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An experiment with ileostomized adult roosters was conducted to determine the ileal digestibility and urinary excretion of D-xylose and L-arabinose. As a reference, D-glucose was included in the experiment. The sugars were tested at graded dietary levels of 2.5, 5.0, 7.5, and 10.0%. Mean ileal digestibility of D-glucose and D-xylose was nearly 100%. Ileal digestibility of L-arabinose decreased linearly (P less than .05) with increasing dose level. The corresponding ileal digestibilities for L-arabinose at dietary levels of 2.5, 5.0, 7.5, and 10.0% were 95.5, 93.6, 80.3, and 74.6%. Both pentose sugars were partly excreted in the urine. The extent of this urinary excretion in percentage of intake increased linearly (P less than .05) as the dietary level increased. In roosters fed the 2.5% D-xylose diet, 7.2% of the D-xylose consumed appeared in the urine. This level increased to 20.2% when roosters were fed a diet containing 10.0% D-xylose. Corresponding values for L-arabinose at these dietary inclusion levels were 8.7 and 16.6%. Chemicals/CAS: arabinose, 147-81-9; glucose, 50-99-7, 84778-64-3; xylose, 25990-60-7, 58-86-6; Arabinose, 147-81-9; Glucose, 50-99-7; Xylose

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB. Molecular Sequence Numbers: GENBANK: M57384, S55527, S55528, S55529, S55530, S55531, S55532, S63909, S69823, Z14057; Chemicals/CAS: Carbohydrate Epimerases, EC 5.1.3; DNA, Bacterial; Phosphotransferases, EC 2.7; Plasmids; Repressor Proteins; xylose isomerase, EC 5.3.1.5; Xylose; xylulokinase, EC 2.7.1.17

Hemicellulose consists primarily of pentose sugars, joined together in a polysaccharide chain with D-xylose as the most abundant component. Ileal digestibility and urinary excretion of D-xylose and associated effects of this pentose sugar on ileal and faecal digestibility of dry matter (DM), organic matter (OM), gross energy (GE) and nitrogen were studied in pigs. Castrated pigs were prepared with a post-valvular T-caecum cannula to measure ileal digestibility. Faecal digestibility was measured in non-cannulated pigs. D-xylose was given at dietary inclusion levels of 100 and 200 g/kg, and the control sugar, D-glucose, at a rate of 200 g/kg diet. Ileal digestibility of D-xylose as well as that of D-glucose was found to be close to 100%. The presence of D-xylose in the diet decreased ileal digesta pH and increased ileal flow of volatile fatty acids, suggesting the occurrence of microbial degradation of D-xylose in the pig small intestine. In pigs fed on the 100 g D-xylose/kg diet, 44.5% of the D-xylose intake appeared in the urine. This percentage increased significantly to 52.6 when pigs were fed on the 200 g D-xylose/kg diet. Ileal and faecal digestibility of DM, OM, GE and N, as well as N retention, decreased significantly in pigs fed on the 200 g D-xylose/kg diet. Chemicals/CAS: Fatty Acids, Volatile; Xylose

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low- affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min-1 mg of dry weight-1. In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2- deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

An experiment was conducted to examine the effects of graded levels (2.5, 5.0, 7.5, 10.0, and 15.0%) of dietary D-xylose or L-arabinose on chick performance. As reference, D-glucose was included in the experiment. A second experiment was performed to determine the AMEn of D-xylose and L-arabinose. Results of Experiment 1 showed a significant linear decrease (P less than .05) in weight gain and efficiency of feed utilization when the dietary level of either D-xylose or L-arabinose was increased. The same was true for daily feed intake of the D-xylose treatments. Water intake was linearly (P less than .05) increased as dietary level of both pentose sugars increased, and, as a result, dry matter content of the droppings decreased. Results of Experiment 2 showed that the AMEn value of either pentose sugar was dose related. The AMEn values for D-xylose at 5 and 10% dietary inclusion were 2,660 and 2,020 kcal/kg, respectively. Those for L-arabinose at these inclusion levels were 2,300 and 1,360 kcal/kg, respectively. Feeding equal dietary levels of either pentose sugar resulted in higher concentrations of xylose than of arabinose in blood plasma. Concentration of glucose in blood was not affected by feeding either D-xylose or L-arabinose. Cecal length and weight were markedly increased by feeding L-arabinose and intermediately by D-xylose.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose- H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D- xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D- xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D- glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a K(i) of 220 μM.

