Identification of Independent and Shared Metabolic Responses to High-Fiber and Antibiotic Treatments in Fecal Metabolome of Grow-Finish Pigs.

Feeding high-fiber (HF) coproducts to grow-finish pigs as a cost-saving practice could compromise growth performance, while the inclusion of antibiotic growth promoters (AGPs) may improve it. The hindgut is a shared site of actions between fiber and AGPs. However, whether the metabolic interactions between them could occur in the digestive tract of pigs and then become detectable in feces have not been well-examined. In this study, wheat middling (WM), a HF coproduct, and bacitracin, a peptide antibiotic (AB), were fed to 128 grow-finish pigs for 98 days following a 2 × 2 factorial design, including antibiotic-free (AF) + low fiber (LF); AF + HF; AB + LF, and AB + HF, for growth and metabolic responses. The growth performance of the pigs was compromised by HF feedings but not by AB. A metabolomic analysis of fecal samples collected on day 28 of feeding showed that WM elicited comprehensive metabolic changes, especially in amino acids, fatty acids, and their microbial metabolites, while bacitracin caused selective metabolic changes, including in secondary bile acids. Limited metabolic interactions occurred between fiber and AB treatments. Moreover, the correlations between individual fecal metabolites and growth support the usage of fecal metabolome as a source of biomarkers for monitoring and predicting the metabolic performance of grow-finish pigs.

Bacterial Diversity and Lactic Acid Bacteria with High Alcohol Tolerance in the Fermented Grains of Soy Sauce Aroma Type Baijiu in North China.

Soy sauce aroma type baijiu (also known as Maotai-flavor baijiu) is one of the most popular types of baijiu in China. Traditionally, it is mainly produced in Southwest China. However, in recent decades, some other regions in China have also been able to produce high-quality soy sauce aroma type baijiu, but their microbial flora characteristics during fermentation are still unclear. Here, the bacterial microbial community structure of fermented grains in different rounds of Lutaichun soy sauce aroma type baijiu produced in North China was studied by high-throughput sequencing technology, and the potential probiotics strains with good characteristics (alcohol tolerance, etc.) were screened. The results showed that lactic acid bacteria were the main bacteria in the process of baijiu fermentation. However, as the number of repeated fermentation rounds increased, the proportion of lactic acid bacteria decreased. Firmicutes (96.81%) were the main bacteria in baijiu fermentation at the phylum level, and Lactobacillus (66.50%) were the main bacteria at the genus level. Finally, two strains with high resistance to alcohol stress, Lactiplantibacillus plantarum LTJ12 and Pediococcus acidilactici LTJ28, were screened from 48 strains of lactic acid bacteria in the fermented grains. The survival rates of L. plantarum LTJ12 and P. acidilactici LTJ28 under the 8% alcohol stress treatment were 59.01% and 55.50%, respectively. To the best of our knowledge, this study is the first to reveal the microbial succession of fermented grains in different rounds of soy sauce aroma type baijiu from North China, and has the benefit of explaining the deep molecular mechanism in the process of baijiu fermentation. In addition, the obtained lactic acid bacteria strains with high alcohol tolerance could be conducive to the development of new products such as active probiotic alcoholic beverages and may have important industrial development prospects also.

Comparative Genomics Analysis Provides New Insights into High Ethanol Tolerance of Lactiplantibacillus pentosus LTJ12, a Novel Strain Isolated from Chinese Baijiu.

