High-throughput screening of glycosynthases using azido sugars for oligosaccharides synthesis.

Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar. We created a diverse library of fucosynthase mutants using semi-random and random error prone mutagenesis and then identified improved fucosynthase mutants with desired activity using two distinct screening methods developed by our group to detect glycosynthase activity (i.e., by detecting azide formed upon completion of fucosynthase reaction); (a) pCyn-GFP regulon method, and (b) Click chemistry method. Finally, we provide some proof-of-concept results illustrating the utility of both these screening methods to rapidly detect products of glycosynthase reactions involving azido sugars as donor groups.

Agl28 and Agl29 are key components of a Halobacterium salinarum N-glycosylation pathway.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

κ-Selenocarrageenan Oligosaccharides Prepared by Deep-Sea Enzyme Alleviate Inflammatory Responses and Modulate Gut Microbiota in Ulcerative Colitis Mice.

κ-Selenocarrageenan (KSC) is an organic selenium (Se) polysaccharide. There has been no report of an enzyme that can degrade κ-selenocarrageenan to κ-selenocarrageenan oligosaccharides (KSCOs). This study explored an enzyme, κ-selenocarrageenase (SeCar), from deep-sea bacteria and produced heterologously in Escherichia coli, which degraded KSC to KSCOs. Chemical and spectroscopic analyses demonstrated that purified KSCOs in hydrolysates were composed mainly of selenium-galactobiose. Organic selenium foods through dietary supplementation could help regulate inflammatory bowel diseases (IBD). This study discussed the effects of KSCOs on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in C57BL/6 mice. The results showed that KSCOs alleviated the symptoms of UC and suppressed colonic inflammation by reducing the activity of myeloperoxidase (MPO) and regulating the unbalanced secretion of inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10). Furthermore, KSCOs treatment regulated the composition of gut microbiota, enriched the genera Bifidobacterium, Lachnospiraceae_NK4A136_group and Ruminococcus and inhibited Dubosiella, Turicibacter and Romboutsia. These findings proved that KSCOs obtained by enzymatic degradation could be utilized to prevent or treat UC.

Structural Diversity among Edwardsiellaceae Core Oligosaccharides.

The Edwardsiella genus presents five different pathogenic species: Edwardsiella tarda, E. anguillarum, E. piscicida, E. hoshinae and E. ictaluri. These species cause infections mainly in fish, but they can also infect reptiles, birds or humans. Lipopolysaccharide (endotoxin) plays an important role in the pathogenesis of these bacteria. For the first time, the chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharides of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri were studied. The complete gene assignments for all core biosynthesis gene functions were acquired. The structure of core oligosaccharides was investigated by ¹H and 13C nuclear magnetic resonance (NMR) spectroscopy. The structures of E. piscicida and E. anguillarum core oligosaccharides show the presence of →3,4)-L-glycero-α-D-manno-Hepp, two terminal ß-D-Glcp, →2,3,7)-L-glycero-α-D-manno-Hepp, →7)-L-glycero-α-D-manno-Hepp, terminal α-D-GlcpN, two →4)-α-D-GalpA, → 3)-α-D-GlcpNAc, terminal ß-D-Galp and →5-substituted Kdo. E. hoshinare core oligosaccharide shows only one terminal ß-D-Glcp, and instead of terminal ß-D-Galp a terminal α-D-GlcpNAc. E. ictaluri core oligosaccharide shows only one terminal ß-D-Glcp, one →4)-α-D-GalpA and do not have terminal α-D-GlcpN (see complementary figure).

Functional effects of human milk oligosaccharides (HMOs).

Human milk oligosaccharides (HMOs) are the third most important solid component in human milk and act in tandem with other bioactive components. Individual HMO levels and distribution vary greatly between mothers by multiple variables, such as secretor status, race, geographic region, environmental conditions, season, maternal diet, and weight, gestational age and mode of delivery. HMOs improve the gastrointestinal barrier and also promote a bifidobacterium-rich gut microbiome, which protects against infection, strengthens the epithelial barrier, and creates immunomodulatory metabolites. HMOs fulfil a variety of physiologic functions including potential support to the immune system, brain development, and cognitive function. Supplementing infant formula with HMOs is safe and promotes a healthy development of the infant revealing benefits for microbiota composition and infection prevention. Because of limited data comparing the effect of non-human oligosaccharides to HMOs, it is not known if HMOs offer an additional clinical benefit over non-human oligosaccharides. Better knowledge of the factors influencing HMO composition and their functions will help to understand their short- and long-term benefits.

