Description

Abstract

Human milk oligosaccharides (HMOs) are essential bioactive components of breast milk that support infant immunity, cognitive development, and long-term health, yet current infant formulas provide only a small fraction of their natural diversity. To address this gap, we developed a synthetic biology platform using a modular bacterial consortium of Streptococcus thermophilus, a GRAS (Generally Recognized As Safe), dairy-adapted, and naturally competent chassis. By dividing the HMO biosynthetic pathway among complementary strains, implementing controlled cell aggregation, and establishing metabolic interdependence, we aim to reduce metabolic burden, enhance intermediate sharing, and maintain population balance. This approach is expected to enable the scalable production of a broader spectrum of HMOs than currently available, improving the nutritional quality of formula and establishing a flexible platform for other complex biomolecules.

The Problem

Breast milk is more than nutrition — it’s a complex biological system. Human Milk Oligosaccharides (HMOs) are its third-most-abundant solid component, with over 200 distinct structures. HMOs are intricate branched carbohydrates whose specific linkages dictate their biological functions [1].

Each structure serves a distinct purpose:

• 2′-Fucosyllactose (2′-FL) — promotes beneficial Bifidobacterium growth [2][3].
• Lacto-N-neotetraose (LNnT) — enhances immune system maturation [3][4].
• Sialylated HMOs — support brain development and pathogen protection [5][6].
• Complex branched HMOs — provide targeted antimicrobial effects [1][7].

HMOs offer numerous health benefits:

Yet today’s infant supply only 2–6 HMO types, leaving ~95% of natural diversity — and many health benefits — unavailable to millions of formula-fed infants worldwide [1]. Studies link limited HMO intake to higher rates of respiratory infection, allergies, necrotizing enterocolitis, and even to long-term risks of obesity and autoimmune disease [7].

Our Innovation: Synthetic Bacterial Consortia

We are developing a safe, cost-effective, and highly scalable bacterial platform for HMO production. Rather than focusing on a single molecule, we are designing a modular system that can be easily adapted to produce a wide spectrum of HMOs, and, in the future, other high-value biomolecules. This approach will make access to HMOs broader, cheaper, and more sustainable, while opening the door to new applications beyond infant nutrition.

A cornerstone of our project is the choice of host organism. The ideal host must:

• Produce target compounds efficiently and allow easy recovery,
• Be straightforward to engineer genetically to build diverse production strains,
• Be recognized as safe for human consumption, especially in infant formulas,
• Grow quickly with minimal input and infrastructure.

After careful evaluation, we selected Streptococcus thermophilus, a well-characterized lactic acid bacterium widely used in the dairy industry. It is GRAS (Generally Recognized As Safe), robust, genetically tractable, and fast-growing in simple media. This makes it an excellent chassis for our envisioned platform.

However, HMOs are complex molecules. Producing them in a single bacterial cell places a heavy metabolic burden, often reducing yields and stability. To overcome this, we are pioneering a division-of-labor strategy: splitting the biosynthetic pathway between different bacterial subpopulations. Each strain handles part of the pathway, reducing stress on individual cells and potentially boosting overall productivity.

This innovation comes with two main challenges: efficient transport and diffusion of pathway intermediates between subpopulations, and maintaining a stable balance among these strains to sustain optimal production. Tackling these challenges will not only advance HMO biosynthesis but also pave the way for a versatile co-culture platform for other complex metabolic pathways.

To address the challenge of transferring intermediates between bacterial subpopulations, we devised two complementary strategies.

First, we aim to split the metabolic pathway in a way that promotes efficient transport of intermediates. By strategically placing specific enzymes at the surface of our engineered cells, we can bring catalytic steps closer to the extracellular environment, facilitating the release and uptake of pathway intermediates between strains.

Second, we are working to maintain close spatial proximity between the different bacterial subpopulations. Proximity can dramatically enhance diffusion of intermediates, but simply forcing cells to aggregate can stress or even harm them. Our solution is to develop a controllable cell–cell adhesion system, allowing us to promote or fine-tune aggregation only when and where it benefits production.

To achieve this, we are exploring the use of surface-displayed alpha-Rep and nanobodies, both targeting GFP and complementary partners as programmable adhesion modules. By decorating different subpopulations with matching binding partners, we can drive specific and reversible interactions between strains. This approach lets us build a synthetic microbial consortium where proximity is engineered, boosting efficiency while preserving cell viability.

Lastly, we needed a strategy to maintain a stable equilibrium between our bacterial subpopulations. Without some form of regulation, one strain could easily outgrow the other, jeopardizing the efficiency of the entire system.

To tackle this, we designed a system based on metabolic interdependency. By engineering complementary nutrient requirements into each strain, we can ensure that neither can thrive without the other. Specifically, we plan to engineer the first strain so that it can metabolize lactose into glucose and galactose but cannot use galactose, while the second strain will be modified to utilize only galactose. This built-in interdependency aims to provide a self-regulating mechanism that helps maintain population balance without external intervention.

