobic surfaces (Xia et al., 2021) [18] and the use of antifouling biocides. However, these methods are often characterized by low stability in sea water(Xia et al., 2021) [18] or toxicity (Ferreira et al., 2021b) [19].
PHYSICAL METHODS
Manual/mechanical removal
Changing the attachment environment
✅ Environmentally friendly
❌ May damage surfaces, limited effectiveness
CHEMICAL METHODS
Superoleophobic surfaces
Copper compounds
Antifouling biocides
✅ Strong antifouling effect
❌ Toxicity & low stability in seawater
BIOLOGICAL METHODS
Natrual predators
Micro-nano topology
Bioactive substance
✅ Eco-friendly, sustainable
❌ Limited effectiveness validation
In summary, while current solutions offer various approaches to combat marine biofouling, they often come with trade-offs in terms of cost, environmental impact, and effectiveness.
New Antifouling Solutions
The entire process of biofouling not only increases surface roughness and resistance but can also lead to equipment failure, increased maintenance costs, and environmental pollution. Therefore, understanding and controlling the formation of biofouling is crucial for many industrial and environmental applications. The following figure showcases the four phases of biofouling formation:
Figure 4. Marine Biofilm Formation: Stages of Substrate Colonization
Marine biofouling progresses through four stages: rapid formation of a conditioning film/biofilm upon immersion, colonization by bacteria and diatoms, settlement of larger organisms such as Ulva spores and barnacle larvae, and eventual development into a complex, stable community dominated by various fouling organisms. (Schultz et al., 2010) [11]
Our approach is fundamentally inspired by the formation mechanism of biofouling and its developmental stages. Recognizing that biofilm establishment initiates the entire fouling cascade, we strategically target inhibition at this earliest phase. By preventing microbial adhesion and biofilm maturation, we address the root cause of biofouling formation—effectively , the foundation that enables larger organisms to colonize surfaces. This proactive strategy offers a sustainable, affordable, and long-term solution to marine fouling challenges.
Zosteric Acid (ZA), a naturally occurring sulfated phenolic compound derived from eelgrass (Zostera marina), exhibits exceptional antifouling properties at low concentrations to resist the attachment of bacteria, fungi, and even macrofouling organisms (table 2.). Its simple molecular structure, negligible ecological toxicity, and low bioaccumulation potential make it an environmentally sound alternative to traditional biocides. It operates by disrupting microbial adhesion and biofilm development through site-blocking interactions, preventing the critical initial attachment phase.
| Classification | Target Organisms | ZA Concentrations | Reduction of Biofilm / % | Reference |
|---|---|---|---|---|
| Bacteria | Lake Erie bacteria | 50 mg/L | 92.5% | (Xu et al., 2005) [40] |
| Bacteria | P. putida | 500 mg/L | 98.2% | (Xu et al., 2005) [40] |
| Fungi | Candida albicans | 10 mg/L | 70% | (Villa et al., 2011) [38] |
| Macrofouling organisms | Balanus eberneus (barnacles) | Flux of 500 μg·cm⁻²·d⁻¹ | 50% | (Haslbeck et al., 1996) [39] |
Table 2. ZA antifouling ability and effective concentrations across organisms.
Some authoritative literature and practical ship research has successfully incorporated Zosteric Acid (ZA) into coatings and verified its anti-fouling properties. Substances such as Slygard 184 silicone rubber (Carlos A. Barrios, 2005) [4], RTV-11 high-filler silicone rubber, and ZSM-5 nano-zeolite (Boopalan M.,2011) [37], when combined with ZA, have demonstrated that regardless of whether the matrix is silicone rubber, epoxy, or other resins, adding ZA to the coating film can significantly reduce the attachment of marine organisms, extending the anti-fouling duration from several months to over ten years.