Slc9a1 plays a vital role in chitosan oligosaccharide transport across the intestinal mucosa of mice.

The mechanism underlying the intestinal transport of COS is not well understood. Here, transcriptome and proteome analyses were performed to identify potential critical molecules involved in COS transport. Enrichment analyses revealed that the differentially expressed genes in the duodenum of the COS-treated mice were mainly enriched in transmembrane and immune function. In particular, B2 m, Itgb2, and Slc9a1 were upregulated. The Slc9a1 inhibitor decreased the transport efficiency of COS both in MODE-K cells (in vitro) and in mice (in vivo). The transport of FITC-COS in Slc9a1-overexpressing MODE-K cells was significantly higher than that in empty vector-transfected cells (P < 0.01). Molecular docking analysis revealed the possibility of stable binding between COS and Slc9a1 through hydrogen bonding. This finding indicates that Slc9a1 plays a crucial role in COS transport in mice. This provides valuable insights for improving the absorption efficiency of COS as a drug adjuvant.

Facile and efficient chemical synthesis of gluco-oligosaccharides (GlcOS) with diverse glycosidic linkages as potential prebiotics to promote the growth of probiotic bacteria.

Glucose-based short-chain oligosaccharides (gluco-oligosaccharides, GlcOS) have been established as functional food ingredients with health-promoting properties. Currently, GlcOS (e.g., isomalto-oligosaccharides, IMOs) are commercially produced via enzymatic processes, which face the challenges of low yield and high cost. Therefore, developing efficient technologies for large-scale production of prebiotic GlcOS is highly desirable. Herein, a facile chemical process was developed to synthesize GlcOS as potential prebiotics via enhanced dehydration condensation of glucose in concentrated sulfuric acid (60-92 %). The maximum GlcOS yield of 83 % was achieved under the optimal condition of 50 % initial glucose loading, 76 % H2SO4, 70 °C, and 20 min. Structural analysis revealed that the synthesized GlcOS are mainly short-chain oligomers with a degree of polymerization (DP) between 2 and 4 (46 % DP 2, 22 % DP 3, 12 % DP 4) and a small percentage of larger oligosaccharides (DP 5-9), which are linked by predominantly α- and ß-(1→6) linkages along with (1→4), (1→ 3), (1→2), and (1↔1) linkages. In vitro fermentation experiments by probiotic Bifidobacterium bifidum ATCC 29521, Bifidobacterium animalis subsp. lactis DSM 10140, and Limosilactobacillus reuteri ATCC 6475 indicated that the GlcOS can be utilized as a carbon source for bacterial growth, and their promotion effect was overall comparable to three commercial prebiotic IMOs. GlcOS were also successfully synthesized from maltose and cellobiose with similar yield and structures to those from glucose, implying the possibility of synthesizing the prebiotic GlcOS directly from inexpensive starch and cellulose.

Radical cation scavenging activity of berberine bridge enzyme-like oligosaccharide oxidases acting on short cell wall fragments.

Oligogalacturonide-oxidases (OGOXs) and cellodextrin-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide-oxidases (OSOXs) that oxidize, respectively, oligogalacturonides (OGs) and cellodextrins (CDs), thereby inactivating their elicitor nature and concomitantly releasing H2O2. Little is known about the physiological role of OSOX activity. By using an ABTS·+-reduction assay, we identified a novel reaction mechanism through which the activity of OSOXs on cell wall oligosaccharides scavenged the radical cation ABTS·+ with an efficiency dependent on the type and length of the oxidized oligosaccharide. In contrast to the oxidation of longer oligomers such as OGs (degree of polymerization from 10 to 15), the activity of OSOXs on short galacturonan- and cellulose-oligomers (degree of polymerization ≤ 4) successfully counteracted the radical cation-generating activity of a fungal laccase, suggesting that OSOXs can generate radical cation scavenging activity in the apoplast with a power proportional to the extent of degradation of the plant cell wall, with possible implications for redox homeostasis and defense against oxidative stress.

Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome.

Introduction: Polycystic Ovary Syndrome (PCOS) is the most common reproductive endocrine disorder among women of reproductive age, which is one of the main causes of anovulatory infertility. Even though the rapidly developed assisted reproductive technology (ART) could effectively solve fertility problems, some PCOS patients still have not obtained satisfactory clinical outcomes. The poor quality of oocytes caused by the abnormal follicular development of PCOS may directly contribute to the failure of ART treatment. Ovarian granulosa cells (GCs) are the most closely related cells to oocytes, and changes in their functional status have a direct impact on oocyte formation. Previous studies have shown that changes in the ovarian microenvironment, like oxidative stress and inflammation, may cause PCOS-related aberrant follicular development by impairing the physiological state of the GCs. Therefore, optimizing the ovarian microenvironment is a feasible method for enhancing the development potential of PCOS oocytes. Methods: In this study, we first detected the expression of inflammatory-related factors (TGF-ß1, IL-10, TNFα, IL-6) and oxidative stress-related factors (HIF-1α and VEGFA), as well as the proliferation ability and apoptosis level of GCs, which were collected from control patients (non-PCOS) and PCOS patients, respectively. Subsequently, human ovarian granulosa cell line (KGN) cells were used to verify the anti-inflammatory and anti-oxidative stress effects of chitosan oligosaccharide (COS) on GCs, as well as to investigate the optimal culture time and concentration of COS. The optimal culture conditions were then used to culture GCs from PCOS patients and control patients. Results: The results showed that GCs from PCOS patients exhibited obvious inflammation and oxidative stress and significantly reduced proliferation and increased apoptosis. Furthermore, COS can increase the expression of anti-inflammatory factors (TGF-ß1 and IL-10) and decrease the expression of pro-inflammatory factors (TNFα and IL-6), as well as promote the proliferation of GCs. Moreover, we found that COS can reduce the level of reactive oxygen species in GCs under oxidative stress by inhibiting the expression of HIF-1α and VEGFA and by suppressing the apoptosis of GCs induced by oxidative stress. Conclusion: We find that inflammation and oxidative stress exist in the GCs of PCOS patients, and COS can reduce these factors, thereby improving the function of GCs.

Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from ß-cyclodextrin by combined action of cyclodextrinase and 4,6-α-glucanotransferase GtfB.

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-α-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-ΔN from Limosilactobacillus reuteri 121 to convert ß-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of α-(1 â†’ 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of α-(1 â†’ 6) linked segments with α-(1 â†’ 4) segments embedded at the reducing end and middle part. The proportion of α-(1 â†’ 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

Human milk whey glycoprotein N-glycans varied greatly among different maternal secretor status.

Human milk glycans are complex carbohydrates, which play a pivotal role in infant health and neonatal development. Maternal secretor status is known to affect free oligosaccharides in milk. Here, the milk N-glycome of secretor (Se+) and nonsecretor (Se-) individuals was qualitatively and quantitatively analyzed by hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry. The total glycosylation, fucosylation, and sialylation of N-glycans was three times higher in the Se+ group compared to the Se- group (p < 0.001) per equal volume of milk. Importantly, 52 out of 63 N-glycans-including the eight most abundant ones-differed greatly between Se+ and Se- individuals (p < 0.05). Moreover, nine N-glycans (H5N3F1, H6N3, H3N5F1, H5N5F1, H5N5F1S1, H5N4F3S1, H6N4F2S1, H6N5F4S1, and H8N7S1) were >10 times more abundant in Se+ milk than in Se- milk. These findings lay a glycomics-basis for designing personalized nutrition supplements for infants.

Enzymatic formation of cyclic maltooligosaccharides for the application of quercetin inclusion complex.

To improve the applicability of quercetin (QCT), we produced a QCT and cycloamylose (CA-QCT) inclusion complex based on the cyclization activity of cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19). The encapsulated QCT was purified using recycling preparative high-performance liquid chromatography, and its formation was analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The water solubility of CA-QCT was 55,000-fold higher than that of QCT. CA-QCT had 97 % stability for one week at pH 8 in a 4 °C water bath. According to a 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay, CA-QCT activity in aqueous solution was 24 times higher than that of an equal amount of QCT in aqueous solution. In an anti-inflammatory assay using lipopolysaccharide-induced RAW264.7 macrophages, CA-QCT in aqueous solution decreased nitric oxide production in a similar manner to QCT in dimethyl sulfoxide (DMSO). Additionally, even under aqueous conditions, CA-QCT more effectively inhibited the production of inflammatory mediators, such as interleukin-1ß, interleukin-6, and cyclooxygenase, compared with QCT dissolved in DMSO.

