Role of UDP-Glycosyltransferase (ugt) Genes in Detoxification and Glycosylation of 1-Hydroxyphenazine (1-HP) in Caenorhabditis elegans.

Caenorhabditis elegans is a useful model organism to study the xenobiotic detoxification pathways of various natural and synthetic toxins, but the mechanisms of phase II detoxification are understudied. 1-Hydroxyphenazine (1-HP), a toxin produced by the bacterium Pseudomonas aeruginosa, kills C. elegans. We previously showed that C. elegans detoxifies 1-HP by adding one, two, or three glucose molecules in N2 worms. Our current study evaluates the roles that some UDP-glycosyltransferase (ugt) genes play in 1-HP detoxification. We show that ugt-23 and ugt-49 knockout mutants are more sensitive to 1-HP than reference strains N2 or PD1074. Our data also show that ugt-23 knockout mutants produce reduced amounts of the trisaccharide sugars, while the ugt-49 knockout mutants produce reduced amounts of all 1-HP derivatives except for the glucopyranosyl product compared to the reference strains. We characterized the structure of the trisaccharide sugar phenazines made by C. elegans and showed that one of the sugar modifications contains an N-acetylglucosamine (GlcNAc) in place of glucose. This implies broad specificity regarding UGT function and the role of genes other than ogt-1 in adding GlcNAc, at least in small-molecule detoxification.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Efficient Biosynthesis of Difucosyllactose via De Novo GDP-l-Fucose Pathway in Metabolically Engineered Escherichia coli.

Difucosyllactose (DFL) is an important component of human milk oligosaccharides (HMOs) and has significant benefits for the growth and development of infants. So far, a few microbial cell factories have been constructed for the production of DFL, which still have problems of low production and high cost. Herein, a high-level de novo pathway DFL-producing strain was constructed by multistep optimization strategies in Escherichia coli BL21star(DE3). We first efficiently synthesized the intermediate 2'-fucosyllactose (2'-FL) in E. coli BL21star(DE3) by the advisable stepwise strategy. The truncated α-1,3/4-fucosyltransferase (Hp3/4FT) was then introduced into the engineered strain to achieve de novo biosynthesis of DFL. ATP-dependent protease (Lon) and GDP-mannose hydrolase (NudK) were deleted, and mannose-6-phosphate isomerase (ManA) was overexpressed to improve GDP-l-fucose accumulation. The regulator RcsA was overexpressed to fine-tune the expression level of pathway genes, thereby increasing the synthesis of DFL. The final strain produced 6.19 g/L of DFL in the shake flask and 33.45 g/L of DFL in the 5 L fermenter, which were the highest reported titers so far. This study provides a more economical, sustainable, and effective strategy to produce the fucosylated human milk oligosaccharides (HMOs).

Rational Design of an α-1,3-Fucosyltransferase for the Biosynthesis of 3-Fucosyllactose in Bacillus subtilis ATCC 6051a via De Novo GDP-l-Fucose Pathway.

3-Fucosyllactose (3-FL) is an important oligosaccharide and nutrient in breast milk that can be synthesized in microbial cells by α-1,3-fucosyltransferase (α-1,3-FucT) using guanosine 5'-diphosphate (GDP)-l-fucose and lactose as substrates. However, the catalytic efficiency of known α-1,3-FucTs from various sources was limited due to their low solubility. To enhance the microbial production of 3-FL, the efficiencies of α-1,3-FucTs were evaluated and in Bacillus subtilis (B. subtilis) chassis cells that had been endowed with a heterologous synthetic pathway for GDP-l-fucose, revealing that the activity of FucTa from Helicobacter pylori (H. pylori) was higher than that of any of other reported homologues. To further improve the catalytic performance of FucTa, a rational design approach was employed, involving intracellular evaluation of the mutational sites of M32 obtained through directed evolution, analysis of the ligand binding site diversity, and protein structure simulation. Among the obtained variants, the FucTa-Y218 K variant exhibited the highest 3-FL yield, reaching 7.55 g/L in the shake flask growth experiment, which was 3.48-fold higher than that achieved by the wild-type enzyme. Subsequent fermentation optimization in a 5 L bioreactor resulted in a remarkable 3-FL production of 36.98 g/L, highlighting the great prospects of the designed enzyme and the strains for industrial applications.

De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

Infant microbiome cultivation and metagenomic analysis reveal Bifidobacterium 2'-fucosyllactose utilization can be facilitated by coexisting species.

The early-life gut microbiome development has long-term health impacts and can be influenced by factors such as infant diet. Human milk oligosaccharides (HMOs), an essential component of breast milk that can only be metabolized by some beneficial gut microorganisms, ensure proper gut microbiome establishment and infant development. However, how HMOs are metabolized by gut microbiomes is not fully elucidated. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigate microbiome responses to 2'-fucosyllactose (2'FL), a prevalent HMO and a common infant formula additive, by establishing individualized microbiomes using fecal samples from three infants as the inocula. Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2'FL. Using metagenomic data, we predict that extracellular fucosidases encoded by co-existing members such as Ruminococcus gnavus initiate 2'FL breakdown, thus critical for B. breve's growth. Using both targeted co-cultures and by supplementation of R. gnavus into one microbiome, we show that R. gnavus can promote extensive growth of B. breve through the release of lactose from 2'FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrates that HMO utilization can vary with an individual's microbiome.