Lactic acid bacteria have received a significant amount of attention due to their probiotic characteristics. The species Lactiplantibacillus plantarum and Lactiplantibacillus pentosus are genotypically closely related, and their phenotypes are so similar that they are easily confused and mistaken. In the previous study, an ethanol-resistant strain, LTJ12, isolated from the fermented grains of soy sauce aroma type baijiu in North China, was originally identified as L. plantarum through a 16S rRNA sequence analysis. Here, the genome of strain LTJ12 was further sequenced using PacBio and Illumina sequencing technology to obtain a better understanding of the metabolic pathway underlying its resistance to ethanol stress. The results showed that the genome of strain LTJ12 was composed of one circular chromosome and three circular plasmids. The genome size is 3,512,307 bp with a GC content of 46.37%, and the number of predicted coding genes is 3248. Moreover, by comparing the coding genes with the GO (Gene Ontology), COG (Cluster of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, the functional annotation of the genome and an assessment of the metabolic pathways were performed, with the results showing that strain LTJ12 has multiple genes that may be related to alcohol metabolism and probiotic-related genes. Antibiotic resistance gene analysis showed that there were few potential safety hazards. Further, after conducting the comparative genomics analysis, it was found that strain LTJ12 is L. pentosus but not L. plantarum, but it has more functional genes than other L. pentosus strains that are mainly related to carbohydrate transport and metabolism, transcription, replication, recombination and repair, signal transduction mechanisms, defense mechanisms and cell wall/membrane/envelope biogenesis. These unique functional genes, such as gene 2754 (encodes alcohol dehydrogenase), gene 3093 (encodes gamma-D-glutamyl-meso-diaminopimelate peptidase) and some others may enhance the ethanol tolerance and alcohol metabolism of the strain. Taken together, L. pentosus LTJ12 might be a potentially safe probiotic with a high ethanol tolerance and alcohol metabolism. The findings of this study will also shed light on the accurate identification and rational application of the Lactiplantibacillus species.

Functional and structural investigation of a novel ß-mannanase BaMan113A from Bacillus sp. N16-5.

Mannan is an important renewable resource whose backbone can be hydrolyzed by ß-mannanases to generate manno-oligosaccharides of various sizes. Only a few glycoside hydrolase (GH) 113 family ß-mannanases have been functionally and structurally characterize. Here, we report the function and structure of a novel GH113 ß-mannanase from Bacillus sp. N16-5 (BaMan113A). BaMan113A exhibits a substrate preference toward manno-oligosaccharides and releases mannose and mannobiose as main hydrolytic products. The crystal structure of BaMan113A suggest that the enzyme shows a semi-enclosed substrate-binding cleft and the amino acids surrounding the +2 subsite form a steric barrier to terminate the substrate-binding tunnel. Based on these structural features, we conducted mutagenesis to engineer BaMan113A to remove the steric hindrance of the substrate-binding tunnel. We found that F101E and N236Y variants exhibit increased specific activity toward mannans comparing to the wild-type enzyme. Meanwhile, the product profiles of these two variants toward polysaccharides changed from mannose to a series of manno-oligosaccharides. The crystal structure of variant N236Y was also determined to illustrate the molecular basis underlying the mutation. In conclusion, we report the functional and structural features of a novel GH113 ß-mannanase, and successfully improved its endo-acting activity by using structure-based engineering.

Effects of three different mannans on obesity and gut microbiota in high-fat diet-fed C57BL/6J mice.

To compare the effects of three mannans, Konjac glucomannan (KGM), guar gum (GG) and locust bean gum (LBG), on obesity and obesity-related metabolic disorders in mice fed with high-fat diet (HFD), and to investigate the potential modulation of gut microbiota, we performed a 14 week study on C57BL/6J mice fed a HFD with/without mannan supplementation. The results showed that supplementing 8% KGM, GG, and LBG to a HFD dramatically reduced the body weight gain and adipose accumulation, attenuated liver injury, and antagonized glycolipid metabolism and inflammation-related parameters of HFD-fed mice in different degrees. However, only LBG had such roles when the supplement dose was reduced to 2%. In addition, it was found that LBG required more time to exert its impacts on weight control and lipid metabolism. Furthermore, 16S rRNA gene sequencing of gut microbiota indicated that mannans with different structures and supplement doses affected the overall structure of the gut microbiota to a varying extent and specifically changed the abundance of some OTUs. Moreover, several OTUs belonging to the genera Muribaculum, Staphylococcus, [Eubacterium] fissicatena group, and Christensenella had a high correlation with obesity and obesity-related metabolic disorders of the host. In summary, all the three mannans had the potential to be used as alternative dietary supplements or functional foods to prevent obesity and obesity-related metabolic disorders induced by a HFD, but the effects of the dose and time varied, and the functions of the mannans were associated with their ability to regulate the gut microbiota.

