[Show abstract][Hide abstract] ABSTRACT: Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol-trans-dihydrodimer and pallidol) produced were tested for antimicrobial activity. The resveratrol-trans-dihydrodimer displayed antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 µM, respectively) and against Gram-negative Escherichia coli (MIC = 123 µM, upon addition of the efflux pump inhibitor Phe-Arg-β-naphthylamide). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometric analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol-trans-dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide and a platform for the development of polyphenolic antimicrobials. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Full-text · Article · Jun 2015 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Natural products have been associated with significant health benefits in preventing and treating various chronic human diseases such as cancer, cardiovascular diseases, diabetes, Alzheimer's disease, and pathogenic infections. However, the isolation, characterization and evaluation of natural products remain a challenge, mainly due to their limited bioavailability. Metabolic engineering and fermentation technology have emerged as alternative approaches for generating natural products under controlled conditions that can be optimized to maximize yields. Optimization of these processes includes the evaluation of factors such as host selection, product biosynthesis interaction with the cell's central metabolism, product degradation, and byproduct formation. This review summarizes the most recent biochemical strategies and advances in expanding and diversifying natural compounds as well as maximizing their production in microbial and plants cells.
Full-text · Article · Feb 2014 · Current Opinion in Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Most natural polyphenols have low antimicrobial activity. In this work, we tried to generate analogues of resveratrol via either enzymatic oligomerization or site-directed mutagenesis of stilbene synthase (STS). Enzymatic oligomerization produced two oligomers, resveratrol-trans-dihydrodimer and Pallidol. The trans-dihydrodimer showed commercially relevant antimicrobial activity against gram-positive bacteria, B. cereus (MIC = 15.0 µM), Listeria (125.0 µM), and S. aureus (MIC = 62.0 µM). Further, we identified it’s mechanism of action to be via altering membrane structure and polarization, and inhibiting DNA synthesis. This was deciphered by transcriptomic, biochemical and flow cytometric analysis. Gene expression microarray and qRT-PCR data from B. cereus indicated down regulation of ABC transporters (ATP binding proteins). Flow cytometry analysis showed depolarization of cell membrane upon treatment with resveratrol-trans-dihydrodimer. A decrease in nucleic DNA content of B. cereus and E. coli upon incubation with the trans-dihydrodimer was also observed by flow cytometry. Further, in vitro activity of DNA gyrase was strongly inhibited by the resveratrol-trans-dihydrodimer.
Next we created 15 mutants of stilbene synthase and tried to create resveratrol analogues by feeding different substrates. Type III polyketide synthases have been studied extensively over the past few decades, but most of these structural-functional studies have been carried out on chalcone synthase (CHS) like enzymes, which carry out a C6-C2 Claisen condensation of their polyketide intermediates. Stilbene synthases share 75-90% sequence similarity with CHS and form the same tetraketide intermediate, but cyclize the product via a C1-C7 aldol condensation. This cyclization requires a thioesterase-like hydrolysis step made possible by the hydrogen bonding network (HBN) present only in STSes, involving residues S338, T132 and E192 and a water molecule. Here we studied the effect of mutating functionally important residues in STS. Similar mutations have been carried out in CHS but we predicted that new products would be formed because of the different cyclization of intermediates by STS. Mutant G256L completely lost its ability to handle the bulky p-coumaroyl-CoA substrate while a T197G mutant was still able to produce resveratrol while forming at the same time an increased amount of a more hydrophobic compound which is yet to be identified.
Finally, we have demonstrated that the enzymatic oligomerization of resveratrol is an attractive strategy for generating effective antimicrobials with potential therapeutic application. And further we plan to study the antimicrobial activity of resveratrol analogues created by mutating the functionally important residues of STS. The mechanism of action of which can be identified by carrying out studies similar to those carried out for the resveratrol-trans-dihydrodimer.
