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.
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, 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 below.
Drawing insights from multiple metabolic engineering studies for ZA production (Jendresen & Nielsen, 2019) [5], (Zhang et al., 2023) [6], high-yield strains have been constructed by overexpressing key enzymes (e.g., KIATPSL, PcAPSK, aroGfbr) (Liu et al., 2021) [7]., modifying variants of SULT1A1 and TAL, and performing targeted gene knockouts (e.g., cysH) to optimize the metabolic pathway for enhanced yield and efficiency.
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 where it is not wanted on surfaces such as ship and submarine hulls, devices such as water inlets, pipework, grates, ponds, and rivers that cause degradation to the primary purpose of that item. The following figure showcases the four phases of biofouling formation:
Figure 1. Marine Biofilm Formation: Stages of Substrate Colonization
The attachment of barnacles to submerged surfaces in the marine environment follows a progressive four-stage process (Sarkar, P. K., 2022) [11]. It begins with Stage I, where an unimpeded substrate is immersed in seawater and rapidly develops a conditioning film, forming a thin biofilm. In Stage II, this surface becomes host to the primary colonizers—mainly bacteria and diatoms—which establish a microbial community. As the biofilm matures, Stage III involves secondary colonization, characterized by the settlement of larger organisms like Ulva spores and barnacle larvae. Finally, in Stage IV, known as tertiary colonization, the surface becomes densely populated with Ulva, algae, and barnacles, forming a complex and stable fouling community. This multi-step process illustrates the ecological succession and increasing biological complexity of marine biofouling.
Figure 2. Fishing Net and Barnacles: A Marine biofouling Scene
In particular, fish farms and marine transport industry have been significantly affected by biofouling. As barnacles and other fouling organisms cling to smooth surfaces, including hulls, fishing nets, and marine life, industries like fishing face increasing challenges. First, biofouling can increase a ship's fuel consumption by 5% to 86%. A medium-sized cargo ship may consume an additional 2 tons of fuel per day due to biofouling, at an extra cost of $1,000. A large tanker may consume an additional five tons of fuel per day, at an extra cost of $2,500[56]. Second, world aquaculture production was 110.2 million tonnes (US$243.5 billion) in 2016, compared to the absence of biofouling, the direct economic loss in the management of biofouling for the aquaculture sector has been predicted to be 5–10% of production costs. (Bannister et al., 2019b)[12] However, the economic cost of biofouling varies greatly at different aquaculture sites, species and companies, in that farmers utilize dissimilar management schemes and cost accounting. Besides, the majority of these indirect effects cannot be quantified, therefore the overall economic cost of marine aquaculture biofouling is obscure but in all likelihood significantly underestimated. (Fitridge et al., 2012)[13]
Most existing chemical antifouling techniques are harmful to the environment. For example, zinc pyrithione (Amara et al., 2017) [10] 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.
Current biofouling mitigation strategies include 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, 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. Biological approaches, including the use of natural predators, bioactive antifouling substances, and micro-nano topologies, show promise, though their implementation is often complex. Despite these advances, trade-offs between efficacy, cost, and environmental impact continue to present significant challenges. However, ZA, as a natural bioactive substance with protective properties, presents a promising solution.
| Current solution | Detail | Advantages | Disadvantages | Reference |
|---|---|---|---|---|
| Physical methods | Manual or mechanical removal |
1. Efficient and fast 2. Cheap 3. Physical removal is relatively environmentally friendly |
1. May damage the surface of the object 2. Difficult to remove completely |
(Li et al., 2023) [30] (The Environmental Literacy Council, 2025) [31] (Frontiers (2024)) [32] |
| Change the attachment environment | Environmental friendly and high sustainability | The effect is uncertain since the attachment of barnacles is affected by a variety of factors | (ScienceDaily: Your Source for the Latest Research News, n.d.) [33] |
|
| Chemical methods | Superoleophobic surface: eg. PDMS\HSS-PZS coating |
1. Able to prevent the adhesion of oil stains on different substrates. Could prevent bacterial attachment and subsequent biofilm formation 2. The coating can be fabricated on large scale |
The stability of most traditional superoleophic coating is unsatisfactory in sea water |
(He et al . ,2021) [17] ( Xia et al. ,2021) [18] (Ferreira et al., 2021b) [19] |
| Copper compounds eg.Copper(I) oxide | effectively prevents the growth of barnacles, algae, and other marine organisms | The heavy metal pollution in the ocean accumulates through the food chain | (Amara et al., 2017b) [37] |
|
| Antifouling biocide | able to prevent the attachment of marcorganisms by killing them. | Toxic to target species and may lead to physiological disturbance of some creatures |
(Ferreira et al., 2021b) [19] (Silva et al., 2021b) [20] |
|
| Biological methods | Natural predator |
1. Environmental friendly and sustainable 2. Avoid bioaccumulation |
1. Risk of invasive species 2. Limited effectiveness time consuming |
(Bannister J. Et al. ,2019) [12] |
| Micro-nano topology |
1. Long term effective 2. Non-toxic |
Only effective on a narrow range of fouling organisms |
(Carman M. L., et al. 2006)[34] (Bers A. V., Wahl M., et al. 2004) [35] |
|
| Bioactive substance |
1. Broad spectrum activity 2. Environmental sustainability, for its low toxicity and good ecosystem compatibility |
1. High cost for production 2. Leaching issues, degrading issues |
(Yebra D. M., et al. 2004) [36] |
Table 1. Advantages and disadvantages of current strategies for antifouling
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. Our project aims to address these challenges by leveraging the unique properties of Zosteric Acid (ZA) as a natural antifouling agent.
