Production Of Human Milk Oligosaccharides Research Articles

An Overview of Sugar Nucleotide-Dependent Glycosyltransferases for Human Milk Oligosaccharide Synthesis.

Human milk oligosaccharides (HMOs) have received increasing attention because of their special effects on infant health and commercial value as the new generation of core components in infant formula. Currently, large-scale production of HMOs is generally based on microbial synthesis using metabolically engineered cell factories. Introduction of the specific glycosyltransferases is essential for the construction of HMO-producing engineered strains in which the HMO-producing glycosyltransferases are generally sugar nucleotide-dependent. Four types of glycosyltransferases have been used for typical glycosylation reactions to synthesize HMOs. Soluble expression, substrate specificity, and regioselectivity are common concerns of these glycosyltransferases in practical applications. Screening of specific glycosyltransferases is an important research topic to solve these problems. Molecular modification has also been performed to enhance the catalytic activity of various HMO-producing glycosyltransferases and to improve the substrate specificity and regioselectivity. In this article, various sugar nucleotide-dependent glycosyltransferases for HMO synthesis were overviewed, common concerns of these glycosyltransferases were described, and the future perspectives of glycosyltransferase-related studies were provided.

Microbial Production of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are complex nonnutritive sugars present in human milk. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. Lower prevalence of breastfeeding around the globe, rising popularity of nutraceuticals, and low availability of HMOs have inspired efforts to develop economically feasible and efficient industrial-scale production platforms for HMOs. Recent progress in synthetic biology and metabolic engineering tools has enabled microbial systems to be a production system of HMOs. In this regard, the model organism Escherichia coli has emerged as the preferred production platform. Herein, we summarize the remarkable progress in the microbial production of HMOs and discuss the challenges and future opportunities in unraveling the scope of production of complex HMOs. We focus on the microbial production of five HMOs that have been approved for their commercialization.

Biosynthesis of Human Milk Oligosaccharides: Enzyme Cascade and Metabolic Engineering Approaches

Human milk oligosaccharides (HMOs) have unique beneficial effects for infants and are considered as the new gold standard for premium infant formula. They are a collection of unconjugated glycans, and more than 200 distinct structures have been identified. Generally, HMOs are enzymatically produced by elongation and/or modification from lactose via stepwise glycosylation. Each glycosylation requires a specific glycosyltransferase (GT) and the corresponding nucleotide sugar donor. In this review, the typical HMO-producing GTs and the one-pot multienzyme modules for generating various nucleotide sugar donors are introduced, the principles for designing the enzyme cascade routes for HMO synthesis are described, and the important metabolic engineering strategies for mass production of HMOs are also reviewed. In addition, the future research directions in biotechnological production of HMOs were prospected.

Enzymatic production of a suite of human milk oligosaccharides directly in milk

Human milk oligosaccharides (HMOs) denote specific glycans in human breast milk. They function as prebiotics, immune modulating, and antimicrobial agents in the gut of breastfed infants, and certain HMOs even promote the cognitive development of the baby. HMOs are virtually absent in cow’s milk and hence in infant formula, which provides a huge incentive for identifying ways in which HMOs can be produced to improve infant formulas. Here, we show that different sialylated and fucosylated HMOs can be generated in cow’s milk via different simultaneous enzymatic transglycosylation reactions catalyzed by an engineered sialidase (EC 3.2.1.18, from Trypanosoma rangeli) and an 1,2-α-L-fucosidase (EC 3.2.1.63, from Tannerella forsinthia) acting on the lactose in the milk and on casein glycomacropeptide, two types of commercially available HMOs, i.e. 2′-fucosyllactose and lacto-N-neotetraose, added to the milk. We also outline the details of the individual reactions in aqueous systems, demonstrate that the enzymatic reactions can be accomplished at 5 °C, and validate the products formed by LC-MS and NMR analysis. Enzymatic production of HMOs directly in milk provides opportunities for enriching milk and infant formulas and extends the use of enzymatic transglycosylation reactions to synthesis of HMOs in milk and eventually in other beverages.

Engineering Pheromone-Mediated Quorum Sensing with Enhanced Response Output Increases Fucosyllactose Production in Saccharomyces cerevisiae.

