Abstract

Visual health disorders—such as dry eyes, night blindness, and myopia—are becoming increasingly prevalent worldwide, particularly among adolescents in East Asia. According to the WHO, at least 2.2 billion people globally suffer from some form of vision impairment, and at least 1 billion of these cases could have been prevented or remain unaddressed. In urban areas of countries like China and South Korea, the situation is especially severe, with reported rates of adolescent myopia reaching as high as 67% and 97%, respectively.

Vitamin A (retinal) is essential for synthesizing rhodopsin, a light-sensitive receptor protein in the retina that is critical for vision. A deficiency in vitamin A can lead to dry eyes and other vision problems. This deficiency is often caused by insufficient dietary intake of β-carotene—a provitamin A carotenoid primarily sourced from vegetables. However, limited vegetable consumption, especially among adolescents, reduces β-carotene intake and contributes to the rising incidence of vision problems.

To provide a scalable and sustainable alternative solution for protecting vision health, our team collaborated across Engineering, Human Practices, Education, Entrepreneurship, and Dry Lab, and came up with the idea of POCO youth personal care, stationery and snacks. Together, we accomplished the following:

1. Successfully introduced a synthetic β-carotene biosynthetic pathway into Saccharomyces cerevisiae with CRISPR-Cas9 genome editing to enable the de novo synthesis of β-carotene. The β-carotene titer produced by S. cerevisiae in shake-flask fermentation reached 128.1 mg/L.

2. Successfully developed and optimized the spatial metabolic engineering toolkit for improved subcellular targeting and pathway efficiency.

3. Successfully applied the spatial metabolic engineering toolkit by fusing the optimized toolkit with target genes, enabling precise localization of carB and carRP to lipid droplets. This compartmentalization significantly enhanced pathway performance, achieving 318.4mg/L β-carotene production in shake flasks.

4. Successfully optimized the fermentation process in a 5-L bioreactor to achieve a higher yield of β-carotene to 1.8 g/L.

5. Successfully developed a color-based monitoring device for β-carotene fermentation, enabling automation in large-scale fermentation and offering a convenient tool for future iGEM teams focused on intelligent bioprocessing.

6. Human practices: we engaged 30+ stakeholders across the entire pipeline—from lab to industry—including public, clinicians, researchers, and legal/industry advisors, Key Opinion Leaders (KOLs), investors ensuring our project is robust and socially relevant.

7. Entrepreneurship: we created POCO as a fun-seeking youth brand that redefines the eye healthcare experience by employing a “personal care and snacks first, supplements later” strategy to make eye health protection enjoyable.

8. Education: we successfully designed initiatives around three core highlights: AI-powered content innovation, participatory game development, and community engagement. These efforts reached more than 30,000 participants across online and offline platforms, inspiring broad awareness and participation.

Issues

1. A Global Public Health Challenge

Vision impairment is remarkably common and growing in prevalence. Global estimates of people affected by vision-impairing eye conditions underscore the urgency of this issue (see Figure 1). Notably, 312 million children and adolescents under the age of 19 are affected by myopia. These conditions—such as myopia, cataract, diabetic retinopathy, and glaucoma—affect hundreds of millions, often with lifelong consequences.

2. Many Cases Are Preventable

Globally, at least 2.2 billion people live with vision impairment. Shockingly, 1 billion of these cases could have been prevented or remain unaddressed (Figure 2)¹. These preventable impairments are often due to lack of access to early diagnosis, affordable care, or essential nutrients like vitamin A.

3. Alarming Regional Trends

There is significant regional and economic variation in vision impairment (Figure 3, adapted from²). Among all regions, East Asia, South-East Asia, and South Asia rank the highest in prevalence—yet much of this vision loss could have been prevented or remains unaddressed.

Again, another study also showed that three Asian regions—South Asia (61.2 M), East Asia (52.9 M), and South-East Asia (20.8 M)—account for 62% of the world’s 216.6 million cases of moderate to severe bilateral distance vision impairment³.

Other studies also pointed out:

• The prevalence of myopia is highest in high-income Asia-Pacific countries (53.4%) and East Asia (51.6%)⁴.

• In urban areas of China and South Korea, adolescent myopia rates have reached 67% and 97%, respectively⁵.

• Dry eye syndrome affects over 30% of adults in some regions of China⁶.

4. β-carotene is essential to protect vision health

These trends underscore the urgent need for accessible, nutrient-based, and preventive solutions to support vision health in everyday life. β-carotene plays a vital role in this context, as it is a precursor to vitamin A — a key nutrient essential for maintaining healthy eyesight.

However, many adolescents today avoid eating β-carotene-rich vegetables such as carrots due to their distinct taste and texture, leading to insufficient intake.