| Products | Method / Property | Release Efficiency | Duration | Antifouling Effect | Reference |
|---|---|---|---|---|---|
| Entrapment of ZA into Silicone Coatings (Sylgard 184) | Direct ZA powder mixing coating | Initial: 437 μg/cm²·day → 1.5 μg/cm²·day | Less than 2 months | Effective in reducing bacterial attachment | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [41] |
| Water solvent coating | Initial: 15.7 μg/cm²·day → 0.2 μg/cm²·day | 5 years | Demonstrates anti-bacterial attachment ability | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [41] | |
| Acetone / water solvent coating | Initial: 0.12 μg/cm²·day → 0.03 μg/cm²·day | 21 years | Bacterial coverage ≈30% of pure silicone coatings, significantly reduced attachment | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [41] | |
| Pyridine / water solvent coating | Initial: 0.64 μg/cm²·day → 0.07 μg/cm²·day | 12 years | Significantly reduces bacterial attachment compared to pure silicone coatings | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [41] | |
| ZA loaded in Zeolite Hybrid Polymer Coatings | ZA: ZSm-5(1:2) + Epoxy | Initial ≤16 μg/cm²·day → <1 μg/cm²·day (steady) | 12 months continuous seawater immersion | 6 mo: almost no bacterial colonisation; 12 mo: very thin biofilm, no macrofouling — far cleaner than biocide-free control | (Boopalan M., 2011) [37] |
Table 3. Coating applications related to Zosteric Acid (ZA).
These results indicate that the incorporation method of Zosteric acid significantly affects its release rate and the service life of the coating. Loading ZA into ZSM-5 nano-zeolite and hybridizing it with epoxy resin, after 12 months of exposure in the South China Sea, only a very thin biofilm was formed, and the electrochemical corrosion rate was reduced by two orders of magnitude, showing long-term protection potential under seawater conditions.
Researches above have demonstrated that Zosteric Acid (ZA) is an eco-friendly compound with notable anti-biofilm properties. Currently, ZA is obtained through two primary methods: extraction from seagrass, which is limited by low natural abundance and high cost, and chemical synthesis, which poses serious environmental concerns due to pollution. There is an urgent need to establish efficient ZA production platforms to facilitate its practical use in eco-friendly ship coatings
Our Solution
To overcome these limitations, our project employs synthetic biology to develop engineered microbial strains for high-yield ZA production, offering a sustainable and efficient alternative for green antifouling applications.Therefore, employing synthetic biology to biosynthesize ZA is not only necessary but the most sustainable and scalable solution to achieve eco-friendly antifouling production. Accordingly, we have conducted extensive research and found that Escherichia coli has been established in the literature as a model organism for ZA production (Zhang et al., 2023)[6]
Figure 5. Metabolic pathway of Zosteric acid
The process is divided into two modules and some crucial enzymes are required.
2. Module II ensures a stable supply of PAPS, a crucial cofactor for SULT1A1. A sulfur metabolism pathway is engineered by knocking out cysH, overexpressing cysC and cysDN, and introducing KIATPSL and PcAPSK. This guarantees sufficient PAPS for ZA biosynthesis.
Our project is fundamentally built upon the landmark study by Jendresen, 2019[5] and Zhang et al., 2023[6] [41], which established the microbial production of Zosteric Acid (ZA) in E. coli . We adopted three principal strategies from these studies:
- Enchancing PAPS pool by knockout of cysH to suppress PAPS drainage through competing routes;
- Increasing PAPS supply via heterologous expression of KIATPSL and PcAPSK;
- Constructing the biosynthetic pathway from L-tyrosine to ZA by introducing TAL and SULT1A1.
In addition, inspired by the exzyme engineering work of Liu et al. (2021) [7], we applied AI-aid and semi-rational design to improve the catalytic efficiency of SULT1A1, while optimizing the co-expression of TAL and SULT1A1 to enhance substrate circulation and boost ZA yield. Meanwhile, we also evaluated the biofilm inhibition capability of the ZA-rich fermentation product using three substrates: 96-well plates, tubes, and slides.These mutilevel approaches allow us to evaluate Zosteric acid antifouling efficiency from laboratory-standard conditions to ocean application-oriented environments.
Conclusion
In summary, our project’s focus is not to demonstrate whether ZA can be incorporated into coatings—that question has been answered by prior studies. Rather, we aim to make ZA commercially feasible and ecologically responsible by developing an optimized biosynthetic (synbio) production route, potentially enabling each tonne of coating to carry an effective dosage. Central to our approach is a conservation-first philosophy: By reducing the use and discharge of harmful chemicals helps alleviate long-term pressures on marine biodiversity, it is possible to achieve a win–win between economic benefits and ecological conservation, and promoting the safe adoption and widespread application of green antifouling technologies in global shipping and marine coatings, delivering both economic viability and measurable benefits for biodiversity protection. In doing so, we contribute to advancing the United Nations Sustainable Development Goals (SDGs), particularly those related to ocean health, biodiversity conservation (SDG 14, SDG 3), and sustainable industry (SDG 8, SDG 9, SDG 4, SDG 12).
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