The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock. © 2009 The Authors.

Zie ook Erratum: FEMS Yeast Research 14(2014)2:368-368, doi:10.1111/1567-1364.12127

The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose-1) and a maximum growth rate of 0.21 h -1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose. Copyright © 2009, American Society for Microbiology. All Rights Reserved. Chemicals / CAS: glucose dehydrogenase, 37250-49-0, 37250-50-3, 37250-84-3, 9028-53-9; hydrolyase, 9044-86-4; oxoglutarate dehydrogenase, 9031-02-1; xylose, 25990-60-7, 58-86-6; aldehyde dehydrogenase, 37353-37-0, 9028-86-8; 2,5-dioxovalerate dehydrogenase, 1.2.1.26; Aldehyde Oxidoreductases, 1.2.-; Bacterial Proteins; D-xylo-aldonate dehydratase, 4.2.1.-; Glucose 1-Dehydrogenase, 1.1.1.47; Hydro-Lyases, 4.2.1.-; Xylose

The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose-1) and a maximum growth rate of 0.21 h -1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression is regulated at the transcriptional level. Using a β-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an efficient inducer of the exlA promoter, whereas sucrose or maltodextrin were not. Upon D-xylose induction, the exlA promoter was threefold more efficient than the frequently used A. niger glucoamylase (glaA) promoter under maltodextrin induction. Detailed induction analyses demonstrated that induction was-dependent on the presence of D-xylose in the medium. Carbon-source-limited chemostat cultures with the uidA reporter strain showed that D-xylose was also a very good inducer in a fermenter, even in the presence of sucrose.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-β-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exlA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli β- glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.

An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PglaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations, PglaA-driven GFP expression by the glucoamylase promoter could be demonstrated in xylose-limited continuous cultures. In these cultivations, the xylose concentration was therefore too low to cause repression. Transient experiments initiated with a maltose pulse did not further induce red-shifted GFP production in xylose-limited continuous cultures. Maltose induction under conditions of xylose repression was microscopically observed and quantified in a flow-through chamber. Red-shifted GFP was first produced after 5 h induction. Finally the strain was characterized in glucose-limited continuous cultures, and here the area of the mycelium stained with cytoplasmic GFP increased with increasing specific growth rate, indicating that GFP can be used as a marker of cellular activity in this type of cultivation.

The aim of this study was to monitor cell wall polysaccharide (CWPs) utilization during fermentation by the human colonic microbiota in the TNO in vitro model of the colon (TIM-2). Chicory root pulp (CRP) and treated (ensiled) CRP (ECRP) containing four times more soluble pectin than CRP, were fermented in the model. After the adaptation phase of the human fecal inoculum to CRP and ECRP for 48 h, both materials were fermented quite rapidly (57% carbohydrate utilization in 2 h). ECRP carbohydrates (85%) were less fermented in 24 h compared to CRP carbohydrates (92%). It was hypothesized that soluble fibers that are readily fermentable and dominantly present in ECRP programmed the microbiota in TIM-2 to fully adapt to these soluble fibers which was not able to change towards the fermentation of insoluble fibers anymore. Consequently, ECRP insoluble fibers were less utilized than CRP insoluble fibers in TIM-2 leading to an overall lower fiber utilization and SCFA production. © 2014 Elsevier Ltd. All rights reserved. Chemicals/CAS: arabinose, 147-81-9; fucose, 3615-37-0, 3713-31-3; galactose, 26566-61-0, 50855-33-9, 59-23-4; glucose, 50-99-7, 84778-64-3; mannose, 31103-86-3, 3458-28-4; pectin, 9000-69-5; rhamnose, 10485-94-6, 3615-41-6; xylose, 25990-60-7, 58-86-6

Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA- dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (α-D-xylopyranosyl, (1,6)-D- glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated α-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria α-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-α-D- xylopyranoside with apparent K(m) and V(max) values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the α-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-α-D-xylopyranoside nor α- glucosides were substrates. Glucose repressed the synthesis of α-xylosidase fivefold, and 80% of this repression was released in an L. pentosus ΔccpA mutant. The α-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted. Chemicals/CAS: alpha-D-xylosidase, EC 3.2.1.-; Bacterial Proteins; Carrier Proteins; Disaccharides; Glycosides; isoprimeverose cation symporter, Lactobacillus; isoprimeverose, 534-98-5; Symporters; Xylose; Xylosidases, EC 3.2.1.-