Human milk, a crucial nutrient for infants, contains a vast array of human milk oligosaccharides (HMOs) that play a significant role in establishing a healthy gut microbiota, preventing diseases, and promoting healthy development. Infant formula, while widely used, often lacks the diversity of HMOs found in breast milk, limiting its benefits. The approximately 200 HMOs present in human milk exhibit structural diversity, influencing their bioactivity and prebiotic effects. Current commercial HMO production relies on microbial fermentation, but this method is limited in its capacity to produce the full range of HMOs at a commercially viable scale. Currently, only a small number of simple HMOs are commercially produced, leaving a vast majority of HMOs understudied. This limitation highlights the need for alternative biological platforms capable of producing a broader diversity of HMOs to enhance the nutritional value of infant formula and explore their potential health benefits in adults. The study aims to leverage the metabolic capabilities of plants as a novel and potentially more efficient platform for the large-scale and cost-effective production of a wide range of HMOs.

Extensive research highlights the significant health benefits associated with HMOs, both for infants and adults. Studies demonstrate HMOs' role in shaping the infant gut microbiota, preventing diseases, and promoting healthy development. In adults, HMOs are being investigated for their prebiotic potential, ability to improve intestinal barrier function, reduce gastrointestinal inflammation, and treat irritable bowel diseases. However, most studies have been limited to a small subset of HMOs due to the challenges in obtaining sufficient quantities of the diverse HMO structures. The limitations of microbial fermentation in producing a wide variety of HMOs at scale have been well-documented in the literature, emphasizing the need for alternative production methods. While microbial platforms are currently the primary source for commercially available HMOs, they are not sufficient to produce the full spectrum of HMOs that are present in human milk. The lack of large-scale production for many HMOs hinders research efforts aimed at exploring their various health benefits and potential applications.

The study employed both transient and stable expression of bacterial HMO biosynthetic enzymes in *Nicotiana benthamiana* plants. For transient expression, *Agrobacterium tumefaciens* strains carrying genes encoding various glycosyltransferases were injected into plant leaves. This allowed for high-throughput screening of biosynthetic pathways. Following transient expression, HMOs were extracted using liquid-liquid extraction, C18 solid-phase extraction (SPE), and porous graphitic carbon (PGC) SPE before characterization by mass spectrometry (MS). The researchers initially focused on producing neutral HMOs, specifically lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), by expressing a pathway involving specific galactosyltransferases and an N-acetylglucosaminyltransferase. Building on this success, they investigated the production of fucosylated HMOs by co-expressing an α-1,2-fucosyltransferase along with the neutral HMO pathway. This resulted in the production of 2ʹ-fucosyllactose (2ʹFL) and lacto-N-fucopentaose I (LNFPI). The production of acidic HMOs was achieved by co-expressing the neutral HMO biosynthetic pathway, sialyltransferases, and a mammalian pathway for the production of CMP-Neu5Ac. This resulted in the generation of 6'-sialyllactose (6'SL) and sialyllacto-N-neotetraose (LSTc), as well as other acidic HMOs. To optimize LNFPI production, the researchers overexpressed nucleotide sugar biosynthetic pathways alongside the LNFPI pathway, finding that overexpression of the GDP-fucose pathway significantly increased LNFPI yield. For stable transformation, two constructs (HMO10 and HMO11) were designed for the constitutive production of 2'FL and LNFPI in transgenic *N. benthamiana*. Quantitative PCR with reverse transcription (RT-qPCR) was used to confirm the expression of transgenes. HMO extraction and purification from plant tissue involved a multi-step process including water extraction, yeast fermentation to remove simple sugars, and a two-step resin adsorption with polyvinylpolypyrrolidone (PVPP) and C18 SPE. The bifidogenic activity of plant-produced HMOs was assessed through growth assays using *Bifidobacterium longum* subsp. *infantis* ATCC 15697 and *Bifidobacterium animalis* subsp. *lactis* ATCC 27536. Technoeconomic analysis (TEA) was conducted using SuperPro Designer to compare the cost of LNFPI production in plants and microbes, considering different ethanol selling prices and incorporating data on reported yields and recovery rates from peer-reviewed publications. Various analytical techniques, including different types of mass spectrometry (MS), high-performance anion exchange chromatography with pulsed amperometric detection, and high-performance liquid chromatography were employed for characterization and quantification of HMOs.

