Marine biofouling—initiated by microbial biofilms and macro-organism adhesion such as barnacles—adds 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 mechanisms even at very low levels. (Cattò et al., 2015) [3], (Barrios et al., 2004) [4]. Low toxicity, biodegradability, and negligible ecological footprint make ZA a potential substitute for traditional antifouling chemicals. Despite this, commercial-scale application has been prevented by the prohibitive expense and ecological damage of chemical synthesis.
We utilize synthetic biology in this project to create engineered Escherichia coli strains that are capable of effective and sustainable biosynthesis of ZA. Here we engineer Escherichia coli for high-titer, low-cost ZA biosynthesis, following the strategies bellow:
Optimizing the E. coli metabolic pathway via gene knockouts (e.g., cysH) , overexpression of enzymes (e.g., KIATPSL, PcAPSK, aroGfbr) and modification of key enzymes (e.g., SULT1A1, TAL), for higher yield and efficiency in production. (Jendresen & Nielsen, 2019) [5], (Zhang et al., 2023) [6], (Liu et al., 2021) [7].
To translate the molecule into coatings, we develop silica-PLGA microcapsules and PVA/alginate double-network hydrogels that achieve a projected 12-month release profile in ASTM D1141 salt-spray–accelerated tests equivalent to ~1 year at 25 °C (Xue & Shi, 2004) [8].
By integrating metabolic engineering, smart materials and stakeholder outreach, we offer a non-toxic, scalable antifouling platform that can reduce hull-fouling fuel penalties by up to 26 % and maintenance costs by ≈30 % (Schultz et al., 2010) [1], (Biofouling, n.d.) [9].
Biofouling, driven by microbial biofilm formation and macro-organism attachment, is a major problem for ships and marine constructions. It is the accumulation of microorganisms, plants, algae, or small animals that is not wanted on surfaces such as ship and submarine hulls, devices such as water inlets, pipework, grates, ponds, and rivers, which can cause degradation to the primary purpose of that item. The following figure showcases the three phases of biofouling formation:
Biofouling attachment occurs in three key phases. First, a biofilm forms on submerged surfaces, starting with microbial colonization that develops into a mature biofilm, releasing chemical and physical cues. Second, barnacle larvae (cyprids)explore the surface, temporarily attaching, testing conditions, and if suitable, secreting cement for permanent settlementfollowed by metamorphosis. Finally, during the interaction phase, larvae detect and respond to ionic signals, quorum sensing molecules, EPS, and electric signals from the biofilm, which guide them to either commit to settlement or reject the surface.
Nonetheless, fish farms and the seafood industry have been significantly affected by biofouling pollution. As barnacles and other pollutant organisms cling to smooth surfaces, including hulls, fishing nets, and marine life, industries like fishing face increasing challenges. On one hand, annual fuel consumption rises from 2,300 tons without biofouling to 2,900 tons with it, representing about a 26% increase, which results in an additional $390,000 in fuel costs each year. On the other hand, farmers incur substantial expenses to clean their equipment for corrosion repair. Compared to the absence of biofouling, the cost of corrosion repairs caused by biofouling has increased by approximately 200%, and the frequency of repairs has doubled. Most existing chemical antifouling techniques are harmful to the environment. For example, zinc pyrithione [2] can become toxic to bivalves and crustaceans after hydrolysis. These toxic chemicals can harm fish and potentially affect the food we consume. Furthermore, chemicals can disrupt the balance of ecosystems, and excessive fouling by organisms can dominate, posing a risk to coral reefs and other natural habitats.
Simply put, marine biofouling isn't solely a problem for the fishing industry. It also impacts the condition of the ocean, the marine life we consume, and individuals employed in ocean professions. With increasing ship traffic and the construction of ocean infrastructure, the problem will only escalate.
Manual or mechanical removal
For facilities such as ships and docks, barnacles can be scraped manually using tools such as shovels and scrapers (Guo et al., 2020a). Small boats can also come into the shipyard and clean up the bottom of the ship after it is exposed. For barnacles on the surface of turtles, sea turtles, and other organisms, you can use a plastic spatula, knife, etc., to carefully scrape off, or soak them in warm water to soften them before scraping (Hopkins et al., 2021). In addition, the high-pressure water gun is also an efficient cleaning tool, using the impact and shear force of the high-pressure water jet to peel the barnacles, suitable for large areas of adhesion (Lee et al., 2022; Guo et al., 2020b).