Enhancing bile tolerance of Lactobacilli is involved in the hypolipidemic effects of liraglutide.

Liraglutide is an analog of human glucagon-like peptide-1 which play essential roles in regulation of glycolipid metabolism. To investigate role of lactic acid bacteria (LAB) in lipid-lowering effect of liraglutide, 40 mice were divided into normal food diet (NFD), high-fat food (HFD), 10.0 mg/kg/d simvastatin-treated HFD (SIM + HFD), 200 and 400 µg/kg/d liraglutide-treated HFD (LL + HFD and HL + HFD) groups for 5 weeks. We found that liraglutide could upregulate cholesterol 7α-hydroxylase (CYP7A1) and LDL-receptor (LDLR), whereas downregulate 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Besides, liraglutide enhance abundance of lactobacillaceae in gut of hyperlipidemic mice and increase bile tolerance ability of LAB by upregulating bile salt hydrolases, and the lysate of liraglutide-sensitive LAB could also directly downregulate HMGCR, the key enzyme in cholesterol synthesis, and inhibit hepatocyte steatosis. These findings might provide new theoretical guidance for clinical application of liraglutide and research and development of antiobesity, hypolipidemic, and cholesterol-lowering drugs or functional foods.

Biological detoxification of fumonisin by a novel carboxylesterase from Sphingomonadales bacterium and its biochemical characterization.

Fumonisins have posed hazardous threat to human and animal health worldwide. Enzymatic degradation is a desirable detoxification approach but is severely hindered by serious shortage of detoxification enzymes. After mining enzymes by bioinformatics analysis, a novel carboxylesterase FumDSB from Sphingomonadales bacterium was expressed in Escherichia coli, and confirmed to catalyze fumonisin B1 to produce hydrolyzed fumonisin B1 by liquid chromatography mass spectrometry for the first time. FumDSB showed high sequence novelty, sharing only ~34% sequence identity with three reported fumonisin detoxification carboxylesterases. Besides, FumDSB displayed its high degrading activity at 30-40 °C within a broad pH range from 6.0 to 9.0, which is perfectly suitable to be used in animal physiological condition. It also exhibited excellent pH stability and moderate thermostability. This study provides a FB1 detoxification carboxylesterase which could be further used as a potential food and feed additive.

Biochemical characterization of a novel halo/organic-solvents/final-products tolerant GH39 xylosidase from saline soil and its synergic action with xylanase.

Xylosidases with tolerance to high concentration of salts, organic solvents, and enzyme hydrolytic products are preferential for industrial application but were rarely reported. In this study, a novel xylosidase XYL21 belong to glycoside hydrolase 39 was characterized with optimal temperature of 45 °C and optimal pH of 5.50. Different to other GH39 xylosidases, XYL21 had excellent tolerance to salts, the activity of which is not inhibited but slightly increased in 0.50-1.50 M NaCl. It is also tolerant to organic solvents, especially retaining 105.18% relative activity even in the presence of 15.00% (v/v) ethanol. Moreover, XYL21 was insensitive to the final lignocellulose hydrolysis products including glucose, xylose, arabinose, mannose and galactose, which retains 111.36% and 53.49% relative activity in 0.30 and 0.90 M xylose, respectively. Further structural modeling analysis indicated that its excellent tolerance may be attributed to its high structural flexibility caused by the high proportion of random coils. Furthermore, XYL21 had a wide substrate specificity to catalyze xylan and xylo-oligosaccharides, and it significantly cooperated with xylanase to improve the hydrolysis efficiency with 1.52-fold. Considering these unique properties, XYL21 is a good candidate for both basic research and various potential industrial applications such as seafood processing and bioethanol production.

