Membrane-bound β-hexosyl transferase from Hamamotoa (Sporobolomyces) singularis in Komagataella (Pichia) pastoris was produced as previously described (13, 14). The standard transgalactosylation reaction utilizing Komagataella (Pichia) pastoris resting cells (harboring membrane-bound enzyme) was initiated by adding standardized amounts of enzyme (1 U g⁻¹ lactose) in 5 mM sodium phosphate buffer (pH 5.0) to a similarly buffered solution containing lactose (200 g liter⁻¹) and N-Acetylglucosamine (25 g liter⁻¹) at 30°C. Reaction products and substrates were analyzed by high-performance liquid chromatography (HPLC) (Shimadzu Corporation, Kyoto, Japan) under isocratic conditions at 65°C and a 0.5-ml min⁻¹ flow rate. The mobile phase was water, and separation was performed by two columns in tandem a Supelco gel Ca++ (Supelco, PA), and HPX-42A (Bio-Rad, CA) columns (300 mm by 7.8 mm) coupled to an SPD-20MA and ELSD-LT II detectors (Shimadzu Corporation, Kyoto, Japan). The column was calibrated using galactosyl-lactose (Carbosynth, Berkshire, United Kingdom), LacNAc, Lactose, N-Acetylglucosamine, Glucose, and Galactose (Sigma-Aldrich, St. Louis, MO). Enzymatic activity was determined using 4-nitrophenyl β-D- glucopyranoside or oNP-Glc as substrate as per the previously described methods (13, 14).

The human equivalent dose (HED) for LacNAc was calculated for the animal study using the methods highlighted by the United States Food and Drug Administration and is based on the approximate body weight of the subject (43). The equation used is as follows:

Where the K_m factor is a number based on body surface area. For this study, we used an animal dose of 1,500 mg kg⁻¹, based on ~30 mg LacNAc fed to a ~20 g mouse per day. Additionally, we used a K_m factor of 3 for mice and 16 to represent a 5 kg human infant (43). Using the formula above, the HED for this study represent an equivalent of 281.25 mg LacNAc per kg body weight in infants. While for a 20 kg child (K_m= 25), the HED would be 180 mg kg⁻¹.

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill (Approved protocol number: 19-084).

A total of 50 6-week-old C57BL/6J mice were co-housed at random in groups of 5–6 animals and fed a defined diet (D17121301; Research Diets INC.) containing no prebiotics to normalize the gut microbiota for 2 weeks. After a 2-week standardization period, fresh stool samples were collected directly from the anus of each animal into a sterile tube. To avoid cage batch effects, animals were moved into paired housing such that no two animals from the same standardization group were co-housed. Each animal was considered one experimental unit. Upon reassigning housing (Figure 2A), animal pairs were split into three distinct groups, each of which began feeding on either the defined control diet (D17121301) (n = 17), GOS diet (D17121302) (n = 17) in which 71.8 g of cellulose per kilogram diet was replaced with 71.8 g of pure GOS, or hGOS (D18121401) (n = 16) where LacNAc represented a 1% (w/w). Composition of each diet is detailed in Supplementary Table 1. Each diet was offered ad libitum for 2 weeks prior to stool sample collection. Individual animal mass and dietary consumption were measured daily during the first 14 days of the dietary study to assess animal growth and food consumption (total food consumed in a cage/2 = individual animal food consumption) between diet groups. After day-14 sample collection, half of the animals in each treatment group were removed for a tangential study and all remaining animals (total=24, n = 8 per diet) continued to consume their respective diets ad libitum, with stool sample collections occurring at three additional time points each ~1-week apart prior to animal sacrifice. At the conclusion of the trial on day 38, each animal was euthanized via CO₂ asphyxiation and cervical dislocation.