Project inspiration and motivation

When I [Pierre] first began assembling this team, I was deeply inspired by a brilliant researcher who happened to be pregnant during a demanding project. She shared her quiet worry: “The very first inequality a baby faces can begin at birth, when a mother wants to give what is best but simply cannot.”

Her situation is far from unique. Medical complications, difficulties with lactation, and the realities of modern work–life balance often make breastfeeding impossible or unsustainable despite the World Health Organization’s recommendation of exclusive breastfeeding for the first six months of life.

In some countries, notably the United States, paid maternity leave is not guaranteed, forcing many parents back to work within weeks.

These families aren’t making a casual choice. They are making the only choice they can. Yet the infant formulas on the market today, though lifesaving, cannot match the biological richness of human milk.

This story touched the other members of the team. It is a little-known problem, but one that affects us all. We wanted to raise awareness of this issue and try to solve it with our ideas and conviction.

A Modular Project

We set out to build a bacterial production platform capable of synthesizing Human Milk Oligosaccharides (HMOs). To achieve this, our project brings together three interconnected pillars: HMO production through division of labour, controlled cell aggregation, and metabolic interdependence.

As a proof of concept, we began by concentrating on one HMO family, the fucosylated HMOs. To access the fucose building blocks, our first step would be to depolymerize fucoidan into fucose monomers using a surface-displayed fucanase, thereby avoiding the need to transport large polysaccharides across the cell membrane. Because S. thermophilus naturally expresses the surface protease HtrA, which could degrade our engineered constructs, we planned to delete htrA in order to safeguard the stability and functionality of our displayed enzymes. In parallel, a second subpopulation would be engineered to express both a fucose permease and a fucosyltransferase, a combination expected to enable the first steps of HMO biosynthesis, with room for future optimization.

Controlled aggregation adds another layer of efficiency. By physically bringing the subpopulations closer together at the onset of production, we aim to enhance the diffusion of intermediates between them. To achieve this, cells display either a nanobody or an α-rep targeting distinct epitopes of the green fluorescent protein (GFP), creating a “molecular glue” that promotes close cell–cell contact.

Finally, metabolic interdependency helps maintain balance between subpopulations. One S. thermophilus strain is engineered to metabolize lactose into glucose and galactose but unable to degrade galactose further (via deletion of the responsible gene), while the complementary strain thrives on galactose. This mutual reliance fosters a stable, self-regulating consortium.

S. thermophilus as a New Chassis for iGEM

Streptococcus thermophilus is an attractive chassis for synthetic biology because it combines a long record of safe use with growing genetic tractability. This lactic acid bacterium holds GRAS status in the United States and QPS status in the European Union, reflecting decades of application in yogurt and cheese production without safety concerns. Comparative genomics confirms the absence of active virulence factors and a very low prevalence of antibiotic resistance genes, reinforcing its suitability for food and health applications [10][11].

Among sequenced strains, S. thermophilus LMD-9 stands out as a reference for engineering. Its complete genome reveals efficient lactose and galactose metabolism, casein proteolysis, and the capacity to produce bioactive compounds such as γ-aminobutyric acid (GABA), all relevant for food biotechnology [12][13]. Importantly, LMD-9 can be transformed by electroporation and, under specific conditions, exhibits natural competence, meaning it can take up and integrate free DNA via homologous recombination, thus providing a route for clean, marker-free genetic modifications [14][15]. In parallel, strong constitutive promoters have been characterized to drive heterologous gene expression and metabolic pathway engineering [16].

This combination of proven safety, food-adapted metabolism, natural competence, and expanding molecular tools makes S. thermophilus a strong candidate chassis for synthetic biology in dairy, probiotic, and nutraceutical applications — even though its genetic toolbox still lags behind that of traditional model organisms like E. coli or Bacillus subtilis [11].

The Impact Of Our Platform

By enabling multi-HMO fermentation at industrial scale, our platform could:

Summary

Human milk oligosaccharides (HMOs) are the third most abundant solid component of breast milk and provide critical benefits for infant immunity, cognitive development, and long-term health, yet current formulas supply only a handful of the more than 200 natural HMO structures. Traditional chemical synthesis cannot replicate this diversity, and single-strain microbial fermentation struggles with the metabolic burden of producing multiple complex HMOs simultaneously. Inspired by a colleague’s personal experience of the “first inequality” facing infants when breastfeeding is not possible, our team set out to close this gap by engineering a safe, scalable bacterial consortium platform for HMO production. By splitting the biosynthetic pathway across complementary strains of Streptococcus thermophilus—a GRAS, dairy-adapted, naturally competent bacterium widely used in yogurt and cheese production—we reduce the stress on individual cells, enhance intermediate sharing through controlled cell aggregation, and maintain population balance via metabolic interdependence. This modular division-of-labor approach is designed to produce a broader range of HMOs than currently available, potentially narrowing the gap between formula and breast milk, while also serving as a flexible platform that could be adapted, with further optimization, for other complex biomolecules in functional foods and biotechnology.

References

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