[Show abstract][Hide abstract] ABSTRACT: Many natural polyphenols possess low antimicrobial activities which can be improved by using them as scaffolds to generate libraries of their oligomers. We have performed the in vitro diversification of resveratrol mediated by soybean peroxidase, and isolated two dimers (resveratrol-trans-dihydrodimer, and pallidol) and tested them as antimicrobials. In particular, resveratrol-trans-dihydrodimer was found to be an effective antimicrobial against Gram positive strains B. cereus (MIC = 15.0 µM), Listeria (125.0 µM), and S. aureus (MIC = 62.0 µM) as well as against Gram negative E. coli (MIC = 122.5 µM; in the presence of efflux pump inhibitor Phe-Arg-β-naphthylamide), while pallidol did not show significant antimicrobial activity. Further we deciphered the mechanism of action of the trans-dihydrodimer by transcriptomic, biochemical and flow cytometry analysis. Gene expression microarray and qRT-PCR data from B. cereus indicated down regulation of ABC transporters (ATP binding proteins). Flow cytometry analysis showed a strong depolarization of cell membrane upon treatment with resveratrol-trans-dihydrodimer, while ATP measurement indicated an accumulation of overall ATP (>2 fold); down regulation of ABC transporters that break down ATP is one of the individual events that contributes for ATP accumulation.
Alternately, a decrease in nucleic DNA content of B. cereus and E. coli upon incubation with the resveratrol-trans-dihydrodimer was also observed by flow cytometry. An in vitro activity assay of DNA gyrase from E. coli showed a strong inhibitory effect caused by the resveratrol-trans-dihydrodimer, which is consistent with previous reports regarding the inhibitory effect of polyphenols on DNA gyrase activity. This enzyme requires ATP as a source of energy for supercoiling DNA and its inhibition stops the breakdown of ATP. This observation suggests that resveratrol-trans-dihydrodimer could be responsible for inhibiting other enzymes that require ATP resulting in the increase of overall ATP. Finally, we have demonstrated that the enzymatic oligomerization of resveratrol is an attractive strategy for generating effective antimicrobials with potential therapeutic application especially due the low toxicity of resveratrol derivatives against mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: Over the years, natural products from plants and their non-natural derivatives have shown to be active against different types of chronic diseases. However, isolation of such natural products can be limited due to their low bioavailability, and environmental restrictions. To address these issues, in vivo and in vitro reconstruction of plant metabolic pathways and the metabolic engineering of microbes and plants have been used to generate libraries of compounds. Significant advances have been made through metabolic engineering of microbes and plant cells to generate a variety of compounds (e.g. isoprenoids, flavonoids, or stilbenes) using a diverse array of methods to optimize these processes (e.g. host selection, operational variables, precursor selection, gene modifications). These approaches have been used also to generate non-natural analogues with different bioactivities. In vitro biosynthesis allows the synthesis of intermediates as well as final products avoiding post-translational limitations. Moreover, this strategy allows the use of substrates and the production of metabolites that could be toxic for cells, or expand the biosynthesis into non-conventional media (e.g. organic solvents, supercritical fluids). A perspective is also provided on the challenges for generating novel chemical structures and the potential of combining metabolic engineering and in vitro biocatalysis to produce metabolites with more potent biological activities.
[Show abstract][Hide abstract] ABSTRACT: Enzymatic oligomerization of Resveratrol to generate novel anti-microbial analogs
Plant polyphenols such as flavonoids and stilbenoids have been associated with several health benefits and thus have been widely studied in recent years. Despite the current controversy regarding their potential for pharmacological applications, recent studies suggest that natural phenols can be converted into active metabolites in vivo, and more attention is required on the in vitro synthesis of analogs to enhance their efficacy. Also, a constant growth of anti-microbial resistance and rapid microbial evolution, underscores the importance of identification of alternatives to generate new and novel antimicrobial compounds. We propose that the antimicrobial activity can be significantly enhanced by generating oligomers of these compounds. In particular, we produced, isolated and characterized a dimer from resveratrol (d-viniferin, the tri-dehydrodimer of resveratrol) from an in vitro enzymatic oligomerization reaction mediated by soybean peroxidase (SBP) in aqueous buffer. Dose response studies in gram- positive and gram- negative bacteria show great antimicrobial potential of d-viniferin as compared to resveratrol. For example, d-viniferin showed MIC and IC50 values of 125 M and 59 M respectively against E. coli (in the presence of efflux pump inhibitor Phe-Arg--naphthylamide, PABN, 25 g/mL), while resveratrol shows MIC and IC50 values of 2000 M and 250 M respectively. Significant improvement was observed against B. cereus with d-viniferin giving MIC and IC50 values of 5 M and 15 M, while for resveratrol MIC and IC50 values were >150 M, all in presence of the efflux pump inhibitor 1-(1-Naphthylmethyl) piperazine (NMP).
