Adaptation of Gut Microbiome to Transgenic Pigs Secreting β-Glucanase, Xylanase, and Phytase

We previously generated transgenic pigs with enhanced growth rate and reduced nutrient loss. However, the composition of their gut microbiome is unknown. In this study, we successfully generated EGFP marker-free transgenic (MF-TG) pigs with high expression levels of microbial β-glucanase, xylanase, and phytase in the parotid gland. We collected intestinal contents from the ileum, cecum and colon of five MF-TG and five wild-type (WT) sows and investigated the gut microbiome of the transgenic pigs via metagenomic analysis. Results showed that the levels of probiotics, such as Lactobacillus reuteri and Streptococcus, were more abundant in the cecum of the MF-TG pigs and higher than those of WT pigs. By contrast, the levels of harmful microorganisms, such as Campylobacter, Chlamydia trachomatis, and Campylobacter fetus, and various unidentified viruses, were higher in the cecum of the WT pigs than those of the MF-TG pigs. By comparing unigenes and the eggNOG database, we found that the microorganisms in the colon of the MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in the WT pigs. Moreover, the microorganisms in the cecum of the MF-TG pigs were active in GlycosylTransferase Family 8 (GT8), Glycoside Hydrolase Family 13 (GH13), and Glycoside Hydrolase Family 32 (GH32). Furthermore, the levels of numerous carbohydrases, such as glucan 1,3-beta-glucosidase, xylan 1,4-beta-xylosidase and exo-1,3-1,4-glucanase, were higher in the cecum of the MF-TG pigs than those of the WT pigs. The results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host. This cooperation could be beneficial for improving feed efficiency.

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Structure of a glycoside hydrolase family 50 enzyme from a subfamily that is enriched in human gut microbiome bacteroidetes

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25189 ◽

2016 ◽

Vol 85 (1) ◽

pp. 182-187 ◽

Cited By ~ 6

Author(s):

Kaleigh Giles ◽

Benjamin Pluvinage ◽

Alisdair B. Boraston

Keyword(s):

Gut Microbiome ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Human Gut ◽

Human Gut Microbiome ◽

Hydrolase Family

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Adaptation of Gut Microbiotas to Transgenic Pigs Secreting β-Glucanase, Xylanase and Phytase

10.21203/rs.3.rs-84926/v1 ◽

2020 ◽

Author(s):

Jianxin Mo ◽

Guoling Li ◽

Guangyan Huang ◽

Haoqiang Wang ◽

Junsong Shi ◽

...

Keyword(s):

Feed Efficiency ◽

Digestive Enzymes ◽

Regulatory Subunit ◽

Wild Type ◽

Transgenic Pigs ◽

Intestinal Microbes ◽

Type D ◽

Aspartate Carbamoyltransferase ◽

Fractional Abundance ◽

Marker Free

Abstract BackgroundThe gut microbiotas play an important role in digestive function and feed efficiency in pigs. However, the effect of exogenous digestive enzymes on the composition and functional contributions of swine intestinal microbes is unclear. The objective of this study was to investigate the change of gut microbiotas in the transgenic pigs secreting microbial digestive enzymes in their salivary glands.MethodsEGFP marker-free transgenic (MF-TG) pigs were generated by deleted the EGFP coding genes in the transgenic pigs we previously generated. Samples of chyme from the ileum, caecum and colon of five MF-TG and five wild-type (WT) sows were collected for investigating the gut microbiomes via metagenomics analyses.ResultsThe levels of probiotics were abundant in the caecum of MF-TG pigs and higher than those of WT pigs. By contrast, the levels of some harmful microorganisms were higher in the caecum of WT pigs than those of MF-TG pigs. In addition, the microorganisms in the colon of MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in WT pigs. Moreover, the levels of numerous carbohydrases in the caecum of MF-TG pigs were higher than those of WT pigs. ConclusionsThe results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host.

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Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

Journal of Biological Chemistry ◽

10.1074/jbc.m117.792598 ◽

2017 ◽

Vol 292 (51) ◽

pp. 21092-21101 ◽

Cited By ~ 4

Author(s):

Alexander Holm Viborg ◽

Takane Katayama ◽

Takatoshi Arakawa ◽

Maher Abou Hachem ◽

Leila Lo Leggio ◽

...

Keyword(s):

Gut Microbiome ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Human Gut ◽

Human Gut Microbiome ◽

Hydrolase Family

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Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers

Applied and Environmental Microbiology ◽

10.1128/aem.01860-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 7223-7232 ◽

Cited By ~ 20

Author(s):

Yuxiang Bai ◽

Rachel Maria van der Kaaij ◽

Hans Leemhuis ◽

Tjaard Pijning ◽

Sander Sebastiaan van Leeuwen ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Lactobacillus Reuteri ◽

Accurate Determination ◽

Reducing Power ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Linear Chains ◽

Dietary Starch ◽

Hydrolase Family

ABSTRACT4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW ofLactobacillus reuteristrains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) inEscherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.

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Xylanases of glycoside hydrolase family 30 – An overview

Biotechnology Advances ◽

10.1016/j.biotechadv.2021.107704 ◽

2021 ◽

Vol 47 ◽

pp. 107704

Author(s):

Vladimír Puchart ◽

Katarína Šuchová ◽

Peter Biely

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 30

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Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

Applied Sciences ◽

10.3390/app11094048 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4048

Author(s):

Javier A. Linares-Pastén ◽

Lilja Björk Jonsdottir ◽

Gudmundur O. Hreggvidsson ◽

Olafur H. Fridjonsson ◽

Hildegard Watzlawick ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Ligand Docking ◽

Glycoside Hydrolase Family ◽

Ligand Interactions ◽

Tim Barrel ◽

Branching Point ◽

The Difference ◽

Dynamics Simulations ◽

And Function ◽

Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

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Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84038-3 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31254-31267

Author(s):

Claire Moulis ◽

Gilles Joucla ◽

David Harrison ◽

Emeline Fabre ◽

Gabrielle Potocki-Veronese ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Polymerization Mechanism ◽

Hydrolase Family

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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