scholarly journals Metabolic Engineering of a Glycerol-Oxidative Pathway in Lactobacillus panis PM1 for Utilization of Bioethanol Thin Stillage: Potential To Produce Platform Chemicals from Glycerol

2014 ◽  
Vol 80 (24) ◽  
pp. 7631-7639 ◽  
Author(s):  
Tae Sun Kang ◽  
Darren R. Korber ◽  
Takuji Tanaka
Keyword(s):  
Lactic Acid ◽  
Triosephosphate Isomerase ◽  
Value Added ◽  
Dependent Manner ◽  
Bioethanol Production ◽  
Thin Stillage ◽  
Content Type ◽  
Platform Chemicals ◽  
Wide Range ◽  
Oxidative Pathway

ABSTRACTLactobacillus panisPM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact thatL. panisPM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly,L. panisPM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes ofEscherichia coli, was introduced intoL. panisPM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production.

scholarly journals Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

2014 ◽  
Vol 58 (12) ◽  
pp. 7072-7082 ◽  
Author(s):  
Horacio Reyes-Vivas ◽  
Ignacio de la Mora-de la Mora ◽  
Adriana Castillo-Villanueva ◽  
Lilian Yépez-Mulia ◽  
Gloria Hernández-Alcántara ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Cytotoxic Effect ◽  
Triosephosphate Isomerase ◽  
Glycolytic Enzyme ◽  
Cysteine Residue ◽  
Enzyme Inactivation ◽  
Dependent Manner ◽  
Drug Resistant ◽  
Content Type

ABSTRACTGiardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains ofGiardia lambliawas evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites.G. lambliatriosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effectivein vitroagainst drug-resistant and drug-susceptible strains ofG. lamblia.

scholarly journals The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

2016 ◽  
Vol 84 (10) ◽  
pp. 2878-2894 ◽  
Author(s):  
Jesús F. T. Miranda-Ozuna ◽  
Mar S. Hernández-García ◽  
Luis G. Brieba ◽  
Claudia G. Benítez-Cardoza ◽  
Jaime Ortega-López ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Triosephosphate Isomerase ◽  
Specific Binding ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Affinity Ligand ◽  
Surface Localization

Triosephosphate isomerase ofTrichomonas vaginalis(TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes,tvtim1andtvtim2, that participates in glucose metabolism.TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associatedTvTIM inT. vaginalisand to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that thetvtimgenes were differentially expressed in response to glucose concentration.tvtim1was overexpressed under glucose-restricted (GR) conditions, whereastvtim2was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization ofTvTIM inT. vaginalis. Affinity ligand assays demonstrated that the recombinantTvTIM1 andTvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r andTvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover,T. vaginaliswas exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate thatTvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor ofT. vaginalis.

scholarly journals D-Alanyl-D-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Shenwei Zhang ◽  
Jee-Hwan Oh ◽  
Laura M. Alexander ◽  
Mustafa Özçam ◽  
Jan-Peter van Pijkeren
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Heterologous Expression ◽  
Genome Editing ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Content Type ◽  
Suicide Vector ◽  
Vancomycin Resistant ◽  
Dose Dependent

ABSTRACTThe peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending ind-alanyl-d-lactate and binds with high affinity to peptidoglycan ending ind-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a natived-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genusLactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCEThe genusLactobacilluscontains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selectedLactobacillusspecies. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance inLactobacillus; 81% ofLactobacillusspecies are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

scholarly journals Versatile Cas9-Driven Subpopulation Selection Toolbox forLactococcus lactis

2018 ◽  
Vol 84 (8) ◽  
Author(s):  
Simon van der Els ◽  
Jennelle K. James ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Mobile Genetic Elements ◽  
Broad Host Range ◽  
Content Type ◽  
Genetic Elements ◽  
Wide Range ◽  
Dairy Fermentation ◽  
Selection Of

ABSTRACTCRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy speciesLactococcus lactis. The Cas9 expression vector pLABTarget, encoding theStreptocccus pyogenesCas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of apepN-targeting derivative of pLABTarget intoL. lactisstrain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in apepNdeletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of apepNdeletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives inL. lactis. Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting inL. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.IMPORTANCEMobile genetic elements inLactococcus lactisand other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.

scholarly journals Manganese Catalyzed Reformation of Ethylene Glycol to Glycolic acid and Lactic Acid