Bacillus subtilis RZ001 improves intestinal integrity and alleviates colitis by inhibiting the Notch signalling pathway and activating ATOH-1.

Intestinal mucosal barriers help the body resist many intestinal inflammatory diseases, such as inflammatory bowel disease (IBD). In this study, we identified a novel bacterium promoting the repair of intestinal mucosa and investigated the potential mechanisms underlying its activity. Culture supernatant of Bacillus subtilis RZ001 upregulated the expression of mucin 2 (MUC2) and tight junction (TJ) proteins in HT-29 cells in vitro. Oral administration of B. subtilis RZ001 may have significantly reduced symptoms such as the dextran sulfate sodium (DSS)-induced decrease in body weight, shortening of colon length and overproduction of proinflammatory factors. The number of goblet cells and levels of MUC2 and TJ proteins were significantly increased in adult mice fed with B. subtilis RZ001. B. subtilis RZ001 cells upregulated the levels of MUC2 in the intestinal organoids. Furthermore, culture supernatant of B. subtilis RZ001 could suppress the Notch signalling pathway and activate the expression of atonal homolog 1 (Atoh1). The transcription factor Atoh1 is required for intestinal secretory cell differentiation and activates transcription of MUC2 via binding to E-boxes on the MUC2 promoter. Taken together, B. subtilis strain RZ001 has the potential for treating IBD. The present study is helpful to elucidate the mechanisms of B. subtilis action.

Structure-based engineering of heparinase I with improved specific activity for degrading heparin.

BACKGROUND: Heparinase I from Pedobacter heparinus (Ph-HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Enzymatic degradation of heparin by heparin lyases not only largely facilitates heparin structural analysis but also showed great potential to produce low-molecular-weight heparin (LMWH) in an environmentally friendly way. However, industrial applications of Ph-HepI have been limited by their poor yield and enzyme activity. In this work, we improve the specific enzyme activity of Ph-HepI based on homology modeling, multiple sequence alignment, molecular docking and site-directed mutagenesis. RESULTS: Three mutations (S169D, A259D, S169D/A259D) exhibited a 50.18, 40.43, and 122.05% increase in the specific enzyme activity and a 91.67, 108.33, and 75% increase in the yield, respectively. The catalytic efficiencies (kcat/Km) of the mutanted enzymes S169D, A259D, and S169D/A259D were higher than those of the wild-type enzyme by 275, 164, and 406%, respectively. Mass spectrometry and activity detection showed the enzyme degradation products were in line with the standards of the European Pharmacopoeia. Protein structure analysis showed that hydrogen bonds and ionic bonds were important factors for improving specific enzyme activity and yield. CONCLUSIONS: We found that the mutant S169D/A259D had more industrial application value than the wild-type enzyme due to molecular modifications. Our results provide a new strategy to increase the catalytic efficiency of other heparinases.

The critical roles of exposed surface residues for the thermostability and halotolerance of a novel GH11 xylanase from the metagenomic library of a saline-alkaline soil.

Thermostable and halotolerant GH11 xylanases are highly desirable in various industrial applications but they were rarely characterized. In this study, we firstly revealed the genetic diversity of GH11 xylanases from the metagenomic DNA of a saline-alkaline soil by constructing a gene fragment library. Twelve distinct xylanase fragment sequences were identified, which shared 62-96% identity to known GH11 xylanases. Among them, Xyn22 with the highest relative abundance was cloned and found to exhibit excellent halotolerance. Xyn22 retained 80 and 58% relative activity in the presence of 3 and 5 M NaCl, respectively, and had no loss of activity after incubation in 3 M NaCl at 37 °C for 1 h. Additionally, the acidic amino acid residues E137 and E139 on the protein's surface were revealed to cooperatively play important roles in its excellent halotolerance and halostabilty. Furthermore, the thermostability of Xyn22 was significantly improved without compromising halotolerance by introducing an aromatic interaction between Y48 and F53. In conclusion, this study not only provides a thermostable and halotolerant GH11 xylanase, but also reveals the key roles of surface-exposed amino acids in its thermostability and halotolerance, which could be used for further molecular modification of other xylanases and similar enzymes.