The significant increase in anti-microbial activity in presence of efflux pump inhibitor PABN in the case of E. coli indicated that efflux pumps are responsible for the extrusion of resveratrol and its analogs from the cell. Thus, d-viniferin was tested against an E. coli strain with a deletion of tolC gene (part of AcrAB-tolC efflux pump) in the presence and absence of PABN. In both cases, significant but similar growth inhibition was observed ([+]PABN, MIC and IC50 values were 1.2 and 3 M respectively; [-]PABN, MIC and IC50 values were 2.5 and 7 M respectively). Moreover, in the absence of PABN both the dimer and monomer had no effect on E. coli at these concentrations. The effect of efflux pump inhibitor on B. cereus was less significant; MIC and IC50 values in the absence of NMP were 12 and 30 M respectively.
Visual evidence discarded the possibility of d-viniferin causing cellular aggregation leading to a lower cell density or count. Also, morphological changes in B. cereus were observed before and after incubation with d-viniferin; there was a decrease in the cell size while cell wall was found to be unaltered and intact.
In summary, it was demonstrated that novel and active antimicrobials from plant polyphenols can be generated in vitro. In particular, d-viniferin has shown great antimicrobial activity and its low toxicity against mammalian cells provides a new insight in the development of antimicrobial with potential therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: The enormous pool of chemical diversity found in nature serves as an excellent inventory for accessing biologically active compounds. This chemical inventory, primarily found in microorganisms and plants, is generated by a broad range of enzymatic pathways under precise genetic and protein-level control. In vitro pathway reconstruction can be used to characterize individual pathway enzymes, identify pathway intermediates, and gain an increased understanding of how pathways can be manipulated to generate natural product analogs. Moreover, through in vitro approaches, it is possible to achieve a diversification that is not restricted by toxicity, limited availability of intracellular precursors, or preconceived (by nature) regulatory controls. Additionally, combinatorial biosynthesis and high-throughput techniques can be used to generate both known natural products and analogs that would not likely be generated naturally. This current opinion review will focus on recent advances made in performing in vitro pathway-driven natural product diversification and opportunities for exploiting this approach for elucidating and entering this new chemical biology space.
Full-text · Article · Mar 2012 · Current opinion in chemical biology
[Show abstract][Hide abstract] ABSTRACT: Enzymatically derived oligophenols from apocynin can be effective inhibitors of human vascular NADPH oxidase (Nox). An isolated trimer hydroxylated quinone (IIIHyQ) has been shown to inhibit endothelial NADPH oxidase with an IC(50) ~30 nM. In vitro studies demonstrated that IIIHyQ is capable of disrupting the interaction between p47(phox) and p22(phox), thereby blocking the activation of the Nox2 isoform. Herein, we report the role of key cysteine residues in p47(phox) as targets for the IIIHyQ. Incubation of p47(phox) with IIIHyQ results in a decrease of ~80% of the protein free cysteine residues; similar results were observed using 1,2- and 1,4-naphthoquinones, whereas apocynin was unreactive. Mutants of p47(phox), in which each Cys was individually replaced by Ala (at residues 111, 196, and 378) or Gly (at residue 98), were generated to evaluate their individual importance in IIIHyQ-mediated inhibition of p47(phox) interaction with p22(phox). Specific Michael addition on Cys196, within the N-SH3 domain, by the IIIHyQ is critical for disrupting the p47(phox)-p22(phox) interaction. When a C196A mutation was tested, the IIIHyQ was unable to disrupt the p47(phox)-p22(phox) interaction. However, the IIIHyQ was effective at disrupting this interaction with the other mutants, displaying IC(50) values (4.9, 21.0, and 2.3μM for the C111A, C378A, and C98G mutants, respectively) comparable to that of wild-type p47(phox).
Full-text · Article · Mar 2012 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37°C, and a significant fraction of particles remain assembled after 30min at 80°C.