Total DNA was extracted from fecal pellets using the Qiagen ClearMag Extraction system on KingFisher Flex Magnetic Bead processing instrument as described (15). Briefly, stool samples were transferred to a screwcap tube containing 10 mg of sterile acid-washed glass beads (0.1–0.5 mm diameter) and 700 μl PM1 solution (Qiagen, Valencia, CA). Samples were homogenized for 5-min at 15 Hz in Qiagen Tissue Lyser II (Qiagen). Bead-beaten samples were treated with IRS-PCR inhibitor remover solution (Qiagen) (3:1; lysate:IRS ratio) overnight at 4°C and transferred to KingFisher Deep-well plate containing ClearMag magnetic beads and binding buffer (Qiagen). Sample plates were subsequently processed on KingFisher Flex instrument to isolate and wash DNA. DNA was quantified with Quant-iT™ PicoGreen® dsDNA reagent (Molecular Probes, Thermo Fisher Scientific, Waltham, MA) and stored at −20°C.

Total DNA was subject to amplification of the V4 variable region of the 16S rRNA gene using primers 515F and 806R (44) with Illumina adaptors. Amplicons were barcoded using Illumina dual-index barcodes [Index 1(i7) and Index 2(i5)], purified using Agencourt® AMPure® XP reagent (Beckman Coulter, Brea, CA) and quantified with Quant-iT™ PicoGreen™ dsDNA Reagent (Molecular Probes, Thermo Fisher Scientific). Libraries were pooled in equimolar amounts and sequenced on HiSeq2500 instrument (Illumina, San Diego, CA).

Analysis of 16S rRNA amplicon sequencing data was carried out using the QIIME2 pipeline as described (45). Briefly, sequences were grouped into Operational Taxonomic Units (OTUs) using UCLUST (46). OTU sequences were aligned, and phylogenetic trees were built (47). Before generating the phylogenetic tree, the overall OTU table was collapsed using the “taxa collapse” plugin in QIIME2. The set of representative sequences was then trimmed to include only one representative sequence for each collapsed OTU. The filtered set of representative sequences was then aligned using MAFFT, and a phylogenetic tree was generated from the alignment using RAxML (48, 49). The phylogenetic tree was finally annotated with presence/absence data using iToL and PhyloToAST (50–52). Alpha and beta diversity metrics were calculated in R 4.0.3 using the phyloseq and vegan packages (53–55). Only data from young animals (6 weeks old) were used in the calculation of diversity metrics. Both the Shannon entropy and inverse Simpson indexes were calculated to ensure an accurate estimation of the true alpha diversity of the samples. Beta diversity was calculated using principal coordinate analysis (PCoA) of the weighted UniFrac distances (56).

Data were evaluated for homogeneity of variance using Levene's test. Statistical significance of alpha diversity was evaluated using a repeated-measures ANOVA followed by Tukey's Honest Significant Difference test to separate means. The 95% confidence ellipses for beta diversity plots were calculated in R 4.0.3 using ggplot2 (57). Beta diversity statistical analyses were performed using the PERMANOVA and PERMDISP functions of the vegan package in R 4.0.3. All statistical analysis results for the alpha and beta diversity analyses can be found in Supplementary Table 2. The α for all statistical tests was fixed at 0.05.

All sequencing data has been submitted to the NCBI repository and can be accessed via the following accession number: PRJNA681811.

The reactions catalyzed by the rBHT enzyme were regio-selective, forming the β-galactosyl linkage at the 4-position of the GlcNAc and the 1-position of D-galactose, synthesizing various glycoconjugates directly from soluble GlcNAc. The obtained products, in addition to GOS, included Gal-β(1, 4)GlcNAc (LacNAc, Figure 1A, panel B) disaccharides and Galβ-(1, 4)Galβ-(1, 4)GlcNAc (Galactosyl-LacNAc, Figure 1A, panel C) trisaccharides, which were produced by two sequential transgalactosylations. Figure 1B shows the kinetics of the reaction performed during 8 days of incubation using rBHT polypeptides (e.g., whole cells displaying membrane-bound enzyme). The enrichment of GOS with LacNAc at a ratio lactose/N-Acetylglucosamine of 8:1 performed for these experiments (200 g/L lactose and 25 g/L GlcNAc) generated 25 g/L of LacNAc and 100 g/L hGOS after 48 h of incubation. At this time point, the reaction was terminated, and the products of the reaction (hGOS) were freeze-dried.

