The extracellular wall-bound β-N-acetylglucosaminidase from Lactobacillus casei is involved in the metabolism of the human milk oligosaccharide lacto-N-triose

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a β-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of β-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3′-N-acetylglucosaminyl-mannose and 3′-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3′-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3′-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3′-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a β-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche.This work was financed by funds of the Spanish Ministry for Economy and Competitiveness (MINECO)/FEDER through projects AGL2014-52996-C2 (1-R and 2-R) and of the Valencian government through project ACOMP/2012/030. G.N.B. was supported by a predoctoral fellowship from the Carolina Foundation and Argentinian Ministry of Education.Peer reviewe

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius

Abstract Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis

Family 1 Glycosyltransferase UGT706F8 from Zea mays Selectively Catalyzes the Synthesis of Silibinin 7-O-β-d -Glucoside

Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and k$_{cat}$ were limited, prohibiting the application of the engineered catalyst. Here, we identified a naturally regioselective silibinin glucosyltransferase, UGT706F8, a family 1 glycosyltransferase from Zea mays. It selectively and efficiently (k$_{cat}$ = 2.1 ± 0.1 s$^{–1}$; KM = 36.9 ± 5.2 $μ$M; TTN = 768 ± 22) catalyzes the quantitative synthesis of silibinin 7-O-β-d-glucoside. We solved the crystal structure of UGT706F8 and investigated the molecular determinants of regioselective silibinin glucosylation. UGT706F8 was the only regioselective enzyme among 18 glycosyltransferases found to be active on silibinin. We found the temperature optimum of UGT706F8 to be 34 °C and the pH optimum to be 7–8. Our results indicate that UGT706F8 is an efficient silibinin glycosyltransferase that enables biocatalytic production of silbinin 7-O-β-d-glucoside

Family 1 Glycosyltransferase UGT706F8 from <i>Zea mays</i> Selectively Catalyzes the Synthesis of Silibinin 7-<i>Ο</i>-<i>β</i>-D-Glucoside

Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and k$_{cat}$ were limited, prohibiting the application of the engineered catalyst. Here, we identified a naturally regioselective silibinin glucosyltransferase, UGT706F8, a family 1 glycosyltransferase from Zea mays. It selectively and efficiently (k$_{cat}$ = 2.1 ± 0.1 s$^{–1}$; KM = 36.9 ± 5.2 $μ$M; TTN = 768 ± 22) catalyzes the quantitative synthesis of silibinin 7-O-β-d-glucoside. We solved the crystal structure of UGT706F8 and investigated the molecular determinants of regioselective silibinin glucosylation. UGT706F8 was the only regioselective enzyme among 18 glycosyltransferases found to be active on silibinin. We found the temperature optimum of UGT706F8 to be 34 °C and the pH optimum to be 7–8. Our results indicate that UGT706F8 is an efficient silibinin glycosyltransferase that enables biocatalytic production of silbinin 7-O-β-d-glucoside

alfa-L-Fucosidasas AlfB y AlfC de Lactobacillus casei: caracterización funcional, cristalización y síntesis de fucosil-oligosacáridos

Comunicación presentadas en la 6ª reunión de la Red temática BAL (Participación de las Bacterias Lácticas en la Salud Humana y en la Calidad Alimentaria), celebrada el 28 y 29 de junio de 2012 en Tarragona.Peer Reviewe