scholarly journals Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

2008 ◽  
Vol 75 (3) ◽  
pp. 652-661 ◽  
Author(s):  
�yvind M. Jakobsen ◽  
Trygve Brautaset ◽  
Kristin F. Degnes ◽  
Tonje M. B. Heggeset ◽  
Simone Balzer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Lysine Production ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
The Impact

ABSTRACT Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V max values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

scholarly journals Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus

2011 ◽  
Vol 77 (17) ◽  
pp. 6020-6026 ◽  
Author(s):  
Ingemar Nærdal ◽  
Roman Netzer ◽  
Trond E. Ellingsen ◽  
Trygve Brautaset
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Feedback Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
Lysine Overproduction

ABSTRACTWe investigated the regulation and roles of six aspartate pathway genes inl-lysine overproduction inBacillus methanolicus:dapG, encoding aspartokinase I (AKI);lysC, encoding AKII;yclM, encoding AKIII;asd, encoding aspartate semialdehyde dehydrogenase;dapA, encoding dihydrodipicolinate synthase; andlysA, encodingmeso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed thatin vivo lysCtranscription was repressed 5-fold byl-lysine and induced 2-fold bydl-methionine added to the growth medium. Surprisingly,yclMtranscription was repressed 5-fold bydl-methionine, while thedapG,asd,dapA, andlysAgenes were not significantly repressed by any of the aspartate pathway amino acids. We show that thel-lysine-overproducing classicalB. methanolicusmutant NOA2#13A52-8A66 has—in addition to ahom-1mutation—chromosomal mutations in thedapGcoding region and in thelysApromoter region. No mutations were found in itsdapA,lysC,asd, andyclMgenes. The mutantdapGgene product had abolished feedback inhibition bymeso-diaminopimelatein vitro, and thelysAmutation was accompanied by an elevated (6-fold)lysAtranscription levelin vivo. Moreover,yclMtranscription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important forl-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increasedl-lysine production levels.

scholarly journals Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Autumn T. LaPointe ◽  
V Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Sindbis Virus ◽  
Virus Disease ◽  
Severe Disease ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Mortality And Morbidity ◽  
The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals The anti-inflammatory peptide Ac-SDKP is released from thymosin-β4 by renal meprin-α and prolyl oligopeptidase

2016 ◽  
Vol 310 (10) ◽  
pp. F1026-F1034 ◽  
Author(s):  
Nitin Kumar ◽  
Pablo Nakagawa ◽  
Branislava Janic ◽  
Cesar A. Romero ◽  
Morel E. Worou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Plasma Concentrations ◽  
Potential Candidate ◽  
Thymosin Β4 ◽  
Wild Type ◽  
Prolyl Oligopeptidase ◽  
Anti Inflammatory

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tβ4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tβ4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tβ4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tβ4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tβ4 was incubated with either meprin-α or POP alone. Incubation of Tβ4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tβ4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tβ4 is mediated by successive hydrolysis involving meprin-α and POP.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

scholarly journals Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

2006 ◽  
Vol 188 (8) ◽  
pp. 3063-3072 ◽  
Author(s):  
Øyvind M. Jakobsen ◽  
Aline Benichou ◽  
Michael C. Flickinger ◽  
Svein Valla ◽  
Trond E. Ellingsen ◽  
...  
Keyword(s):  
Methylotrophic Bacterium ◽  
Primary Metabolism ◽  
Wild Type ◽  
Fructose Bisphosphate ◽  
Methanol Tolerance ◽  
Oxidation Of Methanol ◽  
Bacillus Methanolicus ◽  
Multiple Copies ◽  
Bacterium Bacillus

ABSTRACT The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.

scholarly journals Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

2013 ◽  
Vol 79 (17) ◽  
pp. 5321-5328 ◽  
Author(s):  
Anne Krog ◽  
Tonje Marita Bjerkan Heggeset ◽  
Trond Erling Ellingsen ◽  
Trygve Brautaset
Keyword(s):  
Functional Characterization ◽  
Methylotrophic Bacterium ◽  
Content Type ◽  
Key Enzymes ◽  
Bacillus Methanolicus ◽  
Bacterium Bacillus ◽  
Glutamate Synthesis ◽  
Synthesis And Degradation

ABSTRACTBacillus methanolicuswild-type strain MGA3 secretes 59 g/liter−1ofl-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved inl-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded byyweBand two glutamate synthases (GOGATs) that are encoded by thegltABoperon and bygltA2were found, in contrast toBacillus subtilis, which has two different GDHs and only one GOGAT.B. methanolicushas a glutamine synthetase (GS) that is encoded byglnAand a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by theodhABoperon. TheyweB,gltA,gltB, andgltA2gene products were purified and characterized biochemicallyin vitro. YweB has a lowKmvalue for ammonium (10 mM) and a highKmvalue forl-glutamate (250 mM), and theVmaxvalue is 7-fold higher forl-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similarKmvalues (1 to 1.4 mM) andVmaxvalues (4 U/mg) for bothl-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymesin vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activityin vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium.In vivoexperiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulatingl-glutamate production in this organism.