Protective effects of a novel probiotic strain, Lactococcus lactis ML2018, in colitis: in vivo and in vitro evidence.

Multiple articles have confirmed that an imbalance of the intestinal microbiota is closely related to aberrant immune responses of the intestines and to the pathogenesis of inflammatory bowel diseases (IBDs). Probiotic strains have been identified for the treatment and prevention of IBDs. The aim of this study was to screen a new probiotic strain with anti-inflammatory activity and investigate the potential mechanisms underlying its activity. We identified a new probiotic strain, L. lactis ML2018, that has anti-inflammatory properties and was isolated from traditional fermented food. In an in vitro experiment, L. lactis ML2018 prevented the release of nitric oxide (NO) and the production of inflammatory factors induced by lipopolysaccharides (LPS) in RAW264.7 cells. The in vivo anti-inflammatory effects of L. lactis ML2018 were evaluated using a dextran sulfate sodium (DSS)-induced animal model of colitis. Oral administration of L. lactis ML2018 significantly ameliorated colitis induced by DSS, which included preventing a decrease in body weight, shortening of the colon length and apoptosis of epithelial cells. L. lactis ML2018 could inhibit DSS-induced intestinal inflammation by preventing the overproduction of proinflammatory factors, suppressing the infiltration of macrophages, controlling the fibrosis, improving the integrity of the intestinal epithelial barrier and upregulating the concentrations of short-chain fatty acids (SCFAs). Moreover, L. lactis ML2018 could prevent inflammation by inhibiting the activation of the NF-κB and MAPK signaling pathways. These data suggest that L. lactis ML2018 could have therapeutic potential for treating IBDs.

Galactomannan Degrading Enzymes from the Mannan Utilization Gene Cluster of Alkaliphilic Bacillus sp. N16-5 and Their Synergy on Galactomannan Degradation.

Two glycoside hydrolases encoded by the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16-5 were studied. The recombinant Gal27A (rGal27A) hydrolyzed both galactomannans and oligo-galactomannans to release galactose, while the recombinant Man113A (rMan113A) showed poor activity toward galactomannans, but it hydrolyzed manno-oligosaccharides to release mannose and mannobiose. rGal27A showed synergistic interactions with rMan113A and recombinant ß-mannanase ManA (rManA), which is also from Bacillus sp. N16-5, in galactomannan degradation. The synergy degree of rGal27A and rManA on hydrolysis of locust bean gum and guar gum was 1.13 and 2.21, respectively, and that of rGal27A and rMan113A reached 2.00 and 2.68. The main products of galactomannan hydrolyzed by rGal27A and rManA simultaneously were galactose, mannose, mannobiose, and mannotriose, while those of galactomannan hydrolyzed by rGal27A and rMan113A were galactose and mannose. The yields of mannose, mannobiose, and mannotriose dramatically increased compared with the hydrolysis in the presence of rManA or rMan113A alone.

Transcriptional regulation of the mannan utilization genes in the alkaliphilic Bacillus sp. N16-5.

Bacillus sp. N16-5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate-binding domain of the manno-oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno-oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter.

Heterologous expression in Pichia pastoris and characterization of a novel GH11 xylanase from saline-alkali soil with excellent tolerance to high pH, high salt concentrations and ethanol.