Multiple observational studies also report that β-carotene intake among adolescents is often below recommended levels. For instance, a Swedish cohort study (n=1139) found that average β-carotene intake among 13-14-year-olds failed to meet the Estimated Average Requirement, particularly among girls⁷⁸⁹. A European review of adolescents reported that β-carotene deficiency was 14-19%¹⁰. Comparable findings among Japanese young adults showed green/yellow vegetable consumption averaging only ~60 g/day, resulting in carotenoid intake of ~2850 µg/day—well below dietary targets¹¹.

This dietary gap contributes to vitamin A deficiency, which is the leading cause of preventable childhood blindness, and significantly increases the risk of illness and death from severe infections like diarrhoeal disease and measles¹².

Current Solutions & Problems

Two major approaches—plant-based extraction and chemical synthesis—suffer from significant drawbacks:

• Plant-based extraction relies heavily on agricultural conditions, making it seasonal and land-intensive. The production cycle is slow, often requiring weeks or months, and the yield is low. Moreover, large-scale farming raises sustainability concerns, including water use and soil degradation.

• Chemical synthesis, while industrially scalable, involves complex multistep reactions that require high energy input and the use of hazardous organic solvents. These safety and environmental issues make the final product unsuitable for food or dietary supplements in many regulatory settings.

Table 1 Comparison of β-Carotene Production Methods

Our solution

To overcome these limitations, we harness microbial biosynthesis to produce β-carotene in a faster, more consistent, and environmentally friendly manner. This biological approach enables scalable production independent of climate or arable land, while ensuring safety and compliance for nutritional use—offering a promising alternative to expand access to vitamin A, especially for populations with low dietary intake. The idea of POCO youth personal care, stationery and snacks was created to help.

Experiment design

Step 1:De novo synthesis of β-carotenein_Saccharomyces cerevisiae_

We selected Saccharomyces cerevisiae as the chassis due to its well-defined genetics, ease of cultivation, and proven safety in both food and pharmaceutical applications.

Instead of relying on exogenous precursor supplementation, we aimed to establish a substrate-free, de novo β-carotene biosynthetic pathway. To achieve this, two heterologous genes from Mucor species—phytoene desaturase (carB, GenBank: AJ238028.1) from Mucor lusitanicus and bifunctional phytoene synthase/lycopene cyclase (carRP, GenBank: AJ250827.1) from Mucor circinelloides—were codon-optimized for S. cerevisiae and driven by strong native promoters (PFBA1 and PGAPDH). This design enables the conversion of glucose directly into β-carotene through endogenous precursor flux.

Step 2:CRISPR-Cas9-Mediated Genomic Integration and Marker Recycling

To ensure stable expression of the synthetic pathway, we constructed a full-length integration cassette containing codon-optimized carB and carRP flanked by DPP3 homologous arms and the selectable marker URA3. The cassette was assembled through fusion PCR and Gibson assembly. A CRISPR-Cas9 system was established by integrating Cas9 into the pox1 locus, while a guide RNA targeting DPP3 was co-transformed to facilitate site-specific genome editing. This allowed efficient, marker-assisted chromosomal insertion of the entire β-carotene pathway in Saccharomyces cerevisiae, avoiding plasmid instability and supporting long-term production.

Step 3:Compartmentalization strategy:Developed and optimized a spatial metabolic engineering toolkitvia lipid droplet engineering

To achieve efficient spatial separation of metabolic reactions and minimize cytoplasmic substrate competition, we developed a lipid droplet (LD)-targeted engineering toolkit to enhance β-carotene biosynthesis in Saccharomyces cerevisiae. As natural reservoirs for lipophilic compounds, LDs provide an ideal compartment for sequestering hydrophobic intermediates and final products. Guided by structural modeling and enzymatic activity predictions, we fused LD-targeting signal peptides (HD2) to key pathway enzymes, with systematic optimization of linker usage and fusion positioning. The following sections detail the stepwise development of this strategy:

a. Selection of Lipid Droplet-Targeting Signal Peptides

→ Identification of endogenous ERG7-derived hydrophobic domains for LD localization (HD1, HD2, HD3, HD4).

b. Linker Position Optimization: N-terminal vs. C-terminal

→ Comparative folding analysis to determine the optimal fusion site.b.

c. HD2 Fusion with or without Flexible Linkers

→ Structural modeling to assess whether a flexible linker alleviates steric hindrance.

d. Structure-Guided Fusion with Key Enzymes (CarB & CarRP)

→ Combined DLKcat prediction and Chai Discovery-based docking to select constructs that maximize catalytic activity while enabling LD-targeting.

Step 4:Fermentation scale-up and yield estimation device development

To evaluate production potential at an industrial scale, we designed a fed-batch fermentation process in 5 L bioreactors, optimizing for nutrient feeding, aeration, and temperature control. Additionally, we proposed a colorimetric prediction model based on culture color intensity to non-destructively estimate β-carotene levels during fermentation. This strategy provides a fast and economical method for production monitoring and harvest timing.