Plastic microbeads are deliberately added, that is, solid plastic particles, usually defined as particles with a diameter of no more than 5 millimeters and insoluble in water, and used for exfoliation or cleaning in rinseable personal care products. They are typically made of petroleum-based plastics such as polyethylene (PET), polypropylene, or polystyrene. These tiny particles were widely used in various cosmetics and personal care products, such as facial cleansers, body scrubs, toothpaste, and shampoo, mainly because of their abrasive properties [1].

During use, these microbeads will flow with the wastewater into the sewer and enter the sewage system. However, due to their small size and buoyancy, they cannot be effectively filtered out by regular sewage treatment plants. As a result, a large number of microbeads will be discharged into rivers, lakes and oceans. It is estimated that a single shower can cause approximately 100,000 plastic particles to enter the aquatic environment, while a 150-milliliter cosmetic container may contain as many as 3 million plastic particles [2].

Fig. 1 Transport of MPs on animal trophic levels [2].

Marine organisms ingesting microbeads can suffer from digestive tract blockages, malnutrition, and even death. More critically, microbeads can adsorb toxic substances like heavy metals and environmental hormones, bioaccumulate through the food chain, and eventually enter the human body, potentially impacting the endocrine system, immune function, and long-term health [2]. Although many countries have implemented bans on plastic microbeads in rinse-off cosmetics, enforcement challenges persist, including unclear ingredient labeling and performance shortcomings of alternative materials.

Fig. 2 Some representative health effects of MPs on animals [2].

When we have a clear understanding of the dangers of plastic beads, an urgent question arises: how can we enjoy the convenience and efficacy of daily chemical products while protecting the blue planet and our own health? The answer may lie in the green solution brought by science and technology.

Biodegradable materials can generally be categorized into three groups based on their source and degradation mechanisms: natural polymers (e.g., starch, cellulose), synthetic biodegradable polymers (e.g., PLA, PCL, PBS), and microbially derived polyesters, particularly polyhydroxyalkanoates (PHAs), which have attracted increasing attention for their environmental compatibility and versatile applications.

However, natural-derived microbeads suffer from weak mechanical properties and poor stability, making it difficult to meet the dual requirements of particle uniformity and functionality in cosmetics; synthetic biodegradable materials mostly rely on petroleum-based feedstocks, entail higher costs, and are restricted by limited degradation conditions, compromising their environmental friendliness.

PHAs, which are synthesized by microorganisms under nutrient-limited and carbon-rich are fully bio-based polyesters. These polymers are capable of complete biodegradation in diverse environments, such as marine water, soil, and compost, resulting in harmless by-products like CO₂ and water, which makes them highly eco-friendly [3, 4]. PHAs also exhibit excellent biocompatibility, making them suitable for medical applications including absorbable sutures, drug delivery systems, and tissue engineering scaffolds, where they elicit minimal immune response [5, 6] Moreover, their physicochemical properties can be precisely modulated by adjusting the ratio of constituent monomers (such as PHB and PHV), or by blending with other materials such as polylactic acid (PLA), starch, or nanocellulose, thus broadening their applicability across industries [7]. From a production standpoint, PHAs offer the added advantage of being synthesized using low-cost or waste-derived carbon sources, including glycerol, whey, and agro-industrial residues. This makes PHA production not only more sustainable but also supportive of circular bioeconomy practices [3]. Although their current production cost remains higher than conventional plastics, PHAs are increasingly recognized as one of the most promising alternatives to petrochemical-based polymers due to their exceptional biodegradability, renewable sourcing, and biomedical potential.

Fig.3 The structures and general formula of PHAs

This project aims to utilize synthetic biology methods to construct an efficient and controllable PHA synthesis system in E. coli for producing PHA microbeads with uniform particle size and functionalizability to replace traditional plastic microbeads.

To achieve the production of size-controlled and highly uniform PHA particles in E. coli, we aim to design a modular and programmable system for the efficient production of size-adjustable PHA particles in E. coli.

Our design is based on synthetic biology methods, which include a core PHA biosynthesis module, a particle size regulation module, and a time control logic module.

• Core PHA biosynthesis module: phaCAB gene cluster

The biochemical pathway for PHA synthesis is a multi-step process, completed by three key enzymes through efficient catalysis [7]:

• PhaA (β-keto hydrolase) initiates this pathway by condensing two acetyl coenzyme A molecules to form acetoacetyl coenzyme A and releasing a HS-CoA molecule.
• Subsequently, PhaB (acetoacetyl coenzyme A reductase) uses NADPH as a reducing agent to reduce acetoacetyl coenzyme A to (R)-3-hydroxybutyryl coenzyme A.
• Finally, the PHA synthase (PhaC) polymerizes the (R)-3-hydroxybutyryl coenzyme A monomers into a PHA polymer, and each monomer is released with HS-CoA for high-yield PHA production.

Fig.4 PHB biosynthesis pathway [7].

• Particle size adjustment module: PhaP and ADF3 spider silk proteins:

To precisely control the size and surface properties of the generated PHA particles, we used two different hydrophobic proteins. PhaP (Phasin), a naturally occurring protein associated with PHA particles, spontaneously binds to the core part of PHA. Its expression level is a known key factor influencing the size and quantity of the particles [8].

Fig.5 Schematic of self-assembly of PHA granules [7].

The spider silk protein ADF3, through its hydrophobic region, mediates strong adsorption with the surface of PHA particles via hydrophobic interactions, while its exposed hydrophilic regions can provide spatial obstruction or electrostatic repulsion. The expression level and duration of this protein have a crucial impact on the final particle size and surface properties [9].

• Time control module: Promoters

To separate the PHA synthesis stage and the particle assembly stage (size regulation module) in terms of time to achieve efficiency optimization and control, we achieved this functionality by using different types of promoters.

• In the PHA accumulation stage: the strong-inducible T7 promoter was used to induce the transcription of the phaCAB genome, thereby promoting the flow of a large amount of carbon to PHA synthesis and the formation of intracellular particles [10, 11].
• In the particle assembly stage: we used the cold-inducible cspA promoter [12], the light-on induced system [13], and the arabinose promoter [14] separately to achieve the expression of the hydrophobic protein phaP or the spider silk protein ADF3.

Under the regulation of these various types of promoters, we can achieve size adjustment only after the majority of PHA core synthesis is completed, thereby avoiding premature interference and metabolic burden. This is crucial for reducing resource competition, increasing yield, and achieving programmable control over the size of PHA particles.

Our goal is to develop an efficient solution for producing PHA particles with highly uniform and precisely controllable dimensions. This capability is essential to meet the rigorous material consistency demands of advanced applications, such as in the cosmetics industry, where monodisperse PHA particles can serve as a biodegradable texture enhancer. By leveraging synthetic biology tools, we aim to deliver high-quality, customizable base materials that ensure excellent batch-to-batch reproducibility, thereby supporting the adoption of sustainable biomanufacturing across various sectors.

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[13] https://parts.igem.org/Part:BBa_K3447133

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