Breakthrough: Plants Used to Produce Health-Boosting Human Milk Oligosaccharides

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMJun 18 2024

Human milk contains approximately 200 oligosaccharides, which are important in establishing the infant gut microbiome and laying the foundation for robust immunity and healthy development. The commercial production of human milk oligosaccharides (HMOs) has thus far been conducted through microbial fermentation but has been successful for very few HMOs.

In a recent study published in Nature Food, researchers from the United States reported using a plant-based HMO production platform that utilizes the carbohydrate anabolic ability of plants to produce a variety of HMOs, including acidic, fucosylated, and neutral HMOs.

​​​​​​​Study: Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides. Image Credit: Krysja/Shutterstock.com

Background

Human milk is vital to establishing a healthy gut microbiome in the infant, which determines healthy development and robust immunity. Oligosaccharides are the bioactive components of human milk that are essential for establishing the gut microbiome in infants.

However, nearly 75% of infants are either exclusively fed or supplemented with infant formula, which does not contain all the HMOs required for healthy development.

Studies have also found that HMOs can be beneficial as prebiotics for adults, positively impacting intestinal barrier function gastrointestinal inflammation, and treating irritable bowel disease.

However, the microbial fermentation process used thus far to produce HMOs can only create a minuscule percentage of the diversity of HMOs present in human milk at the scale required to make HMOs commercially available as supplements in food products.

About the Study

In the present study, the researchers leveraged carbohydrate anabolism in plants to design a plant-based platform to produce various HMOs. They used Nicotiana benthamiana to test the transient and stable expression of the plasmids carrying the glycosyltransferase enzymes required for HMO production.

Glycosyltransferases are enzymes that can create glycosidic linkages and are essential for producing HMOs. In humans, HMO biosynthesis occurs in the Golgi apparatus, but microbial enzymes that carry out the process also function in the cytosol.

Therefore, the HMO biosynthesis enzymes from bacteria were localized in the cytosol of plant cells, and the production of neutral, acidic, and fucosylated HMOs was tested.

The transient expression was tested first in N. benthamiana using Agrobacterium tumefaciens as a medium to introduce the genes for HMO biosynthesis into the plant cells.

The leaves that showed transient expression of the HMO biosynthesis pathways were analyzed using liquid-liquid extraction, octyldecylsilane (c18) solid-phase extraction, porous graphitic carbon solid-phase extraction, and mass spectrometry.

Given that complex HMOs, such as the acidic and fucosylated ones, were built on the core structures of neutral HMOs, the researchers first attempted to produce the two types of neutral HMO core structures consisting of lacto-N-tetraose and lacto-N-neotetraose.

Based on the successful generation of the neutral HMOs and the fact that fucosylated HMOs are the most abundant HMOs in human milk, the researchers further explored the ability of the plant platform to add fucose to the neutral HMOs.

Therefore, they additionally expressed α-1,2-fucosyltransferase and the biosynthetic pathway for producing neutral HMOs. Similarly, sialyltransferases were also expressed to produce acidic HMOs.

The production of neutral and complex HMOs was further optimized for large-scale HMO production by creating transgenic N. benthamiana lines expressing the lacto-N-fucopentaose I biosynthetic pathway. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed the transgene expression.

Major Findings

The study showed that a diverse range of HMOs, from neutral HMOs to complex, fucosylated HMOs of high value, such as lacto-N-fucopentaose I could be produced using the plant-based production platform.

The HMOs produced using the transgenic plant platform were also bifidogenic, proving valuable as prebiotic supplements for infant and adult health.

The plant-based platform could also produce various HMOs that could not be produced using microbial fermentation. Additionally, the researchers overexpressed the biosynthetic pathways for nucleotide sugars to optimize the production of specific HMO classes and HMOs.

Furthermore, to assess and preserve the bifidogenic activity of the HMOs produced in plants, a process for extracting and purifying the HMOs from other plant products and crude extracts that could interfere with bacterial growth was also developed and optimized. This process produced a concentrated HMO extract without the phenolic compounds or simple sugars that could prevent bacterial growth.

Process models and cost comparisons to determine the economic viability of producing HMOs using plant-based platforms revealed that the production of lacto-N-fucopentaose I in plants was economically more favorable than producing it through microbial fermentation.

Conclusions

The findings showed that using a transgenic plant-based platform to produce HMOs essential for infant and adult health was a biologically and economically viable option.

Neutral and complex HMOs, including acidic and fucosylated HMOs, were successfully produced using transgenic N. benthamiana. The researchers believe that further engineering could produce all the HMOs in human milk, including branched HMOs.