We conducted an animal experiment with conventional 6-week-old C57BL/6J mice fed a control diet, or modified diets containing GOS or hGOS to assess their impact on the gut microbiome (Figure 2A). Results showed no impact of GOS or hGOS diets on weight or daily food consumption. Each mouse consumed ~3 g of food per day, with no significant differences between the diets (Supplementary Figure 1). Therefore, based on the formulation of each prebiotic diet, we calculated that the dose of prebiotic consumed per day by each mouse was 0 mg/day (on control diet), and ~190 mg/day of total prebiotic (GOS, hGOS). This estimate translates to ~171 mg/day of GOS (GOS diet) and ~30 mg/day of LacNAc (hGOS diet) based on prebiotic formulation data.

After 2 weeks of feeding on diets containing prebiotics, animals exhibited a significant (repeated measures ANOVA p < 0.05) reduction in alpha diversity (Figure 2B). Over the length of the study, diversity of prebiotic-fed animals returned to values comparable to the control diet with no statistically significant differences between groups at day 28. PCoA plots revealed distinct clustering of hGOS-fed animals, which displayed a much tighter dispersion pattern compared to control animals (PERMDISP p = 0.009), suggesting a higher similarity between communities within hGOS-fed than control-fed animals (Figure 2C). Spatial medians were significantly different between groups (PERMANOVA F = 7.6463, p ≤ 0.001).

Taxonomy plots of relative microbial abundance revealed the genus-level variability between animals fed control diets, and those consuming either GOS or hGOS diets over time (Figure 3A). The most dramatic changes in the assembled microbial communities were observed between timepoint 0 and 14 days after introducing prebiotic diets. Changes between the communities within prebiotic-fed animals after 14 days were minimal. Analysis of Composition of Microbiome (ANCOM) used to further explore microbial abundance changes within the communities of prebiotic-fed animals across all time points revealed an increased relative abundance of beneficial microorganisms including Akkermansia, Bifidobacterium, and Bacteroides, along with Allobaculum in both GOS and hGOS diets. The dietary interventions reduced the relative abundance of Butyricicoccus, Clostridium, Turicibacter, and Lachnospiraceae across all time points (Figures 3A,B, Supplementary Figure 2).

Figure 4 shows a phylogenetic tree generated using PhyloToAST that includes 117 unique OTUs detected in at least one of the three diets examined. Of the 117 unique OTUs detected, 76 OTUs were detected in all conditions (Control, GOS, hGOS), and 40 were detected in control and GOS but not in hGOS fed mice, while none was detected in control and hGOS but not GOS fed mice. The majority of the OTUs detected in the control and GOS mice, but not in hGOS mice, belonged to the Firmicutes phylum. Taxa not detected in the hGOS group compared to control and GOS group within the Phylum Firmicutes and Class Bacilli included Lactobacillus reuteri, L. zeae, species of Enterococcus, Brevibacillus, Paenibacillus, Anaerobacillus, Virgibacillus, Facklamia, Unclassified Lactobacillales, Bacillales, Enterococcaceae, and Planococcaceae. Within the Phylum Firmicutes and Class Clostridia, the following were not detected in the hGOS group: Veillonella dispar, Ruminococcus flavefaciens, Faecalibacterium prausnitzi, species of Butyrivibrio, Pseudobutyrivibrio, Lachnospira, Oxobacter, Roseburia, Dialister, Veillonella, Phascolarctobacterium, Anaerotruncus, Blautia and Unclassified Clostridiales, Clostridiaceae, and Veillonellaceae. Only 3 OTUs corresponding to the Phylum Bacteroidetes (Bacteroides eggerthii, B. caccae, and B. fragilis) and 7 Proteobacteria (Burkholderia bryophila, Acinetobacter, Bilophila, Hydrogenophaga, Pseudomonas, Halomonas, and Unclassified Enterobacteriaceae) were not detected in the hGOS group. Supplementary Table 1 presents the mean relative abundance values for each observed OTU in each of the experimental conditions.