scholarly journals Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

2015 ◽  
Vol 81 (12) ◽  
pp. 4173-4183 ◽  
Author(s):  
Akira Watanabe ◽  
Kazumi Hiraga ◽  
Masako Suda ◽  
Hideaki Yukawa ◽  
Masayuki Inui
Keyword(s):  
Heterologous Expression ◽  
Corynebacterium Glutamicum ◽  
Abc Transporter ◽  
Binding Protein ◽  
Functional Characterization ◽  
Product Inhibition ◽  
Gene Encoding ◽  
Content Type ◽  
Industrial Strains ◽  
Efficient Fermentation

ABSTRACTTheCorynebacterium alkanolyticumxylEFGDgene cluster comprises thexylDgene that encodes an intracellular β-xylosidase next to thexylEFGoperon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover forp-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Kifor xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetacpromoter in theCorynebacterium glutamicumxylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylDgene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicumto take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG-encoded xyloside ABC transporter. The finding thatxylEFGimparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

scholarly journals Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Mohammad M. Rahman ◽  
Mayra A. Machuca ◽  
Mohammad F. Khan ◽  
Christopher K. Barlow ◽  
Ralf B. Schittenhelm ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Content Type ◽  
H Pylori ◽  
Substrate Binding Protein ◽  
Broad Specificity

ABSTRACT The gastric pathogen Helicobacter pylori has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by H. pylori requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, H. pylori DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that H. pylori DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the H. pylori protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. IMPORTANCE The World Health Organization listed Helicobacter pylori as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for H. pylori, our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.

scholarly journals A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

2014 ◽  
Vol 58 (7) ◽  
pp. 3934-3941 ◽  
Author(s):  
Hansjürg Engel ◽  
Moana Mika ◽  
Dalia Denapaite ◽  
Regine Hakenbeck ◽  
Kathrin Mühlemann ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Binding Protein ◽  
Population Analysis ◽  
Resistance Testing ◽  
Penicillin Binding Protein ◽  
Secondary Resistance ◽  
Content Type ◽  
Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

scholarly journals Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry

2011 ◽  
Vol 77 (18) ◽  
pp. 6395-6399 ◽  
Author(s):  
Nathalie Boucher ◽  
Kenneth M. Noll
Keyword(s):  
Abc Transporter ◽  
Thermotoga Maritima ◽  
Binding Constants ◽  
Genomic Region ◽  
Genomic Research ◽  
Gene Encoding ◽  
Content Type ◽  
Differential Scanning Fluorimetry ◽  
Beta Glucosidase ◽  
Abc Transporter Genes

ABSTRACTThe chromosome ofThermotoga maritimastrain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound withKd(dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars:T. maritimaMSB8 genomovar TIGR andT. maritimaMSB8 genomovar DSM 3109, respectively.

scholarly journals Adaptive Evolution of Thermotoga maritima Reveals Plasticity of the ABC Transporter Network

2015 ◽  
Vol 81 (16) ◽  
pp. 5477-5485 ◽  
Author(s):  
Haythem Latif ◽  
Merve Sahin ◽  
Janna Tarasova ◽  
Yekaterina Tarasova ◽  
Vasiliy A. Portnoy ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Thermotoga Maritima ◽  
Binding Protein ◽  
Carbon Sources ◽  
Peptide Transport ◽  
Content Type ◽  
Upstream Promoter ◽  
Membrane Spanning ◽  
Vast Network