2'-Fucosyllactose (2'-FL) changes infants gut microbiota composition and their metabolism in a host-free human colonic model.

BACKGROUND: Breast milk is critical for neonates, providing the necessary energy, nutrients, and bioactive compounds for growth and development. Research indicated that human milk oligosaccharides (HMOs) have been shown to shape a beneficial gut microbiota, as well as their metabolism (e.g. short-chain fatty acids). 2'-Fucosyllactose (2'-FL) is one major HMO that composed of 30% of total HMOs. OBJECTIVES: This study aimed to understand the impact of 2'-FL on the composition and metabolism of infant gut microbiota. METHODS: Our study utilized an in-vitro human colonic model (HCM) to investigate the host-free interactions between 2'-FL and infant gut microbiota. To simulate the infant gut microbiota, we inoculated the HCM system with eight representative bacterial species from infant gut microbiota. The effects of 2'-FL on the gut microbial composition and their metabolism were determined through real-time quantitative PCR and liquid-chromatography mass spectrometry (LC/MS). The obtained data were analyzed using Compound Discoverer 3.1 and MetaboAnalyst 4.0. RESULTS: Our study findings suggest that the intervention of 2'-FL in HCM resulted in a significant change in the abundance of representative bacterial species. PCR analysis showed a consistent increase in the abundance of Parabacteroides. distasonis in all three colon sections. Furthermore, analysis of free fatty acids revealed a significant increase in their levels in the ascending, transverse, and descending colons, except for caproic acid, which was significantly reduced to a non-detectable level. The identification of significant extracellular polar metabolites, such as glutathione and serotonin, enabled us to distinguish between the metabolomes before and after 2'-FL intervention. Moreover, correlation analysis revealed a significant association between the altered microbes and microbial metabolites. CONCLUSIONS: In summary, our study demonstrated the impact of 2'-FL intervention on the defined composition of infant gut microbiota and their metabolic pathways in an in vitro setting. Our findings provide valuable insights for future follow-up investigations into the role of 2'-FL in regulating the growth and development of infant gut microbiota in vivo.

High-Yield Synthesis of 2'-Fucosyllactose from Glycerol and Glucose in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) is vital for the growth and development of newborns. In this study, we developed a synthesis pathway for 2'-FL in Escherichia coli BL21 (DE3). Then, we optimized the solubility of α-1,2-fucosyltransferase, thereby enhancing the production yield of 2'-FL. Based on this finding, we further enhanced the expression of guanosine inosine kinase Gsk and knocked out the isocitrate lyase regulator gene iclR. This strategy reduced the formation of byproduct acetate during the metabolic process and alleviated carbon source overflow effects in the strain, resulting in further improvement of the yield of 2'-FL. In a 3 L bioreactor, employing fed-batch fermentation with glycerol and glucose as substrates, the engineered strain BWLAI-RSZL exhibited impressive 2'-FL titers of 121.9 and 111.56 g/L, along with productivity levels of 1.57 and 1.31 g/L/h, respectively. The reported 2'-FL titers reached a groundbreaking level, irrespective of the carbon source employed (glycerol or glucose), highlighting the significant potential for large-scale industrial synthesis of 2'-FL.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30 min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization.

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Interaction of lectin Sambucus nigra with sialylated trisaccharides in the presence of osmolytes. Chronopotentiometric sensing.

Trisaccharides bind to their interaction partners-lectins relatively weakly, which makes detection of their complexes challenging. In this work, we show that an osmolyte presence improves the distinguishing complexes of lectin Sambucus nigra with trisialyllactoses with various binding affinities. The addition of osmolyte, non-binding sugar mannose significantly improved the precision of binding experiments performed using chronopotentiometric stripping at the electrode surface and fluorescence analysis in solution. Osmolytes minimized nonspecific interactions between binding sugar and lectin. Obtained findings can be utilized in any in vitro methods studying interactions of carbohydrates, respectively their conjugates with proteins. The study of carbohydrate interactions appears important since they play essential roles in a variety of biological processes including carcinogenesis.

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.

Metabolic Engineering of De Novo Pathway for the Production of 2'-Fucosyllactose in Escherichia coli.

2'-Fucosyllactose (2'-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2'-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and ß-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2'-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2'-FL (0.98 g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2'-FL (18.22 g/L) was obtained, corresponding to a yield of 0.25 g/g glycerol and a substrate conversion of 0.88 g/g lactose.

Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli.

