scholarly journals Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

2012 ◽  
Vol 78 (17) ◽  
pp. 6280-6284 ◽  
Author(s):  
Franziska Schäfer ◽  
Judith Schuster ◽  
Birgit Würz ◽  
Claus Härtig ◽  
Hauke Harms ◽  
...  
Keyword(s):  
Secondary Alcohol ◽  
Enantiomeric Excess ◽  
Short Chain ◽  
Wild Type ◽  
Knockout Mutant ◽  
Biotechnological Potential ◽  
Content Type ◽  
Mononuclear Iron ◽  
Tertiary Alcohols ◽  
Fuel Oxygenate

ABSTRACTThe Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strainAquincola tertiaricarbonisL108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas anmdpJknockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.

scholarly journals Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

2011 ◽  
Vol 78 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Sarah Lebeer ◽  
Ingmar Claes ◽  
Hanne L. P. Tytgat ◽  
Tine L. A. Verhoeven ◽  
Eyra Marien ◽  
...  
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Lipoteichoic Acid ◽  
Good Survival ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Knockout Mutant ◽  
Phenotypic Analysis ◽  
Adhesion Properties ◽  
Content Type ◽  
Adhesive Interactions

ABSTRACTLactobacillus rhamnosusGG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently,spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of aspaCBApilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus ofL. rhamnosusGG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, thespaCBAmutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, anL. rhamnosusGG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutantL. rhamnosusGG cells are used. Taken together, our data suggest thatL. rhamnosusGG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.

scholarly journals A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

2012 ◽  
Vol 78 (24) ◽  
pp. 8595-8600 ◽  
Author(s):  
Xiuzhen Gao ◽  
Xi Chen ◽  
Weidong Liu ◽  
Jinhui Feng ◽  
Qiaqing Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enantiomeric Excess ◽  
Acid Synthesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Amino Acid Synthesis ◽  
Content Type

ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.

scholarly journals Novel Role for the Streptococcus pneumoniae Toxin Pneumolysin in the Assembly of Biofilms

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Joshua R. Shak ◽  
Herbert P. Ludewick ◽  
Kristen E. Howery ◽  
Fuminori Sakai ◽  
Hong Yi ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Early Time ◽  
Wild Type ◽  
Knockout Mutant ◽  
Content Type ◽  
Biofilm Development ◽  
Almost All

ABSTRACTStreptococcus pneumoniaeis an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms byS. pneumoniaeis important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence ofS. pneumoniaeand is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that aluxSknockout mutant was unable to form early biofilms and expressed lessplymRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation.S. pneumoniae plyknockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply’s role in biofilm formation appears to be independent of its hemolytic activity, asS. pneumoniaeserotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly ofS. pneumoniaebiofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development.IMPORTANCEThe bacteriumStreptococcus pneumoniae(commonly known as the pneumococcus) is commonly carried in the human nasopharynx and can spread to other body sites to cause disease. In the nasopharynx, middle ear, and lungs, the pneumococcus forms multicellular surface-associated structures called biofilms. Pneumolysin is an important toxin produced by almost allS. pneumoniaestrains, extensively studied for its ability to cause damage to human tissue. In this paper, we demonstrate that pneumolysin has a previously unrecognized role in biofilm formation by showing that strains without pneumolysin are unable to form the same amount of biofilm on plastic and human cell substrates. Furthermore, we show that the role of pneumolysin in biofilm formation is separate from the hemolytic activity responsible for tissue damage during pneumococcal diseases. This novel role for pneumolysin suggests that pneumococcal vaccines directed against this protein should be investigated for their potential impact on biofilms formed during carriage and disease.

scholarly journals A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila

2011 ◽  
Vol 10 (3) ◽  
pp. 423-434 ◽  
Author(s):  
Mariela L. Tomazic ◽  
Sebastián R. Najle ◽  
Alejandro D. Nusblat ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Wild Type ◽  
Knockout Mutant ◽  
Transfer Event ◽  
Content Type ◽  
Ethyl Group ◽  
Fatty Acid Hydroxylase ◽  
Eukaryotic Proteins ◽  
The One

ABSTRACT The gene TTHERM_00438800 ( DES24 ) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C 29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C 29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known.

scholarly journals Formation of Alkenes via Degradation oftert-Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp.

2011 ◽  
Vol 77 (17) ◽  
pp. 5981-5987 ◽  
Author(s):  
Franziska Schäfer ◽  
Liudmila Muzica ◽  
Judith Schuster ◽  
Naemi Treuter ◽  
Mònica Rosell ◽  
...  
Keyword(s):  
Amyl Alcohol ◽  
Bacterial Degradation ◽  
Side Reactions ◽  
Alkyl Ether ◽  
Low Oxygen ◽  
Content Type ◽  
Mononuclear Iron ◽  
Relative Contribution ◽  
Fuel Oxygenate ◽  
Oxygen Concentrations

ABSTRACTBacterial degradation pathways of fuel oxygenates such as methyltert-butyl andtert-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. InAquincola tertiaricarbonisL108,Methylibium petroleiphilumPM1, andMethylibiumsp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of thetert-alkyl ether metabolitestert-butyl andtert-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Bothtert-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In resting-cell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putativetert-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection ofmdpJKgenes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.

