The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.(S)-reticuline ͉ magnoflorine ͉ scoulerine

The secondary metabolites of higher plants include diverse chemicals, such as alkaloids, isoprenoids and phenolic compounds (phenylpropanoids and flavonoids). Although these compounds are widely used in human health and nutrition, at present they are mainly obtained by extraction from plants and extraction yields are low because most of these metabolites accumulate at low levels in plant cells. Recent advances in synthetic biology and metabolic engineering have enabled tailored production of plant secondary metabolites in microorganisms, but these methods often require the addition of expensive substrates. Here we develop an Escherichia coli fermentation system that yields plant alkaloids from simple carbon sources, using selected enzymes to construct a tailor-made biosynthetic pathway. In this system, engineered cells cultured in growth medium without additional substrates produce the plant benzylisoquinoline alkaloid, (S)-reticuline (yield, 46.0 mg l−1 culture medium). The fermentation platform described here offers opportunities for low-cost production of many diverse alkaloids.

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the ␣-(132) linkage of 2-fucosyllactose, and a gene encoding 1,2-␣-L-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal ␣-(132)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2-fucosyllactose was determined to be inversion by using 1 H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).␣-L-Fucosyl residues are frequently found at the nonreducing termini of various glycoconjugates, including blood group substances, milk oligosaccharides, gastric and submaxillary mucins, and serum glycoproteins (30, 37). The results of recent studies indicate that such terminal fucosyl residues attached by

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal␤1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal␤1,3GlcNAc␤1,3Gal␤1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified ␤-linked lacto-Nbiose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

The infant’s gut microbiome is generally rich in the Bifidobacterium genus. The mother’s milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of Bifidobacterium in infants. Given these results, two HMO molecules, 2′-fucosyllactose and lacto-N-neotetraose, have recently been industrialized to fortify formula milk. As of now, however, our knowledge about the HMO consumption pathways in infant gut-associated bifidobacteria is still incomplete. The recent studies indicate that HMO assimilation abilities significantly vary among different Bifidobacterium species and strains. Therefore, to truly maximize the effects of prebiotic and probiotic supplementation in commercialized formula, we need to understand HMO consumption behaviors of bifidobacteria in more detail. In this review, we summarized how different Bifidobacterium species/strains are equipped with varied gene sets required for HMO assimilation. We then examined the correlation between the abundance of the HMO-related genes and bifidobacteria-rich microbiota formation in the infant gut through data mining analysis of a deposited fecal microbiome shotgun sequencing dataset. Finally, we shortly described future perspectives on HMO-related studies.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.