Engineering dynamic control of gene expression is desirable because many engineered functions interfere with endogenous cellular processes that have evolved to facilitate growth and survival. Minimizing conflict between growth and production phases can therefore improve product titers in microbial cell factories. We developed an autoinduced gene expression system by rewiring the Saccharomyces cerevisiae pheromone response pathway. To ameliorate growth reduction due to the early onset response at low population densities, α-pheromone of Kluyveromyces lactis (Kα) instead of S. cerevisiae (Sα) was expressed in mating type "a" yeast. Kα-induced expression of pathway genes was further enhanced by the transcriptional activator Gal4p expressed under the control of the pheromone-responsive FUS1 promoter (Pfus1). As a demonstration, the engineered circuit combined with the deletion of the endogenous galactose metabolic pathway genes was applied to the production of human milk oligosaccharides, 2'-fucosyllactose (2'-FL) and 3-fucosllactose (3-FL). The engineered strains produced 3.37 g/L 2'-FL and 2.36 g/L 3-FL on glucose with a volumetric productivity of 0.14 and 0.03 g/L·h-1 in batch flask cultivation, respectively. These represented 147 and 153% increases over the control strains on galactose wherein the respective pathway genes are expressed under GAL promoters only. Further fed-batch fermentation achieved titers of 32.05 and 20.91 g/L for 2' and 3-FL, respectively. The genetic program developed here thus represents a promising option for implementing dynamic regulation in yeast and could be used for the production of biochemicals that may place a heavy metabolic burden on cell growth.

Biosynthesis of Lacto-N-fucopentaose I in Escherichia coli by metabolic pathway rational design

Lacto-N-fucopentaose I (LNFP I), a member of the fucosylated human milk oligosaccharides (HMOs) family, has received widespread attention because of its importance in infant health. However, constructing a low-cost microbial cell factory for high-efficient production of complex fucosylated HMOs remains challenging. Herein, an efficient engineered strain for LNFP I biosynthesis was reported by metabolic pathway rational design in Escherichia coli BL21(DE3). The fed-batch cultivation of engineered strains produced 2.11 g/L LNFP I, and the conversion rate of lacto-N-tetraose (LNT) to LNFP I reached 32.55 %, the highest level reported so far. The LNFP I-produced platform established here is broadly suitable for complex and branched HMOs production.

In silico analysis of the human milk oligosaccharide glycome reveals key enzymes of their biosynthesis

Human milk oligosaccharides (HMOs) form the third most abundant component of human milk and are known to convey several benefits to the neonate, including protection from viral and bacterial pathogens, training of the immune system, and influencing the gut microbiome. As HMO production during lactation is driven by enzymes that are common to other glycosylation processes, we adapted a model of mucin-type GalNAc-linked glycosylation enzymes to act on free lactose. We identified a subset of 11 enzyme activities that can account for 206 of 226 distinct HMOs isolated from human milk and constructed a biosynthetic reaction network that identifies 5 new core HMO structures. A comparison of monosaccharide compositions demonstrated that the model was able to discriminate between two possible groups of intermediates between major subnetworks, and to assign possible structures to several previously uncharacterised HMOs. The effect of enzyme knockouts is presented, identifying β-1,4-galactosyltransferase and β-1,3-N-acetylglucosaminyltransferase as key enzyme activities involved in the generation of the observed HMO glycosylation patterns. The model also provides a synthesis chassis for the most common HMOs found in lactating mothers.

Utilization of industrial citrus pectin side streams for enzymatic production of human milk oligosaccharides

This study reports the enzymatic upgrading of fucosylated xyloglucan from depectinized citrus residues into 2′-fucosyllactose, a fucosylated human milk oligosaccharide. Alkaline and enzymatic xyloglucan extractions were compared. Of the original fucose present in the depectinized residues of lemon and orange, 35–36% and 48–51% were extracted as fucosylated xyloglucan by enzyme- or alkaline treatment, respectively. Furthermore, the enzymatically extracted xyloglucan structures had a narrower molecular weight distribution around 1 kDa, contrary to a more polydisperse distribution of the alkaline extracted xyloglucans, ranging from 1 to 500 kDa. The applicability of the fucosylated-xyloglucan extracts in transfucosylation reactions, was determined by use of a selected fungal fucosidase, resulting in yields of 10.2–11.4% enzymatic extracts, and 6.5–7.4% for alkaline extracts (orange and lemon respectively). The results demonstrate that depectinized citrus side streams are a useful source of fucosylated xyloglucan, preferably extracted by an enzyme catalyzed approach.

