Production Of Human Milk Oligosaccharides Research Articles

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.

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α-l-fucosidases is a new route for manufacturing biomimetic HMOs. Seven α-l-fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α-l-1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto-N-fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto-N-tetraose as acceptor with a 39% yield. α-l-Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2′-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively.

Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations

Human milk oligosaccharides (HMO) are almost unique constituents of breast milk and are not found in appreciable amounts in cow milk. Due to several positive aspects of HMO for the development, health, and wellbeing of infants, production of HMO would be desirable. As a result, scientists from different disciplines have developed methods for the preparation of single HMO compounds. Here, we review approaches to HMO preparation by (chemo-)enzymatic syntheses or by whole-cell biotransformation with recombinant bacterial cells. With lactose as acceptor (in vitro or in vivo), fucosyltransferases can be used for the production of 2′-fucosyllactose, 3-fucosyllactose, or more complex fucosylated core structures. Sialylated HMO can be produced by sialyltransferases and trans-sialidases. Core structures as lacto-N-tetraose can be obtained by glycosyltransferases from chemical donor compounds or by multi-enzyme cascades; recent publications also show production of lacto-N-tetraose by recombinant Escherichia coli bacteria and approaches to obtain fucosylated core structures. In view of an industrial production of HMOs, the whole cell biotransformation is at this stage the most promising option to provide human milk oligosaccharides as food additive.

Role of human milk oligosaccharides in Group B Streptococcus colonisation.

Group B Streptococcus (GBS) infection is a major cause of morbidity and mortality in infants. The major risk factor for GBS disease is maternal and subsequent infant colonisation. It is unknown whether human milk oligosaccharides (HMOs) protect against GBS colonisation. HMO production is genetically determined and linked to the Lewis antigen system. We aimed to investigate the association between HMOs and infant GBS colonisation between birth and postnatal day 90. Rectovaginal swabs were collected at delivery, as well as colostrum/breast milk, infant nasopharyngeal and rectal swabs at birth, 6 days and days 60–89 postpartum from 183 Gambian mother/infant pairs. GBS colonisation and serotypes were determined using culture and PCR. 1H nuclear magnetic resonance spectroscopy was used to characterise the mother's Lewis status and HMO profile in breast milk. Mothers who were Lewis-positive were significantly less likely to be colonised by GBS (X2=12.50, P<0.001). Infants of Lewis-positive mothers were less likely GBS colonised at birth (X2=4.88 P=0.03) and more likely to clear colonisation between birth and days 60–89 than infants born to Lewis-negative women (P=0.05). There was no association between Secretor status and GBS colonisation. In vitro work revealed that lacto-N-difucohexaose I (LNDFHI) correlated with a reduction in the growth of GBS. Our results suggest that HMO such as LNDFHI may be a useful adjunct in reducing maternal and infant colonisation and hence invasive GBS disease. Secretor status offers utility as a stratification variable in GBS clinical trials.

Enzymatic production of HMO mimics by the sialylation of galacto-oligosaccharides

Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that exhibit a wide range of biological activities. In this report, we describe an efficient, Multi-Enzyme One-Pot strategy to produce HMO mimics by the sialylation of galacto-oligosaccharides (GOSs), which are often added to infant formula as an inexpensive alternative to HMOs. In this system, the sialyltransferase donor, cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), was generated in situ using a CMP-sialic acid synthetase. The sialylated GOSs were obtained by one-step purification after digesting CMP using the alkaline phosphatase PhoA to cytidine and inorganic phosphate. Although the synthesized α2,3-, α2,6- and α2,3/8-sialyl-GOSs exhibit different sialylation levels and patterns, all of these mixtures can be fermented by Bifidobacterium longum subsp. infantis ATCC 15697 but not by Bifidobacterium adolescentis ATCC 15703. The sialidase NanH2, which is unique to the former strain, hydrolyzed all of the synthesized HMO mimics.

Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity

Casein glycomacropeptide (cGMP) and lactose, which are purified (or semi-purified) components obtained from side streams from dairy industry operations, were used as substrates for enzyme catalyzed production of 3′-sialyllactose, a model case compound for human milk oligosaccharides (HMOs). The enzyme employed was a mutated sialidase, Tr6, derived from Trypanosoma rangeli, and expressed in Pichia pastoris after codon-optimization. The Tr6 contained 6 point mutations and exhibited trans-sialidase activity. The Tr6 trans-sialidase reaction conditions were tuned for maximizing Tr6 catalyzed 3′-sialyllactose production by optimizing pH, temperature, acceptor, and donor concentrations using response surface designs. At the optimum reaction conditions, the Tr6 catalyzed the transfer of sialic acid from cGMP to lactose at high efficiency without substantial hydrolysis of the 3′-sialyllactose product. The robustness of the Tr6 catalyzed reaction was verified at 5L-scale providing a yield of 3.6g 3′-sialyllactose at an estimated molar trans-sialylation yield of 50% on the 3′-sialyl in cGMP. Lacto-N-tetraose and lacto-N-fucopentaoses also functioned as acceptor molecules demonstrating the versatility of the Tr6 trans-sialidase for catalyzing sialyl-transfer for generating different HMOs. The data signify the applicability of enzymatic trans-sialylation on dairy side-stream components for production of human milk oligosaccharides.

Lacto-N-Tetraose, Fucosylation, and Secretor Status Are Highly Variable in Human Milk Oligosaccharides From Women Delivering Preterm

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.

Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.

A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria.

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.