Change the attachment environment
Prevent the attachment of barnacles by changing the physical properties, such as surface energy and color of the attachment. For example, the use of low surface energy materials such as fluoropolymers and silicone resins makes barnacles difficult to adhere (Swain & Schultz, 1996; Brady & Singer, 2000). It is also possible to change the color of the attachment base, as barnacle larvae have a preference for certain colors and avoid colors that attract them, such as orange and green (Maki et al., 1992; Satheesh & Wesley, 2010).
Marine antifouling coatings
Insecticide-containing antifouling coatings are the most commonly used types in the market, including hydrated self-polishing antifouling coatings (Gong et al.,2025), hydrolyzed self-polishing antifouling coatings, and hybrid antifouling coatings (Zhang et al.,2023). Insecticide-free antifouling coatings, such as silicone, organofluorine, and other low surface energy antifouling coatings (Gong et al.,2024), as well as biomimetic antifouling coatings (Gong et al.,2023), electrolytic chemical antifouling coatings, nano antifouling coatings (He et al.,2021), etc., are also being developed and applied. These coatings prevent barnacles from adhering by releasing antifoulants, changing the surface properties of the coating (He et al, 2021), etc.
Chemical cleanup
Use specialized barnacle cleaners, but these cleaners are usually more toxic and should be used with caution, and are mainly used for cleaning specific equipment or small areas. Barnacles can also be prevented by electrolysis of seawater to produce substances such as sodium hypochlorite in seawater cooling, circulation systems, and seawater piping systems on offshore platforms and ports.
Use of Natural Predators
In some breeding areas, natural enemies of barnacles can be introduced to control their populations, such as starfish, crabs, certain fish and nudibranch-like nudibranchs, which prey on barnacles or their larvae.
Antifouling of bioactive substances
Bioactive substances are extracted from natural and pollution-free chili peppers and other organisms, and compounded with organic clay to make antifouling agents, which can prevent barnacles from adhering. Biomimetic antifouling coatings can also be developed to prevent barnacles from attaching by mimicking the characteristics of antifouling organisms in the ocean.
Micro-nano topology
-Shark skin bionic grooves
The shark skin shield scale structure is simulated (20-200 μm gradient grooves), the diatom attachment rate decreases by 83% when the depth-to-width ratio is >1.5, and passive desorption is realized through water shear. Wettability reflects the ability or tendency of a liquid to wet a solid surface and then diffuse over a given solid surface. Wettability can be studied by measuring the contact angle between the substrate and a given liquid. Young's equation describes the equilibrium between the solid, liquid, and gas phases on an ideally smooth surface. (See the formula below.)Where γSG, γSL, and γLG are the surface energies of solid/gas, solid/liquid, and liquid/gas, respectively (Li et al., 2023).
Natural Antifouling Agent Extraction
-Macroalgae Active Substance
The U.S. Army extracts antifouling agent from marine macroalgae (only 1 μg/mL), which inhibits microorganisms, algae, and barnacles in a broad spectrum and is environmentally non-toxic.
-D-type amino acid chiral material
Xiangyu Li's team at Northeastern University developed a metal-organic framework (MOF) coating that interferes with bacterial community sensing through a chiral structure to achieve self-triggered antifouling and biofilm dispersal (W. He et al., 2024).
Enzyme-catalyzed antifoulingn
-Haloperoxidase (HPO) mimetic material
Fujian Normal University Zhu Hu team synthesized copper-dipeptide hybrid nanomaterials (HH-Cu), which maintain high activity at seawater pH (7.8-8.2) and low temperature (0-4℃), with a sterilization rate of >90%, and no attenuation of activity in 10 times of repeated use.
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.
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.1). 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.
Some authoritative literature and practical ship research have successfully incorporated Zosteric Acid (ZA) into coatings and verified its anti-fouling properties. Substances such as Slygard 184 silicone rubber (Carlos A. Barrios, 2005), RTV-11 high-filler silicone rubber, and ZSM-5 nano-zeolite (Boopalan M.,), 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.