Transporter Engineering Enables the Efficient Production of Lacto-N-triose II and Lacto-N-tetraose in Escherichia coli.

Lacto-N-triose (LNT II) and lacto-N-tetraose (LNT) are human milk oligosaccharides (HMOs) with various potential functions for infants. HMO production by Escherichia coli fermentation has attracted attention in recent years. However, little is known about the cellular export of HMOs. In this study, we identified four endogenous E. coli transporter genes (setA, setB, ydeA, and mdfA), overexpression of which significantly increased the efficiency of LNT II production. The setA-enhanced strain accumulated 34.2 g/L LNT II in a 3 L bioreactor. In the production of LNT, which uses LNT II as an intermediate, disruption of setA remarkably decreased the LNT II accumulation and enhanced the titer of LNT. Furthermore, by heterologous expression of extracellular β-1,3-N-acetylglucosaminidase from Bifidobacterium bifidum, which degrades LNT II, we eliminated LNT II completely. This study shows that regulation of sugar efflux transporters in E. coli can increase the production of HMOs and decrease the amounts of undesired byproducts.

Human Milk Oligosaccharides: A Comprehensive Review towards Metabolomics.

Human milk oligosaccharides (HMOs) are the third most represented component in breast milk. They serve not only as prebiotics but they exert a protective role against some significant neonatal pathologies such as necrotizing enterocolitis. Furthermore, they can program the immune system and consequently reduce allergies and autoimmune diseases’ incidence. HMOs also play a crucial role in brain development and in the gut barrier’s maturation. Moreover, the maternal genetic factors influencing different HMO patterns and their modulation by the interaction and the competition between active enzymes have been widely investigated in the literature, but there are few studies concerning the role of other factors such as maternal health, nutrition, and environmental influence. In this context, metabolomics, one of the newest “omics” sciences that provides a snapshot of the metabolites present in bio-fluids, such as breast milk, could be useful to investigate the HMO content in human milk. The authors performed a review, from 2012 to the beginning of 2021, concerning the application of metabolomics to investigate the HMOs, by using Pubmed, Researchgate and Scopus as source databases. Through this technology, it is possible to know in real-time whether a mother produces a specific oligosaccharide, keeping into consideration that there are other modifiable and unmodifiable factors that influence HMO production from a qualitative and a quantitative point of view. Although further studies are needed to provide clinical substantiation, in the future, thanks to metabolomics, this could be possible by using a dipstick and adding the eventual missing oligosaccharide to the breast milk or formula in order to give the best and the most personalized nutritional regimen for each newborn, adjusting to different necessities.

Toward Sequencing the Human Milk Glycome: High-Resolution Cyclic Ion Mobility Separations of Core Human Milk Oligosaccharide Building Blocks.

Human milk oligosaccharides (HMOs) are an unconjugated class of glycans that have been implicated for their role in promoting the healthy development of the brain-gut axes of infants. Production of HMOs is ever-changing and specifically tailored for each infant in response to various biological factors (e.g., cognitive development, diseases, or allergies). While every HMO consists of up to only five monosaccharides, their structures can be composed of many possible glycosidic linkage positions and corresponding α/β anomericities, linear or branched chains, and potential fucosylation/sialylation modifications, thus leading to a tremendous degree of isomeric heterogeneity. With limited availability of authentic standards for every putative HMO structure (estimated to be >200 total), new analytical methods are needed for their accurate characterization. Complete sequencing of the human milk glycome would enable a better understanding of their infant-specific biological roles and potentially lead to their widespread incorporation into infant formula. Herein, we explore the use of our high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based platform for the separation of core disaccharide and trisaccharide isomer building blocks as a first step toward the sequencing of larger HMOs. By utilizing the flexible capabilities of the cIMS array, separation pathlengths were extended up to 40 m, thus enabling the resolution of all seven sets of sialylated, fucosylated galactosyllactose and lactosamine HMO building block isomers. Additionally, we assessed the utility of pre-/post-cIMS tandem mass spectrometry (MS/MS) and tandem cIMS (cIMS/cIMS) for the characterization of HMOs based on their diagnostic fragmentation patterns and mobility fingerprints. We anticipate that our presented cIMS-MS-based methodology will enable the better characterization of larger, unknown HMOs when incorporated into an overall workflow that also includes online liquid chromatography and enzymatic hydrolyses.

Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis.

Human milk oligosaccharides (HMOs) are one of the important ingredients in human milk, which have attracted great interest due to their beneficial effect on the health of newborns. The large-scale production of HMOs has been researched using engineered microbial routes due to the availability, safety, and low cost of host strains. In addition, the development of molecular biology technology and metabolic engineering has promoted the effectiveness of HMOs production. According to current reports, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), and some fucosylated HMOs with complex structures have been produced via the engineered microbial route, with 2'-FL having been produced the most. However, due to the uncertainty of metabolic patterns, the selection of host strains has certain limitations. Aside from that, the expression of appropriate glycosyltransferase in microbes is key to the synthesis of different HMOs. Therefore, finding a safe and efficient glycosyltransferase has to be addressed when using engineered microbial pathways. In this review, the latest research on the production of HMOs using engineered microbial routes is reported. The selection of host strains and adapting different metabolic pathways helped researchers designing engineered microbial routes that are more conducive to HMOs production.

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.Graphical abstract

High-Titer De Novo Biosynthesis of the Predominant Human Milk Oligosaccharide 2'-Fucosyllactose from Sucrose in Escherichia coli.

Human milk oligosaccharides (HMOs) are unique components of human breast milk. Their large-scale production by fermentation allows infant formulas to be fortified with HMOs, but current fermentation processes require lactose as a starting material, increasing the costs, bioburden, and environmental impact of manufacturing. Here we report the development of an Escherichia coli strain that produces 2'-fucosyllactose (2'-FL), the most abundant HMO, de novo using sucrose as the sole carbon source. Strain engineering required the expression of a novel glucose-accepting galactosyltransferase, overexpression of the de novo UDP-d-galactose and GDP-l-fucose pathways, the engineering of an intracellular pool of free glucose, and overexpression of a suitable α(1,2)-fucosyltransferase. The export of 2'-FL was facilitated using a sugar efflux transporter. The final production strain achieved 2'-FL yields exceeding 60 g/L after fermentation for 84 h. This efficient strategy facilitates the lactose-independent production of HMOs by fermentation, which will improve product quality and reduce the costs of manufacturing.

Recent advances and challenges in microbial production of human milk oligosaccharides

Human milk oligosaccharides (HMOs) are one of the major differences between livestock milk and human milk, and the prebiotic functions of HMOs have been verified through in vitro and clinical trials. The most abundant HMOs include 2′-fucysollactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT); their application and synthesis have attracted wide attentions. In recent years, the biotechnological production of 2′-FL, 3-FL, LNnT and LNT have emerged based on techniques such as whole-cell catalysis and fermentation. In particular, the development of metabolic engineering and synthetic biology methods and strategies have facilitated efficient biosynthesis of these HMOs. However, these advantages have not been systematically reviewed yet. In this review, we first discuss the structures and applications of HMOs; secondly, strategies of microbial synthesis of the most abundant 2′-FL, 3-FL, LNnT and LNT are summarized and compared. Finally, challenges and perspectives of efficient microbial production of HMOs as well as strategies for overcoming the challenges are discussed. This review reveals the whole picture of recent development in HMOs microbial synthesis and can further facilitate the understanding of limiting factors, and further propose a few directions to promote the development of efficient production hosts.

Enzymatic transfucosylation for synthesis of human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a family of structurally distinct carbohydrate oligomers present in human milk. HMOs protect breastfed babies against infection and promote the development of infant health and cognition. In the gut, fucosylated HMOs in particular function as decoy receptors that intercept epithelial attachment of enteric pathogens and hence help reduce infection. Infant formulae made from bovine milk are essentially devoid of HMOs, which creates a large impetus for biosynthetic production of HMOs. Certain microbial α-L-fucosidases (EC 3.2.1.51, EC 3.2.1.111), specifically various retaining α-L-fucosidases of glycoside hydrolase family 29 (GH29), are capable of catalysing transfucosylation. The use of GH29 α-L-fucosidases to promote transfucosylation reactions thus represents a strategy for biocatalytic synthesis of fucosylated HMOs. The purpose of this review is to present the current knowledge on the use of such α-L-fucosidases for synthesis of fucosylated HMOs by enzymatic transfucosylation. We summarize the available data obtained for both wild type and engineered microbial α-L-fucosidases, discuss enzyme and substrate sources, and review factors governing transglycosylation performance, particularly the use of protein engineering. We describe the mechanistic reaction details of α-l-fucosidase transfucosylation, and examine details of enzyme mutation strategies promoting transfucosylation. Finally, we list recommendations for future reaction targets based on currently abundant substrate sources.