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

Combinatorial metabolic engineering and tolerance evolving of Escherichia coli for high production of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL) is an important functional ingredient of advanced infant formula. Here, Escherichia coli MG1655 was engineered for achieving high 2'-FL production. The expressions of 2'-FL synthesis pathway genes were finely regulated with single or multi copies according to rate-limiting enzyme diagnosis. On this basic, the branch pathway genes were deleted, and the overexpression of the 2'-FL efflux protein SetA and the fructose-1,6-bisphosphatase GlpX were tuned. The resulting strain produced 46.06 ± 1.28 g/L 2'-FL in a 5-L fermenter. Furtherly, adaptive laboratory evolution was conducted. A rpoC gene mutation was obtained which could improve the cell tolerance and the 2'-FL production up to 61.06 ± 1.93 g/L, with the highest productivity of 1.70 g/L/h among E. coli strains by now. Taken together, this work provides a combinatorial strategy to improve 2'-FL accumulation including rational fine-tuning pathway genes expressions and irrational adaptive laboratory evolution. This study should be helpful for constructing high level 2'-FL producers.

Biosynthesis of lactosucrose by a new source of ß-fructofuranosidase from Bacillus methanolicus LB-1.

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The ß-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B. methanolicus LB-1 under the optimized conditions: 10 g/L of glucose, 5 g/L of yeast extract, initial medium pH at 7.0, 37 °C, 24 h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45 kDa. Biosynthesis of LS was performed using the purified ß-fructofuranosidase, and production of LS reached 110 g/L under the optimized reaction conditions: pH at 7.0, 37 °C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28 h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30 min of elution, confirming that B. methanolicus LB-1 ß-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of ß-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

Occurrence, functional properties, and preparation of 3-fucosyllactose, one of the smallest human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are receiving wide interest and high attention due to their health benefits, especially for newborns. The HMOs-fortified products are expected to mimic human milk not only in the kinds of added oligosaccharides components but also the appropriate proportion between these components, and further provide the nutrition and physiological effects of human milk to newborns as closely as possible. In comparison to intensively studied 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) has less attention in almost all respects. Nerveless, 3-FL naturally occurs in breast milk and increases roughly over the course of lactation with a nonnegligible content, and plays an irreplaceable role in human milk and delivers functional properties to newborns. According to the safety evaluation, 3-FL shows no acute oral toxicity, genetic toxicity, and subchronic toxicity. It has been approved as generally recognized as safe (GRAS). Biological production of 3-FL can be realized by enzymatic and cell factory approaches. The α1,3- or α1,3/4-fucosyltransferase is the key enzyme for 3-FL biosynthesis. Various metabolic engineering strategies have been applied to enhance 3-FL yield using cell factory approach. In conclusion, this review gives an overview of the recent scientific literatures regarding occurrence, bioactive properties, safety evaluation, and biotechnological preparation of 3-FL.

Module-Guided Metabolic Rewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli with Lactose De Novo Pathway.

Fucosyllactose (FL) has garnered considerable attention for its benefits on infant health. In this study, we report an efficient E. coli cell factory to produce 2'/3-fucosyllactose (2'/3-FL) with lactose de novo pathway through metabolic network remodeling, including (1) modification of the PTSGlc system to enhance glucose internalization efficiency; (2) screening for ß-1,4-galactosyltransferase (ß-1,4-GalT) and introduction of lactose synthesis pathway; (3) eliminating inhibition of byproduct pathways; (4) constructing antibiotic-free and inducer-free FL strains; and (5) up-regulating the expression of genes in the GDP-l-fucose module. The final engineered strains BP10-3 and BP11-3 produced 4.36 g/L for 2'-FL and 3.23 g/L for 3-FL in shake flasks. In 3 L bioreactors, fed-batch cultivations of the two strains produced 40.44 g/L for 2'-FL and 30.42 g/L for 3-FL, yielding 0.63 and 0.69 g/g glucose, respectively. The strategy described in this work will help to engineer E. coli as a safe chassis for other lactose-independent HMOs production.

Synthesis of the trisaccharide repeating unit of Stenotrophomonas maltophilia O6 antigen through step-wise and one-pot approaches.

Synthetic conjugate vaccines are an important area of research for the prevention and occurrence of diseases caused by Gram-negative bacteria. For the development of such vaccines, access to the pure and homogeneous oligosaccharide fragments of the bacterial cell surface polysaccharides are necessary. Stenotrophomonas maltophilia is a typical opportunistic Gram-negative bacteria that causes severe pulmonary and other infections; often in hospitalized patients. With the emergence of multidrug resistant strains and increased virulence, new therapeutic strategies are needed to combat the threat. Herein, we report the syntheses of the trisaccharide repeating unit of S. maltophilia O6 antigen through stepwise and one-pot assemblies of the trisaccharide. The target trisaccharide was appended with a 2-aminoethyl linker that could provide the opportunity for conjugation to carrier proteins for the synthesis of vaccine candidates.

Strategies for Enhancing Microbial Production of 2'-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide.

Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2'-Fucosyllactose (2'-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2'-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2'-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2'-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2'-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2'-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2'-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2'-FL are suggested.