Total Synthesis of a Trehalose-Containing Lipooligosaccharide Analogue from Mycobacterium linda.

A short and efficient methodology has been developed to synthesize an analogue of a lipooligosaccharide from Mycobacterium linda isolated from Crohn's disease. The total synthesis of the tetrasaccharide was achieved via a convergent [2 + 2] glycosylation approach. The key features of the synthesis involve the selective functionalization of a trehalose core via highly regioselective acylations and regioselective glycosylations. The synthesis was completed via a longest linear sequence of 14 steps in a 14.2% overall yield.

Specific protection mechanism of oligosaccharides on liposomes during freeze-drying.

Liposomes have been received much attention during the past decades as bioactive compounds carriers in food field. However, the application of liposomes is extremely limited by the structural instability during processing such as freeze-drying. In addition, the protection mechanism of lyoprotectant for liposomes during freeze-drying remains controversial. In this study, lactose, fructooligosaccharide, inulin and sucrose were used as lyoprotectants for liposomes and the physicochemical properties, structural stability and freeze-drying protection mechanism were explored. The addition of oligosaccharides could significantly suppress the changes in size and zeta potential, and the amorphous state of liposomes was negligible changed from XRD. The Tg of the four oligosaccharides, especially for sucrose (69.50 °C) and lactose (95.67 °C), revealed the freeze-dried liposomes had formed vitrification matrix, which could prevent liposomes from fusion via increasing the viscosity and reducing membrane mobility. The decrease in Tm of sucrose (147.67 °C) and lactose (181.67 °C), and the changes in functional group of phospholipid and hygroscopic capacity of lyophilized liposomes indicated oligosaccharides replaced water molecules to interact with phospholipids by hydrogen bonds. It can be concluded that the protection mechanism of sucrose and lactose as lyoprotectant was attributed to the combination of vitrification theory and water replacement hypothesis, while the water replacement hypothesis was dominated by fructooligosaccharide and inulin.

Low FODMAP Diet Relieves Visceral Hypersensitivity and Is Associated with Changes in Colonic Microcirculation in Water Avoidance Mice Model.

(1) Background: Irritable bowel syndrome (IBS) is a global public health problem, the pathogenesis of which has not been fully explored. Limiting fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) can relieve symptoms in some patients with IBS. Studies have shown that normal microcirculation perfusion is necessary to maintain the primary function of the gastrointestinal system. Here, we hypothesized that IBS pathogenesis might be related to abnormalities in colonic microcirculation. A low-FODMAP diet could alleviate visceral hypersensitivity (VH) by improving colonic microcirculation; (2) Methods: C57BL/6 mice were raised to establish an IBS-like rodent model using water avoidance (WA) stress or SHAM-WA as a control, one hour per day for ten days. The mice in the WA group were administered different levels of the FODMAP diet: 2.1% regular FODMAP (WA-RF), 10% high FODMAP diet (WA-HF), 5% medium FODMAP diet (WA-MF), and 0% low FODMAP diet (WA-LF) for the following 14 days. The body weight and food consumption of the mice were recorded. Visceral sensitivity was measured as colorectal distention (CRD) using the abdominal withdrawal reflex (AWR) score. Colonic microcirculation was assessed using laser speckle contrast imaging (LCSI). Vascular endothelial-derived growth factor (VEGF) was detected using immunofluorescence staining; (3) Results: The threshold values of CRD pressure in the WA-RF, WA-HF, and WA-MF groups were significantly lower than those in the SHAM-WA group. Moreover, we observed that colonic microcirculation perfusion decreased, and the expression of VEGF protein increased in these three groups of mice. Interestingly, a low-FODMAP dietary intervention could reverse this situation. Specifically, a low-FODMAP diet increased colonic microcirculation perfusion, reduced VEGF protein expression in mice, and increased the threshold of VH. There was a significant positive correlation between colonic microcirculation and threshold for VH; (4) Conclusions: These results demonstrate that a low-FODMAP diet can alter VH by affecting colonic microcirculation. Changes in intestinal microcirculation may be related to VEGF expression.