The study successfully demonstrated the production of all three major classes of HMOs (neutral, fucosylated, and acidic) in *N. benthamiana* plants using engineered biosynthetic pathways. The plant-based system produced a wider diversity of HMOs than current microbial platforms, including complex oligosaccharides not previously achievable through microbial means. Overexpression of specific nucleotide sugar biosynthetic pathways was shown to effectively optimize the production of both individual HMOs (e.g., LNFPI) and entire HMO classes. Specifically, overexpression of the GDP-fucose pathway increased LNFPI production by 32.9% compared to the control. The optimized purification method effectively removed interfering compounds from plant extracts, resulting in an HMO-rich extract suitable for bioactivity assays. Plant-produced HMOs exhibited similar selective bifidogenic activity as HMOs isolated from human milk, promoting the growth of *Bifidobacterium longum* subsp. *infantis* ATCC 15697 while not affecting the growth of *Bifidobacterium animalis* subsp. *lactis* ATCC 27536. Technoeconomic analysis indicated that plant-based production of LNFPI is economically favorable compared to microbial production, especially given the lower yields currently achievable through microbial methods. The minimum selling price (MSP) for LNFPI from plants was significantly lower than that from microbial systems, ranging from US$4.9 kg⁻¹ to US$18.4 kg⁻¹ depending on ethanol selling price, while microbial production resulted in an MSP of US$45.0 kg⁻¹. Stable transgenic *N. benthamiana* lines were also developed and shown to produce LNFPI and 2'FL, although yields were lower than in transiently expressed tissue. Transient expression proved to be a useful platform for testing HMO biosynthetic genes and small-scale production of HMOs for functional validation.

The findings demonstrate the feasibility and potential advantages of using plants as a platform for large-scale HMO production. The ability to produce a greater diversity of HMOs, including complex structures, addresses a critical limitation of current microbial approaches. The cost-effectiveness and scalability of plant-based production, as indicated by the technoeconomic analysis, offer a significant advantage over microbial methods for complex HMOs. The successful purification of HMOs from plants and demonstration of their bifidogenic activity validates their potential as a prebiotic supplement for both infants and adults. The successful generation of stable transgenic lines sets the stage for future efforts focused on optimizing HMO yields and developing high-yielding plant varieties suitable for commercial production. The findings open avenues for future research on a wider range of HMOs, potentially leading to the discovery of new HMOs with specific health benefits and applications.

This study successfully demonstrates the production of diverse human milk oligosaccharides (HMOs) in plants, addressing the limitations of current microbial production methods. Plant-based production offers a cost-effective and scalable alternative, generating a wider range of HMOs, including complex structures previously inaccessible. The bifidogenic activity of plant-produced HMOs is validated, highlighting their prebiotic potential. Future research should focus on further optimizing HMO yields in plants, exploring different plant species, and developing efficient industrial-scale purification processes to fully realize the potential of plant-based HMO production for infant nutrition and broader health applications.

While the study successfully demonstrates the proof-of-concept for plant-based HMO production, some limitations exist. The yields of HMOs in stably transformed plants were lower compared to transient expression systems, suggesting that further optimization of the genetic constructs and plant growth conditions is necessary for achieving high yields at a commercial scale. The technoeconomic analysis is based on theoretical models and may not fully capture the complexities of industrial-scale production. Additional research is needed to optimize the purification process and ensure the scalability of the method for industrial application. Finally, long-term studies are needed to fully evaluate the health benefits of plant-produced HMOs in humans.