scholarly journals Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

2015 ◽  
Vol 81 (24) ◽  
pp. 8469-8477 ◽  
Author(s):  
Hualei Wang ◽  
Wenyuan Gao ◽  
Huihui Sun ◽  
Lifeng Chen ◽  
Lujia Zhang ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Hot Spots ◽  
Enantiomeric Excess ◽  
Random Mutagenesis ◽  
Burkholderia Cenocepacia ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
High Concentration ◽  
Content Type ◽  
Optically Pure

ABSTRACTThe nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzingo-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained fromBurkholderia cenocepaciastrain J2315 (BCJ2315) in hydrolyzingo-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2%eefor the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system,o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7%ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.

scholarly journals Virulence Traits of a Serogroup C Meningococcus and IsogeniccssAMutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Roberta Colicchio ◽  
Chiara Pagliuca ◽  
Susanna Ricci ◽  
Elena Scaglione ◽  
Denis Grandgirard ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Corpus Callosum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Lethal Dose ◽  
Sialic Acids ◽  
Wild Type ◽  
Knockout Mutant ◽  
Content Type

ABSTRACTIn serogroup CNeisseria meningitidis, thecssA(siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine intoN-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogeniccssAknockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of thecssAmutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in thecorpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of thecorpus callosumin the mouse model of meningococcal meningitis.

scholarly journals 3′ Untranslated Region-Dependent Degradation of theaceAmRNA, Encoding the Glyoxylate Cycle Enzyme Isocitrate Lyase, by RNase E/G in Corynebacterium glutamicum

2012 ◽  
Vol 78 (24) ◽  
pp. 8753-8761 ◽  
Author(s):  
Tomoya Maeda ◽  
Masaaki Wachi
Keyword(s):  
Corynebacterium Glutamicum ◽  
Untranslated Region ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Rnase E ◽  
Wild Type ◽  
Knockout Mutant ◽  
Content Type ◽  
In The Wild ◽  
The Glyoxylate Cycle

ABSTRACTWe previously reported that theCorynebacterium glutamicumRNase E/G encoded by therneGgene (NCgl2281) is required for the 5′ maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased inrneGknockout mutant cells grown on sodium acetate as the sole carbon source. Rifampin chase experiments showed that the half-life of theaceAmRNA was about 4 times longer in therneGknockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level ofaceAmRNA was approximately 3-fold higher in therneGknockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3′ rapid amplification of cDNA ends suggested that RNase E/G cleaves theaceAmRNA at a single-stranded AU-rich region in the 3′ untranslated region (3′-UTR). ThelacZfusion assay showed that the 3′-UTR renderedlacZmRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle inC. glutamicum.

scholarly journals Facultative methanotrophs – diversity, genetics, molecular ecology and biotechnological potential: a mini-review

Microbiology ◽  
2020 ◽  
Vol 166 (10) ◽  
pp. 894-908
Author(s):  
Muhammad Farhan Ul Haque ◽  
Hui-Juan Xu ◽  
J. Colin Murrell ◽  
Andrew Crombie
Keyword(s):  
Molecular Ecology ◽  
Significant Proportion ◽  
Vital Role ◽  
Short Chain ◽  
Atmospheric Methane ◽  
Biotechnological Potential ◽  
Content Type ◽  
Methane Oxidizing Bacteria ◽  
Metabolic Versatility ◽  
Carbon Compounds

Methane-oxidizing bacteria (methanotrophs) play a vital role in reducing atmospheric methane emissions, and hence mitigating their potent global warming effects. A significant proportion of the methane released is thermogenic natural gas, containing associated short-chain alkanes as well as methane. It was one hundred years following the description of methanotrophs that facultative strains were discovered and validly described. These can use some multi-carbon compounds in addition to methane, often small organic acids, such as acetate, or ethanol, although Methylocella strains can also use short-chain alkanes, presumably deriving a competitive advantage from this metabolic versatility. Here, we review the diversity and molecular ecology of facultative methanotrophs. We discuss the genetic potential of the known strains and outline the consequent benefits they may obtain. Finally, we review the biotechnological promise of these fascinating microbes.

scholarly journals Biocatalytic Reductive Amination by Native Amine Dehydrogenases to Access Short Chiral Alkyl Amines and Amino Alcohols

2021 ◽  
Vol 1 ◽  
Author(s):  
Laurine Ducrot ◽  
Megan Bennett ◽  
Adam A. Caparco ◽  
Julie A. Champion ◽  
Andreas S. Bommarius ◽  
...  
Keyword(s):  
Active Sites ◽  
Reductive Amination ◽  
Scale Up ◽  
Enantiomeric Excess ◽  
Short Chain ◽  
Chiral Amines ◽  
Wild Type ◽  
Preparative Scale ◽  
Alkyl Amines ◽  
Substrate Concentrations

Small optically active molecules, and more particularly short-chain chiral amines, are key compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-biocatalytic production on a commercial scale is already established, mainly through lipase-catalyzed resolutions leading to ChiPros™ products among others. Nevertheless, their biocatalytic synthesis remains challenging for very short-chain C4 to C5 amines due to low enantiomeric excess. To complement the possibilities recently offered by transaminases, this work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, MsmeAmDH, MicroAmDH, and MATOUAmDH2) were shown to be efficient for the synthesis of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol (99.4%), and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale-up experiments were successfully performed at 150 mM substrate concentrations for the synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-MOIPA”. Modeling studies provided some preliminary results explaining the basis for the challenging discrimination between similarly sized substituents in the active sites of these enzymes.

Sign in / Sign up

Export Citation Format

Share Document