ABSTRACTThermotoga maritimais a hyperthermophilic anaerobe that utilizes a vast network of ABC transporters to efficiently metabolize a variety of carbon sources to produce hydrogen. For unknown reasons, this organism does not metabolize glucose as readily as it does glucose di- and polysaccharides. The leading hypothesis implicates the thermolability of glucose at the physiological temperatures at whichT. maritimalives. After a 25-day laboratory evolution, phenotypes were observed with growth rates up to 1.4 times higher than and glucose utilization rates exceeding 50% those of the wild type. Genome resequencing revealed mutations in evolved cultures related to glucose-responsive ABC transporters. The native glucose ABC transporter, GluEFK, has more abundant transcripts either as a result of gene duplication-amplification or through mutations to the operator sequence regulating this operon. Conversely, BglEFGKL, a transporter of beta-glucosides, is substantially downregulated due to a nonsense mutation to the solute binding protein or due to a deletion of the upstream promoter. Analysis of the ABC2 uptake porter families for carbohydrate and peptide transport revealed that the solute binding protein, often among the transcripts detected at the highest levels, is predominantly downregulated in the evolved cultures, while the membrane-spanning domain and nucleotide binding components are less varied. Similar trends were observed in evolved strains grown on glycerol, a substrate that is not dependent on ABC transporters. Therefore, improved growth on glucose is achieved through mutations favoring GluEFK expression over BglEFGKL, and in lieu of carbon catabolite repression, the ABC transporter network is modulated to achieve improved growth fitness.

scholarly journals d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

2011 ◽  
Vol 77 (8) ◽  
pp. 2582-2588 ◽  
Author(s):  
Marco A. J. Siemerink ◽  
Wouter Kuit ◽  
Ana M. López Contreras ◽  
Gerrit Eggink ◽  
John van der Oost ◽  
...  
Keyword(s):  
Experimental Data ◽  
Heterologous Expression ◽  
Metabolic Network ◽  
Clostridium Acetobutylicum ◽  
Clostridium Beijerinckii ◽  
Racemic Mixture ◽  
Gene Encoding ◽  
Content Type ◽  
Batch Cultures

ABSTRACTAcetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions,Clostridium acetobutylicumATCC 824 (and strains derived from it) generates bothd- andl-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR fromClostridium beijerinckiiNCIMB 8052 was functionally expressed inC. acetobutylicumunder the control of two strong promoters, the constitutivethlpromoter and the late exponentialadcpromoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mMd-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of bothd-2,3-butanediol andmeso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain ofC. acetobutylicum.

scholarly journals Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Hoi Yee Keung ◽  
Tsz Kai Li ◽  
Lok To Sham ◽  
Man Kit Cheung ◽  
Peter Chi Keung Cheung ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Abc Transporter ◽  
Transport System ◽  
Binding Protein ◽  
Atp Binding ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Bifidobacterium Breve ◽  
Abc Transport ◽  
Atp Binding Protein

ABSTRACT Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.

scholarly journals Sulfate-Binding Protein (Sbp) from Xanthomonas citri: Structure and Functional Insights

2017 ◽  
Vol 30 (7) ◽  
pp. 578-588 ◽  
Author(s):  
Cristiane Tambascia Pereira ◽  
Cássia Roesler ◽  
Jéssica Nascimento Faria ◽  
Melissa Regina Fessel ◽  
Andrea Balan
Keyword(s):  
Abc Transporter ◽  
Structural Changes ◽  
Binding Protein ◽  
Functional Characterization ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Xanthomonas Citri ◽  
Canker Disease

The uptake and transport of sulfate in bacteria is mediated by an ATP-binding cassette transporter (ABC transporter) encoded by sbpcysUWA genes, whose importance has been widely demonstrated due to their relevance in cysteine synthesis and bacterial growth. In Xanthomonas citri, the causative agent of canker disease, the expression of components from this ABC transporter and others related to uptake of organic sulfur sources has been shown during in vitro growth cultures. In this work, based on gene reporter and proteomics analyses, we showed the activation of the promoter that controls the sbpcysUWA operon in vitro and in vivo and the expression of sulfate-binding protein (Sbp), a periplasmic-binding protein, indicating that this protein plays an important function during growth and that the transport system is active during Citrus sinensis infection. To characterize Sbp, we solved its three-dimensional structure bound to sulfate at 1.14 Å resolution and performed biochemical and functional characterization. The results revealed that Sbp interacts with sulfate without structural changes, but the interaction induces a significant increasing of protein thermal stability. Altogether, the results presented in this study show the evidence of the functionality of the ABC transporter for sulfate in X. citri and its relevance during infection.

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