Yusuke Tomabechi

Acceptor specificity in the transglycosylation reaction using Endo-M

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M. Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary and secondary hydroxyl groups.

Chemo-enzymatic synthesis of glycosylated insulin using a GlcNAc tag

Artificial insulin with an N-linked oligosaccharide was synthesized by a chemo-enzymatic method using endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). GlcNAc-modified insulin was prepared by the reaction of the carboxymethyl glycoside of GlcNAc and 3 amino groups of bovine insulin using a dimethylphosphinothioic mixed anhydride (Mpt-MA) method. A transglycosylation reaction of the GlcNAc-modified insulin using Endo-M gave mono-transglycosylated insulin predominantly. We determined the transglycosylation site of the mono-transglycosylated insulin.

Laboratory-scale production of (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids, using a bacterial-based method

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery. Graphical abstract High amount of pure (S)-reticuline.

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes. Graphical Abstract BbhII encodes a sulfoglycosidase which is involved in the mucin degradation pathway of Bifidobacterium bifidum.

Microbial production of novel sulphated alkaloids for drug discovery

Natural products from plants are useful as lead compounds in drug discovery. Plant benzylisoquinoline alkaloids (BIAs) exhibit various pharmaceutical activities. Although unidentified BIAs are expected to be of medicinal value, sufficient quantities of such BIAs, for biological assays, are sometimes difficult to obtain due to their low content in natural sources. Here, we showed that high productivity of BIAs in engineered Escherichia coli could be exploited for drug discovery. First, we improved upon the previous microbial production system producing (S)-reticuline, an important BIA intermediate, to obtain yields of around 160 mg/L, which was 4-fold higher than those of the previously reported highest production system. Subsequently, we synthesised non-natural BIAs (O-sulphated (S)-reticulines) by introducing human sulphotransferases into the improved (S)-reticuline production system. Analysis of human primary cells treated with these BIAs demonstrated that they affected a biomarker expression in a manner different from that by the parent compound (S)-reticuline, suggesting that simple side-chain modification altered the characteristic traits of BIA. These results indicated that highly productive microbial systems might facilitate the production of scarce or novel BIAs and enable subsequent evaluation of their biological activities. The system developed here could be applied to other rare natural products and might contribute to the drug-discovery process as a next-generation strategy.

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum

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.

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex N-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.