A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 °C and it was stable after incubation for 1 h at 30 °C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 °C for 10 min. In addition, 5 mM ß-Mercaptoethanol, Cu2+, Co2+, and Mn2+ increased the xylanase activity by 22.3%, 8.8%, 7.1%, and 4.4%, respectively. Significantly, 93.4% and 59.8% of the optimal activity was retained in the presence of 2% and 10% (v/v) ethanol, respectively. Under optimal conditions, the Km,Vmax, and Kcat value of Xyn11-1 for beechwood xylan were 3.7 mg ml-1, 101.0 µmol min-1 mg-1 and 42.1 s-1, respectively. Xyn11-1 is a strict endo-ß-1,4-xylanase, its main enzymatic products being xylotetraose and xylopentaose. Xyn11-1 is the first reported GH11 xylanase isolated from saline-alkali soil, and has excellent tolerance of high pH, high salt concentrations and ethanol, which indicates its great potential for basic research and industrial applications.

Rapid biodegradation of aflatoxin B1 by metabolites of Fusarium sp. WCQ3361 with broad working temperature range and excellent thermostability.

BACKGROUND: Contamination of food and feed by aflatoxin B1 (AFB1) poses serious economic and health problems worldwide, so the development of biological methods for effective AFB1 degradation is strongly required. RESULTS: Among three AFB1-degrading microorganisms isolated from moldy peanut, Fusarium sp. WCQ3361 could remove AFB1 extremely effectively, with a degradation ratio of 70.20% after 1 min and 95.38% after 24 h. Its degradation ratio was not much affected by temperature change (0-90 °C) and it also displayed excellent thermostability, maintaining 99.40% residual activity after boiling for 10 min. Since protease K could reduce the AFB1 degradation ratio by 55.15%, it is proposed that the effective component for AFB1 degradation is a protein. The AFB1 degradation ability of Fusarium sp. WCQ3361 was further verified by feed stock detoxification and the MTT test with HepG2 cells. In addition, no degradation products were detected by preliminary liquid chromatography/mass spectrometry, suggesting that AFB1 might be metabolized to products with different chemical characteristics from AFB1. CONCLUSION: Fusarium sp. WCQ3361 is the first reported AFB1 degradation fungus belonging to the genus Fusarium with broad working temperature range, excellent thermostability and high activity, which provides a potential highly useful solution for dealing with AFB1 contamination in the human diet and animal feed. © 2016 Society of Chemical Industry.

Bioconversion of conjugated linoleic acid by Lactobacillus plantarum CGMCC8198 supplemented with Acer truncatum bunge seeds oil.

Conjugated linoleic acid (CLA) isomers, c9, t11-CLA and t10, c12-CLA, have been proved to exhibit excellent biomedical properties for potential use in anti-cancer applications and in reducing obesity. Acer truncatum Bunge (ATB), which is rich in unsaturated fatty acids, including oleic acid, linoleic acid, and nervonic acid, is a new resource for edible oil. In the present study, we developed a new method for producing two CLA isomers from ATB-seed oil by fermentation using Lactobacillus plantarum CGMCC8198 (LP8198), a novel probiotics strain. Polymerase chain reaction results showed that there was a conserved linoleate isomerase (LIase) gene in LP8198, and its transcription could be induced by ATB-seed oil. Analyses by gas chromatography-mass spectrometry showed that the concentration of c9, t11-CLA and t10, c12-CLA in ATB-seed oil could be increased by about 9- and 2.25-fold, respectively, after being fermented by LP8198.

Gene expression pattern analysis of a recombinant Escherichia coli strain possessing high growth and lycopene production capability when using fructose as carbon source.