2021 ◽  
Author(s):  
Satyadeep Waiba ◽  
BIplab Maji
Keyword(s):  
Lactic Acid ◽  
Ethylene Glycol ◽  
Glycolic Acid ◽  
Value Added ◽  
Hydrogen Gas ◽  
Catalyst Loading ◽  
Pure Hydrogen ◽  
Platform Chemicals ◽  
Dehydrogenative Coupling ◽  
The Reformation

Conversion of readily available feedstocks to valuable platform chemicals via a sustainable catalytic pathway has always been one of the key focuses of synthetic chemists. Cheaper, less toxic, and more abundant base metals as a catalyst for performing such transformations provide an additional boost. In this context, herein, we report a reformation of readily available feedstock, ethylene glycol, to value-added platform molecules, glycolic acid, and lactic acid. A bench stable base metal complex {[HN(C2H4PPh2)2]Mn(CO)2Br}, Mn-I, known as Mn-PhMACHO, catalyzed the reformation of ethylene glycol to glycolic acid at 140 oC in high selectivity with a turnover number TON = 2400, surpassing previously used homogeneous catalysts for such a reaction. Pure hydrogen gas is evolved without the need for an acceptor. On the other hand, a bench stable Mn(I)-complex, {(iPrPN5P)Mn(CO)2Br}, Mn-III, with a triazine backbone, efficiently catalyzed the acceptorless dehydrogenative coupling of ethylene glycol and methanol for the synthesis of lactic acid, even at a ppm level of catalyst loading, reaching the TON of 11,500. Detailed mechanistic studies were performed to elucidate the involvements of different manganese(I)-species during the catalysis.

scholarly journals Pseudomonas Strains Naturally Associated with Potato Plants Produce Volatiles with High Potential for Inhibition of Phytophthora infestans

2014 ◽  
Vol 81 (3) ◽  
pp. 821-830 ◽  
Author(s):  
Lukas Hunziker ◽  
Denise Bönisch ◽  
Ulrike Groenhagen ◽  
Aurélien Bailly ◽  
Stefan Schulz ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Phytophthora Infestans ◽  
Headspace Analysis ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Content Type ◽  
Zoospore Release ◽  
Wide Range ◽  
Bacterial Volatiles

ABSTRACTBacteria emit volatile organic compounds with a wide range of effects on bacteria, fungi, plants, and animals. The antifungal potential of bacterial volatiles has been investigated with a broad span of phytopathogenic organisms, yet the reaction of oomycetes to these volatile signals is largely unknown. For instance, the response of the late blight-causing agent and most devastating oomycete pathogen worldwide,Phytophthora infestans, to bacterial volatiles has not been assessed so far. In this work, we analyzed this response and compared it to that of selected fungal and bacterial potato pathogens, using newly isolated, potato-associated bacterial strains as volatile emitters.P. infestanswas highly susceptible to bacterial volatiles, while fungal and bacterial pathogens were less sensitive. CyanogenicPseudomonasstrains were the most active, leading to complete growth inhibition, yet noncyanogenic ones also produced antioomycete volatiles. Headspace analysis of the emitted volatiles revealed 1-undecene as a compound produced by strains inducing volatile-mediatedP. infestansgrowth inhibition. Supplying pure 1-undecene toP. infestanssignificantly reduced mycelial growth, sporangium formation, germination, and zoospore release in a dose-dependent manner. This work demonstrates the high sensitivity ofP. infestansto bacterial volatiles and opens new perspectives for sustainable control of this devastating pathogen.

scholarly journals Immobilization of Antibacterial Dihydropyrrol-2-ones on Functional Polymer Supports To Prevent Bacterial InfectionsIn Vivo

2011 ◽  
Vol 56 (2) ◽  
pp. 1138-1141 ◽  
Author(s):  
Kitty Ka Kit Ho ◽  
Nerida Cole ◽  
Renxun Chen ◽  
Mark D. P. Willcox ◽  
Scott A. Rice ◽  
...  
Keyword(s):  
Staphylococcal Infection ◽  
Infection Model ◽  
Dependent Manner ◽  
Pathogenic Potential ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Polymer Supports ◽  
Wide Range ◽  
Life Threatening

ABSTRACTAntibiotic-resistantStaphylococcus aureusis of great concern, as it causes a wide range of life-threatening infections. The current study demonstrates that dihydropyrrolone (DHP)-coated polyacrylamide substrates are effective in reducing the number of culturable clinical isolates ofS. aureusin vitroin a dose-dependent manner and are able to reduce the pathogenic potential of staphylococcal infection in a subcutaneous infection model. Covalently bound DHPs therefore show great potential for use as an antimicrobial strategy in device-related applications.