α-L-Fucosidases and their applications for the production of fucosylated human milk oligosaccharides.

α-L-Fucosidases (EC 3.2.1.51), catalyzing the hydrolysis of fucosides and/or the transfer of fucosyl residue, have been characterized and modified into a trans-fucosylation mode or, further, engineered to function as "fucosynthase", which can be employed for the enzymatic synthesis of bioactive glycans, including fucosylated human milk oligosaccharides (HMOs). More than half of HMOs are fucosylated and have attracted ever-increasing interest because of their excellent physiological functions on breast-fed infants. To date, the characterization of novel fucosidases and molecular modification of these enzymes have been extensively studied to efficiently synthesize valuable fucosylated compounds. Herein, we discuss the advantages and challenges of different strategies for the production of HMOs and compare various donor/acceptor substrates used for the synthesis of fucosylated HMOs and their biomimetics. The implementation of trans-fucosylation patterns investigated in this paper via well-designed fucosidase mutants and proper reaction conditions may lead to development of an excellent platform, serving both fundamental studies and industrial-scale processes, for valuable carbohydrates synthesis.Key Points• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.

A safety evaluation of mixed human milk oligosaccharides in rats

Human milk oligosaccharides (HMOs) are indigestible carbohydrates representing the third largest fraction of solutes in human breastmilk. They provide valuable prebiotic and anti-pathogenic functions in breastfed infants, but are not yet included in most infant formula products. Recent biotechnological advances now facilitate large-scale production of HMOs, providing infant formula manufacturers with the ability to supplement their products with HMOs to mimic human breastmilk. Although the safety of individual HMOs has been confirmed in preclinical toxicological studies, the safety of HMO mixtures has not been tested. We therefore performed bacterial reverse mutation and in vitro micronucleus tests and conducted a repeated-dose oral toxicity study in rats with a mixture of five HMOs (HMO MIX I), containing 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL). HMO MIX I was not genotoxic and did not induce adverse effects in the repeated dose study. The no-observed-adverse-effect-level (NOAEL) for HMO MIX I in this study is 10% in the diet (equivalent to 5.67 g HMO MIX I/kg bw/day for males and 6.97 g HMO MIX I/kg bw/day for females). Our results provide strong evidence for the safety of HMO MIX I in infant products and general foods.

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

Biosynthesis of a Functional Human Milk Oligosaccharide, 2'-Fucosyllactose, and l-Fucose Using Engineered Saccharomyces cerevisiae.

2'-fucosyllactose (2-FL), one of the most abundant human milk oligosaccharides (HMOs), has received much attention due to its health-promoting activities, such as stimulating the growth of beneficial gut microorganisms, inhibiting pathogen infection, and enhancing the host immune system. Consequently, large quantities of 2-FL are on demand for food applications as well as in-depth investigation of its biological properties. Biosynthesis of 2-FL has been attempted primarily in Escherichia coli, which might not be the best option to produce food and cosmetic ingredients due to the presence of endotoxins on the cell surface. In this study, an alternative route to produce 2-FL via a de novo pathwayusing a food-grade microorganism, Saccharomyces cerevisiae, has been devised. Specifically, heterologous genes, which are necessary to achieve the production of 2-FL from a mixture of glucose and lactose, were introduced into S. cerevisiae. When the lactose transporter (Lac12), de novo GDP-l-fucose pathway (consisting of GDP-d-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (WcaG)), and α1,2-fucosyltransferase (FucT2) were introduced, the resulting engineered strain (D452L-gwf) produced 0.51 g/L of 2-FL from a batch fermentation. In addition, 0.41 g/L of l-fucose was produced when α-l-fucosidase was additionally expressed in the 2-FL producing strain (D452L-gwf). To our knowledge, this is the first report of 2-FL and l-fucose production in engineered S. cerevisiae via the de novo pathway. This study provides the possibility of producing HMOs by a food-grade microorganism S. cerevisiae and paves the way for more HMO production in the future.