OBJECTIVE: Escherichia coli K12f-pACLYC has a high capability for growth and lycopene production when using fructose as carbon source and the transcription of genes involved was compared in glucose-grown and fructose-grown cells. RESULTS: Escherichia coli K12f-pACLYC was grown on 10 g fructose l(-1) and reached 4.6 g DCW l(-1) with lycopene at 192 mg g DCW(-1), values that are 3-fold and 7-fold higher than when growing on glucose. Gene transcription profiles of fructose-grown and glucose-grown cells were compared. 384 differentially expressed genes (DEGs) with fold changes ≥4 were identified, and the transcription of genes involved in fructose uptake and metabolism, pyruvate catabolism, tricarboxylic acid cycle and oxidative phosphorylation varied significantly. These changes enhanced the metabolic flux into the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cylcle and coupled to oxidative phosphorylation. These enhanced activities provide more precursors, cofactors and energy needed for growth lycopene production. CONCLUSION: The high capability of E. coli K12f-pACLYC for growth and lycopene production when growing on fructose is due to transcriptional regulation, and the relevant genes were identified.

A Novel Manno-Oligosaccharide Binding Protein Identified in Alkaliphilic Bacillus sp. N16-5 Is Involved in Mannan Utilization.

ManH, a novel substrate-binding protein of an ABC transporter, was identified from the mannan utilization gene cluster of Bacillus sp. N16-5. We cloned, overexpressed, and purified ManH and measured its binding affinity to different substrates by isothermal titration calorimetry. ManH binds to mannotriose, mannotetraose, mannopentose, and galactosyl-mannotriose with dissociation constants in the micromolar range. Deletion of manH led to decreased growth ability of the strain when cultivated in medium with manno-oligosaccharides or mannan as the carbon source. ManH belongs to a manno-oligosaccharide transporter and plays an important role in mannan utilization by Bacillus sp. N16-5.

[Characterization of solute-binding protein XynE of the xylooligosaccharide transporter from Bacillus sp. N16-5].

UNLABELLED: The alkaliphilichemicellulolytic bacterium Bacillus sp. N16-5 was isolated from Lake Wudunao in Inner Mongolia. It has a broad substrate spectrum and exhibits a capacity to utilize complex carbohydrates such as galactomannan, xylan and pectin. Previous transcriptional analysis of differential carbohydrate utilization by Bacillus sp. N16-5 has identified a putative gene cluster related to xylan utilization. It contains a putative xylo-oligosaccharide ATP- binding cassette (ABC) transporter encoded by xynEFG gene cluster. OBJECTIVE: xynE gene is predicted to encode an extracellular solute-binding protein of the ABC transporter. Here, the physiological roles of xynE on the xylan utilization was investigated by gene deletion. METHODS: We obtained the xynE deficient strain N16-5 (ΔxynE) through homologous recombination using a temperature sensitive shuttle vector pNNB194. The effects of xynE on xylan utilization by N16-5 were detected by comparing the growth profiles on xylan of the wild type and mutant strains as well as the variation of reducing sugars concentration in the medium during cultivation. We further verified the phenotype by constructing the complementary strain. Moreover, the substrate specificity of XynE was illustrated by the HPLC analysis results of the xylan medium components, which was supplemented with the growth profiles of the wild type strain and N16-5 (ΔxynE) strain on xylose. RESULTS: Compared with the wild type strain, strain N16-5 (ΔxynE) had a delayed exponential phase, obtained a lower maximum optical intensity OD600 value, and presented the accumulation and depletion of reducing sugars during cultivation. The complementary strain retrieved the phenotype of wild type strain, and grown slightly better than it. HPLC analysis showed that N16-5 ( ΔxynE) strain degraded the xylan substrates more slowly than wild type, xylo- oligosaccharides like xylotetraose, xylotriose and xylobiose began to accumulate after 16 h cultivation. Moreover it still maitained a large number of the degradation product xylobiose in the medium after 60 h. When cultured in xylose medium, strain N16-5 (ΔxynE) performed similar growth profile with the wild type strain. CONCLUSION: XynE played an important role in rapidly and effectively utilizing xylan in Bacillus sp. N16-5 and specifically related with xylo-oligosaccharide uptake.