scholarly journals The experimentally obtained functional impact assessments of GT>GC 5’ splice site variants differ markedly from those predicted

2019 ◽  
Author(s):  
Jian-Min Chen ◽  
Jin-Huan Lin ◽  
Emmanuelle Masson ◽  
Zhuan Liao ◽  
Claude Férec ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Genes ◽  
Wild Type ◽  
Human Genetic Disease ◽  
Functional Impact ◽  
Prediction Tools ◽  
Human Disease Genes ◽  
The Impact

ABSTRACTGT>GC 5’ splice site (or +2T>C) variants have been frequently reported to cause human genetic disease. However, although we have demonstrated that GT>GC variants in human disease genes may not invariably be pathogenic, none of the currently available splicing prediction tools appear to be capable of reliably distinguishing those GT>GC variants that generate wild-type transcripts from those that do not. Recently, SpliceAI, a novel deep residual neural network tool, has been developed for splicing prediction. Methodologically distinct from previous approaches that either rely on human-engineered features and/or which focus on short nucleotide windows adjoining exon-intron boundaries, SpliceAI assesses splicing determinants by evaluating 10,000 nucleotides of flanking contextual sequence to predict the functional role in splicing of each position in the pre-mRNA transcript. Herein, we evaluated the performance of SpliceAI in the context of three datasets of GT>GC variants, all of which had been characterized functionally in terms of their impact on mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 disease-causing GT>GC variants and the “in vitro” dataset of 103 GT>GC substitutions. The third dataset comprised 12 BRCA1 GT>GC variants that were recently analyzed by saturation genome editing. We processed all GT>GC variants using the default settings of SpliceAI. Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of the analyzed GT>GC variants revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue is that the impact of GT>GC (as well as GT>GA or +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

scholarly journals A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Sebastián Sasías ◽  
Adriana Martínez-Sanguiné ◽  
Laura Betancor ◽  
Arací Martínez ◽  
Bruno D'Alessandro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
High Frequency ◽  
Salmonella Enterica ◽  
Wild Type ◽  
Content Type ◽  
Naturally Occurring ◽  
Salmonella Enterica Serovar Dublin

ABSTRACTSalmonella entericaserovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to preventSalmonelladissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) ofS. Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory propertiesin vitroandin vivo. The aim of this work was to elucidate the underlying cause of the absence of flagella inS. Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in thefliEgene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only whenfliAwas artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences infliEadjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected inS. Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

scholarly journals Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation

2019 ◽  
Vol 30 (1) ◽  
pp. 4-16 ◽  
Author(s):  
Xiaoyan Gong ◽  
Yong Liao ◽  
Annette Ahner ◽  
Mads Breum Larsen ◽  
Xiaohui Wang ◽  
...  
Keyword(s):  
Physiological Role ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Specific Effects ◽  
Ubiquitin Conjugation ◽  
Protein Array Analysis ◽  
The Impact

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

scholarly journals FSMP-07. CYSTATHIONINE-Γ-LYASE DRIVES ANTIOXIDANT DEFENSE IN CYSTEINE-RESTRICTED IDH1 MUTANT ASTROCYTOMAS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i17-i17
Author(s):  
Andrés Cano-Galiano ◽  
Anais Oudin ◽  
Fred Fack ◽  
Maria-Francesca Allega ◽  
David Sumpton ◽  
...  
Keyword(s):  
Antioxidant Defense ◽  
De Novo ◽  
Idh1 Mutation ◽  
Wild Type ◽  
Isocitrate Dehydrogenase 1 ◽  
Astrocytic Gliomas ◽  
The Impact ◽  
Reducing Potential

Abstract Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzymes are crucial for the generation of reducing potential, yet the impact of the mutation on the cellular antioxidant system is not understood. Here, we investigate how glutathione (GSH) levels are maintained in IDH1 mutant gliomas, despite an altered NADPH/NADP balance. We find that IDH1 mutant astrocytomas specifically upregulate cystathionine γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis. Genetic and chemical interference with CSE in patient-derived glioma cells carrying the endogenous IDH1 mutation, sensitized tumor cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with reduced GSH synthesis as shown by in vitro and in vivo serine tracing and led to delayed tumor growth in mice. Thus we show that IDH1 mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis to maintain the antioxidant defense, which uncovers a novel metabolic vulnerability in this dismal disease.

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