[Show abstract][Hide abstract] ABSTRACT: Ionic liquids (ILs) have emerged as attractive solvents for lignocellulosic biomass pretreatment in the production of biofuels and chemical feedstocks. However, the high cost of ILs is a key deterrent to their practical application. Here, we show that acetate based ILs are effective in dramatically reducing the recalcitrance of corn stover toward enzymatic polysaccharide hydrolysis even at loadings of biomass as high as 50% by weight. Under these conditions, the IL serves more as a pretreatment additive rather than a true solvent. Pretreatment of corn stover with 1-ethyl-3-methylimidizolium acetate ([Emim] [OAc]) at 125 ± 5°C for 1 h resulted in a dramatic reduction of cellulose crystallinity (up to 52%) and extraction of lignin (up to 44%). Enzymatic hydrolysis of the IL-treated biomass was performed with a common commercial cellulase/xylanase from Trichoderma reesei and a commercial β-glucosidase, and resulted in fermentable sugar yields of ∼80% for glucose and ∼50% for xylose at corn stover loadings up to 33% (w/w) and 55% and 34% for glucose and xylose, respectively, at 50% (w/w) biomass loading. Similar results were observed for the IL-facilitated pretreatment of switchgrass, poplar, and the highly recalcitrant hardwood, maple. At 4.8% (w/w) corn stover, [Emim][OAc] can be readily reused up to 10 times without removal of extracted components, such as lignin, with no effect on subsequent fermentable sugar yields. A significant reduction in the amount of IL combined with facile recycling has the potential to enable ILs to be used in large-scale biomass pretreatment.
Full-text · Article · Dec 2011 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Substantial evidence suggests that soluble prefibrillar oligomers of the Aβ42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aβ isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aβ oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aβ oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aβ soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aβ oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aβ oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aβ oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aβ fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aβ oligomers (which are indistinguishable in size and morphology to Aβ soluble oligomers) or promote aggregation of freshly disaggregated Aβ peptide; thus revealing that they are specific for remodeling toxic Aβ conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aβ-mediated toxicity.
[Show abstract][Hide abstract] ABSTRACT: Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment. The unique solvating properties of RTILs foster the disruption of the 3D network structure of lignin, cellulose, and hemicellulose, which allows high yields of fermentable sugars to be produced in subsequent enzymatic hydrolysis. In the current review, we summarize the physicochemical properties of RTILs that make them effective solvents for lignocellulose pretreatment including mechanisms of interaction between lignocellulosic biomass subcomponents and RTILs. We also highlight several recent strategies that exploit RTILs and generate high yields of fermentable sugars suitable for downstream biofuel production, and address new opportunities for use of lignocellulosic components, including lignin. Finally, we address some of the challenges that remain before large-scale use of RTILs may be achieved.
Full-text · Article · Jun 2011 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Effective pretreatment of lignocellulosic biomass is vital to its bioconversion to a usable liquid fuel. A growing body of work has focused on using room temperature ionic liquids (RTILs) to pretreat lignocellulose for subsequent fermentation. However, little is known about the physicochemical parameters of RTILs that promote effective pretreatment. In this work we examine the relationship between the Kamlet–Taft α, β, and π* solvent polarity parameters of different RTILs ([Emim][OAc], [Bmim][OAc], and [Bmim][MeSO4]) and effective pretreatment of lignocellulosic biomass. We find the β parameter is an excellent predictor of pretreatment efficacy. Acetate containing RTILs (β > 1.0) remove >32% of lignin from maple wood flour and significantly reduce cellulose crystallinity, resulting in >65% glucose yields after 12 h cellulase hydrolysis. Pretreatment in [Bmim][MeSO4] (β = 0.60) results in the removal of only 19% of the wood flour's lignin with no decrease in crystallinity, and no improvement in sugar yield over untreated wood flour. The addition of water and the dilution of the acetate anion with the methyl sulfate anion decrease the β value and subsequently have a negative impact on ligninextraction, cellulose crystallinity, and sugar yields.
[Show abstract][Hide abstract] ABSTRACT: Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC(50)=31nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47(phox) and p22(phox). To that end, while apocynin was unable to block the interaction of his-tagged p47(phox) with a surface immobilized biotinylated p22(phox) peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC(50)=1.6microM). These results provide evidence that peroxidase-generated AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.