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.
Here's a reference to the metabolic engineering of Escherichia coli for the production of zosteric acid (Zhang et al., 2023).
SULT1A1, TAL, KIATPSL, and PcAPSK are four key enzymes that are crucial for the synthesis of zosteric acid.
SULT1A1 is a highly efficient sulfotransferase with an extremely high substrate utilization rate for p-coumaric acid. It uses PAPS as the sulfate donor to form sulfated compounds. TAL catalyzes the non-oxidative deamination of L-Tyr to biosynthesize p-hydroxyphenylacetic acid (pHCA). KIATPSL and PcAPSK are two heterogeneous expression enzymes. The recombinant strain constructed with KIATPSL-PcAPSK can significantly increase the yield of ZA and reduce the production of pHCA.
In summary, our project is not about "proving whether ZA can be applied to coatings"; literature has already provided sufficient answers. Our core goal is to increase the yield and reduce the cost of ZA through the biosynthetic (biosyn) route, making this green anti-fouling molecule truly feasible for large-scale commercial application. The biological method can break free from the expensive raw materials and unstable supply of chemical extraction, thus making it affordable for each ton of coating to carry a sufficient dosage of ZA.
Our wetlab strategy is fundamentally built upon the landmark study by DTU (Jendresen et al., 2019), where microbial production of Zosteric Acid (ZA) in E. coli had been realized. We drew upon three principal strategies from this work:
1. PAPS pool enhancement by knockout of cysH to suppress PAPS drainage through competing routes
2. Overexpression of CysDN/cysC to increase PAPS
3. Introduction of TAL and SULT1A1 to construct the L-tyrosine → ZA biosynthetic pathway
We developed this platform by adding more modules from Zhang et al., 2023. This work established the first biosynthetic pathway for Zosteric Acid (ZA) in E. coli through systematic metabolic engineering. It involves the overexpression of PAPS synthases, KIATPSL, and PcAPSK, to maximize the PAPS supply.
We drew upon (Liu et al., 2021) regarding enzyme engineering approaches focused on PcAPSK. It enlightens us to employ rational fusion and AI-augmented enzyme engineering to increase catalytic flux in crucial enzymes like TAL and SULT1A1, boosting efficiency beyond the reference baseline.
The iterative construction and optimization process for the E. coli strain is divided into 3 modules and 6 cycles, as outlined below:
Cycle 0: Reduce the waste of PAPS, a key precursor of ZA
Knock out cysH. cysH encodes PAPS reductase, which diverts PAPS to sulfite for cysteine biosynthesis.
Knocking out cysH blocks this competing pathway. Thus, it can conserve the sulfate donor so that SULT1A1 can efficiently sulf-ate pHCA. Thereby boosting Zosteric Acid titres.
Cycle 1: Initial strain construction (Module I)
Module I: It constitutes the minimal Zosteric-Acid biosynthetic cassette in E. coli:
Introduce exogenous genes to enable the heterologous expression of sulfotransferase SULT1A1 and tyrosine ammonia-lyase (TAL) to ensure that ZA can be produced.
Cycle 2: Enhance PAPS accumulation (Module II)
Plasmid 2A: Overexpression of cysDN and cysC enhances ATP-to-PAPS conversion efficiency.
Plasmid 2B: Heterologous expression of KIATPSL and PcAPSK: Introduce exogenous enzymes to further boost PAPS synthesis.
Plasmid 2C: combine all the endogenous and exogenous enzymes to investigate whether there will be an even larger yield of PAPS with a higher efficiency
Cycle 3: Enhance the key enzyme for L-tyrosine supply (Module III)
Overexpression of aroGfbr: Strengthen key enzymes in the aromatic amino acid biosynthesis pathway.
Overexpression of aroGfbr in the plasmid constructed in cycle 1: an attempt to reach a higher yield of ZA.
Cycle 4: Validate synergistic effects of all modules and design fusion protein
The fusion protein, which could be a flexible linker, might increase the yield of ZA
Cycle 5: Optimizing TAL and SULT1A1 in combination with dry lab(modelling)
Using the methods of mathematical modeling and AI prediction, TAL and SULT1A1 may be optimized to make them have a stronger affinity and higher catalytic efficiency.