scholarly journals Factors and “illusions” influencing the choice of PFI-type public private partnerships

2019 ◽  
Vol 15 (3) ◽  
pp. 222-238
Author(s):  
Ronald McQuaid
Keyword(s):  
Risk Sharing ◽  
Full Range ◽  
Good Practice ◽  
Value Added ◽  
Local Economy ◽  
Public Private Partnerships ◽  
Content Type ◽  
Alternative Funding ◽  
Published Evidence ◽  
Wide Range

Purpose The purpose of this paper is to critically assess some of the micro- and macro-economic reasons for using public finance initiative types of public–private partnerships (PPPs) and how a lack of transparency may result in an “illusion” of making optimal, rational decisions related to them. A series of balances that decision makers need to make in order to choose whether or not to use PPPs are set out, as well as 15 potential “illusions” may affect such decisions. Design/methodology/approach This paper synthesizes published evidence and develops a framework for analyzing PPPs. Findings A wide range of factors influence the choice of PPPs, including: budget enlargement; efficiency and value for money; certainty of expenditure and delivery; flexibility; financing costs; risk sharing; procurement process and transaction costs; legacy and public assets; and the wider impacts on the local economy. However, reasons why PPPs can provide improved infrastructure and services may not be realised due to in-built incentives, behavioural biases and implementation shortcomings. Necessary support for PPPs includes strong, robust and transparent regulatory and governance systems, the dissemination of good practice to all partners, consideration of alternative funding models and high-quality advice and training. Research limitations/implications The paper sets out a number of reasons for using PPPs, and also assesses potential drawbacks and identifies areas where greater research is required. A number of potential “illusions” are identified, whereby decisions may be affected by factors not explicitly or transparently considered, hence giving the decision an “illusion” being rational. Practical implications PPPs are significantly influenced by the socio-economic, legal, legislative and financial system they are embedded in. A clear process for approving projects and recognising all the costs and benefits of PPPs is needed, including developing criteria and instruments to measure each phase of a PPP and its overall value added to the economy and society over its lifetime. Full transparency, having suitable support and explicitly taking account of potential “illusions” affecting decisions, could lead to different decisions, including the decision not to progress the project or to use alternative funding and development methods. Social implications Decisions on PPPs should be based on a clear and transparent long-term basis which includes the perspectives of the full range of stakeholders to help improve the appropriate operation and social sustainability of a PPP. Originality/value The paper sets out some key arguments for and against the use of PPPs in different circumstances, including why non-optimal decisions may be made.

scholarly journals PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariusz Madej ◽  
Zuzanna Nowakowska ◽  
Miroslaw Ksiazek ◽  
Anna M. Lasica ◽  
Danuta Mizgalska ◽  
...  
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Secretion System ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Content Type ◽  
Essential Component ◽  
Wide Range ◽  
Cargo Proteins ◽  
Type Ix Secretion System

ABSTRACT Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

scholarly journals CHEMICAL VALORIZATION OF CELLULOSE FROM LIGNOCELLULOSIC BIOMASS: A STEP TOWARDS SUSTAINABLE DEVELOPMENT

2021 ◽  
Vol 55 (3-4) ◽  
pp. 207-222
Author(s):  
RAMANDEEP KAUR ◽  
PUNEET KAUR
Keyword(s):  
Lignocellulosic Biomass ◽  
Levulinic Acid ◽  
Fossil Fuel ◽  
Value Added ◽  
Integrated Approach ◽  
Synthesis Methods ◽  
Platform Chemicals ◽  
Wide Range ◽  
Modified Cellulose ◽  
Value Added Products

"The potential of non-edible lignocellulosic biomass paves the path to sustainable economy. A large number of valueadded products have been synthesized by the fractionation of the major components of biomass, i.e. cellulose, hemicelluloses and lignin. Cellulose, the most abundant biopolymer on earth, serves as a starting material for the synthesis of various platform chemicals, such as sorbitol, 5- hydroxylmethylfurfural (HMF), dimethylfuran and levulinic acid. Hydrogels and aerogels fabricated from cellulose, modified cellulose or nanocellulose have proved valuable in a wide range of such as biomedical, food and technological applications. Cellulose-based polymers or bioplastics also emerged as an alternative to fossil fuel-based polymers. In this review, chemical paths to valorize plant cellulose for producing various value-added products have been discussed. The major challenge for valorization is the development of novel and green synthesis methods with simultaneous focus on an integrated approach."

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