Pre-, pro-, syn-, and Postbiotics in Infant Formulas: What Are the Immune Benefits for Infants?

The first objective of infant formulas is to ensure the healthy growth of neonates and infants, as the sole complete food source during the first months of life when a child cannot be breastfed. Beyond this nutritional aspect, infant nutrition companies also try to mimic breast milk in its unique immuno-modulating properties. Numerous studies have demonstrated that the intestinal microbiota under the influence of diet shapes the maturation of the immune system and influences the risk of atopic diseases in infants. A new challenge for dairy industries is, therefore, to develop infant formulas inducing the maturation of immunity and the microbiota that can be observed in breastfed delivered vaginally, representing reference infants. Streptococcus thermophilus, Lactobacillus reuteri DSM 17938, Bifidobacterium breve (BC50), Bifidobacterium lactis Bb12, Lactobacillus fermentum (CECT5716), and Lactobacillus rhamnosus GG (LGG) are some of the probiotics added to infant formula, according to a literature review of the past 10 years. The most frequently used prebiotics in published clinical trials are fructo-oligosaccharides (FOSs), galacto-oligosaccharides (GOSs), and human milk oligosaccharides (HMOs). This review sums up the expected benefits and effects for infants of pre-, pro-, syn-, and postbiotics added to infant formula regarding the microbiota, immunity, and allergies.

Purification and biochemical characterization of a novel chitosanase cloned from the gene of Kitasatospora setae KM-6054 and its application in the production of chitooligosaccharides.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30 °C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2-4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs.

The effect of chitosan oligosaccharides on the shelf-life and quality of fresh wet noodles.

In this study, the effects of chitosan oligosaccharides (COS) on the microbial stability and quality properties of fresh wet noodles were evaluated. The addition of COS prolonged the shelf-life of fresh wet noodles at 4 °C by 3-6 days and effectively inhibited the growth of acidity value. However, the presence of COS increased the cooking loss of noodles significantly (P < 0.05) and decreased the hardness as well as tensile strength significantly (P < 0.05). The enthalpy of gelatinization (ΔH) was decreased by COS in the differential scanning calorimetry (DSC) analysis. Meanwhile, the addition of COS decreased the relative crystallinity of starch (from 24.93 % to 22.38 %) without changing the type of X-ray diffraction pattern, revealing that COS weakened the structural stability of starch. In addition, COS was observed to impair the development of compact gluten network by confocal laser scanning micrographs. Further, the free-sulfhydryl groups content and sodium dodecyl sulphate-extractable protein (SDS-EP) values of cooked noodles increased significant (P < 0.05), confirming the obstruction on the polymerization of gluten proteins during the hydrothermal process. Although COS adversely affected the quality of noodles, it was outstanding and feasible for the preservation of fresh wet noodles.

De novo biosynthesis of 2'-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1,2-fucosyltransferase from Azospirillum lipoferum.

Human milk oligosaccharides are complex, indigestible oligosaccharides that provide ideal nutrition for infant development. Here, 2'-fucosyllactose was efficiently produced in Escherichia coli by using a biosynthetic pathway. For this, both lacZ and wcaJ (encoding ß-galactosidase and UDP-glucose lipid carrier transferase, respectively) were deleted to enhance the 2'-fucosyllactose biosynthesis. To further enhance 2'-fucosyllactose production, SAMT from Azospirillum lipoferum was inserted into the chromosome of the engineered strain, and the native promoter was replaced with a strong constitutive promoter (PJ23119). The titer of 2'-fucosyllactose was increased to 8.03 g/L by introducing the regulators rcsA and rcsB into the recombinant strains. Compared to wbgL-based strains, only 2'-fucosyllactose was produced in SAMT-based strains without other by-products. Finally, the highest titer of 2'-fucosyllactose reached 112.56 g/L in a 5 L bioreactor by fed-batch cultivation, with a productivity of 1.10 g/L/h and a yield of 0.98 mol/mol lactose, indicating a strong potential in industrial production.