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 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) [42] |
| P.putida | 500mg/L | 98.2% | (Xu et al., 2005) [42] | |
| Fungi | Candida albicans | 10mg/L | 70% | (Villa et al., 2011) [40] |
| Macrofouling organisms | Balanus eberneus (barnacles) | Flux of 500 μg-1cm-2d-1 | 50% | (Haslbeck et al., 1996) [41] |
Table 2. ZA antifouling ability and effective concentration
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) [38], 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 zosteric acid into silicone coatings Sylgard 184 | Direct ZA powder mixing coating | Initial: 437μg/cm²·day, then reduce to 1.5μg/cm²·day | Less than 2 months | Effective in reducing bacterial attachment | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [43] |
| Water solvent coating | Initial: 15.7μg/cm²·day, then reduce to 0.2μg/cm²·day | 5 years | Demonstrates anti-bacterial attachment ability | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [43] | |
| Acetone/water solvent coating | Initial: 0.12 μg/cm²·day, then reduce to 0.03μg/cm²·day | 21 years | Bacterial coverage is about 30% of that of pure silicone coatings, significantly reducing bacterial attachment | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [43] | |
| Pyndine/water solvent coating | Initial: 0.64μg/cm²·day, then 0.07μg/cm²·day | 12 years | Significantly reduces bacterial attachment compared to pure silicone coatings | (Carlos A. Barrios, 2005) [4], (Barrios & Carlos A., 2004) [43] | |
| ZA loaded in Zeolite Hybrid Polymer Coatings | ZA: ZSm-5(1:2)+Epoxy | Initial: ≤ 16 μg cm⁻²·day, then fall to < 1 μg cm⁻²·day (steady state) | 12 months continuous seawater immersion test | After 6 months: almost no bacterial colonisation; after 12 months: only a very thin biofilm and no macro-fouling, far cleaner than biocide-free control | (Boopalan M., 2011) [38] |
Table 3. Coating applications related to 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.
Here's a reference to the metabolic engineering of Escherichia coli for the production of zosteric acid (Zhang et al., 2023) [39]
.
Figure 3. Metabolic pathway of Zosteric acid
The process is divided into three modules and some crucial enzymes are required.
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.
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.
3. Module III boosts intracellular L-tyrosine levels. We overexpressed key genes in the shikimate pathway, such as aroG, which relieves feedback inhibition of L-Tyr on the substrate, and introduced amino acid mutation. These modifications ensure a steady supply of L-tyrosine for upstream steps.
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 by enhancing the accumulation of PAPS.
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) [44], 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) [41] 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) [45] 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, it 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 sulfate 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.
To enhance the biosynthetic efficiency and specificity of Zosteric Acid (ZA), our modeling team focused on structural-functional optimization and fusion expression design of two key enzymes: Tyrosine Ammonia Lyase (TAL) and Sulfotransferase 1A1 (SULT1A1). TAL catalyzes the conversion of L-tyrosine to p-hydroxycinnamic acid (pHCA), while SULT1A1 sulfates pHCA to produce ZA—together forming the core steps of the ZA biosynthetic pathway.
For TAL and SULT1A1, we adopted a dual-track mutagenesis strategy:
We first aligned enzyme sequences from various sources to identify the approximate positions of conserved and variable regions. Amino acid sequences in FASTA format were retrieved from NCBI, and multiple sequence alignment was performed using Clustal Omega and FA preliminarily identify mutable regions. Then, using Jalview for further comparison, we identified the saturable mutation sites. In parallel, AutoDock Vina combined with PyMOL was used to identify binding sites. By cross-referencing the binding sites with the initially selected variable regions, we pinpointed residues located in both the “binding region” and “mutational region” for saturation mutagenesis design.
A structure-based conservation analysis was conducted using ProtSSN (Protein Structure Similarity Network) to construct the structural similarity network of the target enzymes and obtain conservation scores for all residues. These scores were then used as a reference in subsequent FoldX free energy calculations to support the structural stability ranking of different mutations. This helped assess whether highly stable mutations fell within highly conserved regions, thereby enhancing the biological interpretability and risk assessment of the mutagenesis strategy. Based on FoldX ΔΔG values, a set of mutations with favorable stability was selected, and all possible triple, quadruple, and quintuple mutation combinations were generated as key candidates for future experimental validation and high-throughput screening.
To optimize the Zosteric Acid biosynthetic pathway, we designed a TAL–SULT1A1 fusion protein to reduce the diffusion distance of intermediates and enhance the efficiency of the cascade reaction. The fusion construct was organized linearly with TAL at the N-terminus and SULT1A1 at the C-terminus, with various types of linker peptides inserted in between to assess impacts on conformation, foldability, and functional stability.
We selected both a flexible linker (GGGGS) and a rigid linker (EAAAK), each in three lengths (5 aa, 10 aa, and 15 aa), to investigate the spatial coupling effect. After predicting protein folding using AlphaFold, we employed FoldX to calculate the overall structural stability change (ΔΔG) of the fusion proteins. This allowed us to identify linker configurations that offered both high structural stability and favorable spatial orientation, serving as priority templates for subsequent experimental construction.
Figure 6. Protein folding predictions for the fusion constructs (TAL-(GGGGS)₃-SULT1A1)
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