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Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc2, chitin deacetylase to convert GlcNAc2 into GlcN2 and acetate, and glucosaminidase to cleave GlcN2 into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc2 or GlcN2 to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Graphical abstract Key Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.

Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.

β-N-Acetylglucosaminidases (GlcNAcases) hydrolyse N-acetylglucosamine-containing oligosaccharides and proteins. These enzymes produce N-acetylglucosamine (GlcNAc) and have a wide range of promising applications in the food, energy, and pharmaceutical industries, such as synergistic degradation of chitin with endo-chitinases and using GlcNAc to produce sialic acid, bioethanol, single-cell proteins, and pharmaceutical therapeutics. GlcNAcases also play an important role in the dynamic balance of cellular O-linked GlcNAc levels, catabolism of ganglioside storage in Tay-Sachs disease, and bacterial cell wall recycling and flagellar assembly. In view of these important biological functions and the wide range of industrial applications of GlcNAcases, this review aims to provide a better understanding of various advances for these enzymes. It focuses on enzymatic properties of GlcNAcases, including substrate specificity, catalytic activity, pH optimum, temperature optimum, thermostability, the effects of various metal ions and organic reagents, and transglycosylation.

α-Linked N-acetylglucosamine is one of the major glyco-epitopes in O-glycan of gastroduodenal mucin. Here, we identified glycoside hydrolase (GH) family 89 α-N-acetylglucosaminidase, termed AgnB, from Bifidobacterium bifidum JCM 1254, which is essentially specific to GlcNAcα1-4Gal structure. AgnB is a membrane-anchored extracellular enzyme consisting of a GH89 domain and four carbohydrate-binding module (CBM) 32 domains. Among four CBM32 domains, three tandem ones at C-terminus showed to bind porcine gastric mucin, suggesting that these domains enhance the enzyme activity by increasing affinity for multivalent substrates. AgnB might be important for assimilation of gastroduodenal mucin by B. bifidum and also applicable to production of prebiotic oligosaccharides from porcine gastric mucin.

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40–46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 × 106 Da.

Glucosamine (GlcN), an amino sugar, is a compound derived from substitution of a hydroxyl group of a glucose molecule with an amino group. GlcN and its acetylated derivative, N-acetylglucosamine (GlcNAc), have been widely used in food, cosmetics, and pharmaceutical industries and are currently produced by acid hydrolysis of chitin (a linear polymer of GlcNAc) extracted from crab and shrimp shells. Microbial fermentation by filamentous fungi or recombinant Escherichia coli, as an alternative method for the production of GlcN and GlcNAc, is attracting increasing attention because it is an environmentally friendly process. Although the microbial production of GlcN and GlcNAc is hampered by low yield and high production cost, considerable advances have been made in recent years. Here we review the applications, commercial market, and production of GlcN and GlcNAc, with emphasis on the metabolic and process engineering strategies used to improve GlcN and GlcNAc production by recombinant microbes.