scholarly journals Archaeal Phospholipid Biosynthetic Pathway Reconstructed inEscherichia coli

Archaea ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Takeru Yokoi ◽  
Keisuke Isobe ◽  
Tohru Yoshimura ◽  
Hisashi Hemmi
Keyword(s):  
Recombinant Proteins ◽  
Biosynthetic Pathway ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Ionization Mass ◽  
Inescherichia Coli ◽  
Methanosarcina Acetivorans ◽  
E Coli ◽  
Endogenous Enzymes

A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells ofEscherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen,Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins.In vitroradioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously inE. coli. Introduction of the 4 genes intoE. coliresulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes ofE. coli.

scholarly journals Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryo Yoshida ◽  
Hisashi Hemmi
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Membrane Lipids ◽  
Extreme Environments ◽  
Halophilic Archaea ◽  
Coli Strain ◽  
Glycerol Moiety ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix

Abstract Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

scholarly journals Modification of an Engineered Escherichia Coli by a Combinatorial Strategy to Improve 3,4-Dihydroxybutyric Acid Production

2021 ◽  
Author(s):  
Yidi Liu ◽  
Xinlei Mao ◽  
Baoqi Zhang ◽  
Jinping Lin ◽  
Dongzhi Wei
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Biosynthetic Pathway ◽  
Enzyme Assays ◽  
Aldehyde Dehydrogenases ◽  
E Coli ◽  
Combinatorial Strategy ◽  
Competing Pathways ◽  
Synthesis Of Materials

Abstract Objectives: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multi-functional C4 platform compound with wide applications in the synthesis of materials and pharmaceuticals. Currently, although the biosynthetic pathway for the production of 3,4-DHBA has been developed, low productivity still hampers its use on large scales. Here, a non-natural four-steps biosynthetic pathway was established in recombinant E. coli with a combinatorial strategy.Results: Firstly, several aldehyde dehydrogenases (ALDHs) were screened and characterized for catalyzing the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA through in vitro enzyme assays. Secondly, a recombinant E. coli was successfully constructed to generate 3,4-DHBA from D-xylose by introducing the pathway containing BsGDH, YagF, PpMdlC and ALDH into E. coli with 3.04 g/L 3,4-DHBA obtained. Then, disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP genes contributed to increase the accumulation of 3,4-DHBA by 87%. Final, fusion expression of PpMdlC and YagF resulted in an enhancement of 3,4-DHBA titer (7.71 g/L), as the highest titer reported so far.Conclusions: These results showed that deleting competing pathways and constructing fusion protein could significantly improve the 3,4-DHBA titer in engineered E. coli.

scholarly journals Modulation Effects of Curcumin on Erythrocyte Ion-Transporter Activity

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Prabhakar Singh ◽  
Syed Ibrahim Rizvi
Keyword(s):  
Oxidative Stress ◽  
Membrane Lipids ◽  
Curcuma Longa ◽  
Membrane Lipid ◽  
Membrane Transporters ◽  
Protective Mechanism ◽  
Lipid Hydroperoxides ◽  
Beneficial Effects

Curcumin ((1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), the yellow biphenolic pigment isolated from turmeric (Curcuma longa), has various medicinal benefits through antioxidation, anti-inflammation, cardiovascular protection, immunomodulation, enhancing of the apoptotic process, and antiangiogenic property. We explored the effects of curcuminin vitro(10−5 M to 10−8 M) andin vivo(340 and 170 mg/kg b.w., oral) on Na+/K+ATPase (NKA), Na+/H+exchanger (NHE) activity, and membrane lipid hydroperoxides (ROOH) in control and experimental oxidative stress erythrocytes of Wistar rats. As a result, we found that curcumin potently modulated the membrane transporters activity with protecting membrane lipids against hydro-peroxidation in control as well as oxidatively challenged erythrocytes evidenced by stimulation of NKA, downregulation of NHE, and reduction of ROOH in the membrane. The observed results corroborate membrane transporters activity with susceptibility of erythrocyte membrane towards oxidative damage. Results explain the protective mechanism of curcumin against oxidative stress mediated impairment in ions-transporters activity and health beneficial effects.

Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos

Zygote ◽  
2014 ◽  
Vol 23 (5) ◽  
pp. 732-741 ◽  
Author(s):  
Beatriz C. S. Leão ◽  
Nathália A. S. Rocha-Frigoni ◽  
Elaine C. Cabral ◽  
Marcos F. Franco ◽  
Christina R. Ferreira ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Profile ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Maldi Ms ◽  
Charge Ratio ◽  
Alkyl Ether ◽  
Bovine Embryos ◽  
The Impact

SummaryThis study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2–20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase ‘e’ and ‘p‘, respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

scholarly journals In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli

2021 ◽  
Author(s):  
Filipe Silva ◽  
Sara Santos ◽  
Roberto Meyer ◽  
Eduardo Silva ◽  
Carina Pinheiro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Target Protein ◽  
E Coli ◽  
Recombinant Protein Purification ◽  
Intracellular Processing ◽  
Specific Protease ◽  
A Site

Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, are useful for simplifying the recombinant protein purification process, for screening molecules that fail to remain soluble after tag removal, and to promote higher yields of soluble target protein. Herein, we review controlled intracellular processing (CIP) systems, tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular protein processing regarding system design features, significant advantages and limitations of the various strategies.

scholarly journals Membrane Interactivity of Non-steroidal Anti-inflammatory Drugs: A Literature Review

2020 ◽  
pp. 1-30
Author(s):  
Hironori Tsuchiya ◽  
Maki Mizogami
Keyword(s):  
Membrane Fluidity ◽  
Membrane Lipids ◽  
Direct Interaction ◽  
Membrane Lipid ◽  
Lipid Phase ◽  
Lipid Bilayer Membranes ◽  
Inflammatory Drugs ◽  
Anti Inflammatory

Background: Although the mode of action of non-steroidal anti-inflammatory drugs (NSAIDs) has been exclusively referred to as inhibition of cyclooxygenase, their broad pharmacological and toxicological spectra are not necessarily interpreted by the direct interaction with such enzyme proteins. Aims: Since NSAIDs have the common amphiphilic structure, they have the possibility of acting on membrane-constituting lipids. In order to gain insights into the additional mechanism of NSAIDs, we reviewed their membrane interactivity to modify the physicochemical properties of membranes. Methodology: We retrieved scientific articles from PubMed/MEDLINE, Google Scholar and ACS Publications by searching databases from 1990 to 2019. Research papers published in English in the internationally recognized journals and on-line journals were cited with preference to more recent publications. Collected articles were reviewed by title, abstract and text for relevance. Results: Results of the literature search indicated that NSAIDs structure-specifically cause the in vitro and in vivo interactions with artificial and biological membranes to change membrane fluidity, lipid phase transition and permeability. The features and potencies of their membrane interactivity vary depending on drug concentration, medium pH and membrane lipid composition. In addition to membrane proteins, NSAIDs act on membrane lipids to exhibit the anti-inflammatory and anti-tumor activity by interacting with lipid bilayer membranes at relatively low concentrations to decrease membrane fluidity and thereby affect the enzymatic activity of membrane-associated proteins and to exhibit the gastrointestinal and cardiovascular toxicity by interacting with membranous phospholipids at relatively high concentrations to increase membrane fluidity and thereby impair the membrane-relevant biofunctions. Other diverse effects of NSAIDs may also be related to their membrane interactions. Conclusion: NSAIDs share the membrane interactivity common to them as one of possible pharmacological and toxicological mechanisms.            

scholarly journals Construction of a Convenient Screening Method for P-Hydroxybenzoate Hydroxylase To Enable Efficient Gallic Acid and Pyrogallol Biosynthesis

2021 ◽  
Author(s):  
Zhenya Chen ◽  
Tongtong Chen ◽  
Shengzhu Yu ◽  
Yi-Xin Huo
Keyword(s):  
Gallic Acid ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Biological Activities ◽  
Screening Method ◽  
Docking Simulation ◽  
Biosynthetic Pathways ◽  
E Coli ◽  
Hydroxybenzoate Hydroxylase

Abstract BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis of GA and pyrogallol has been emerged as an ecofriendly method to replace the traditional chemical synthesis. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high active PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM-1s-1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the mechanism explanation of the high activity. Expression of this mutant in E. coli BW25113 (F’) can generate 830 ± 33 mg/L GA from 1000 mg/L 3,4-DHBA. After that, we utilized this mutant to assemble a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (F’)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high active PobA mutant. Assembling this mutant into GA and pyrogallol biosynthetic pathways achieved the de novo production of these two compounds. Besides, this mutant has great potential for GA or pyrogallol derivatives production. The screening method could be used for other GA biosynthesis-related enzymes.

scholarly journals Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

2021 ◽  
Author(s):  
Kanade Tatsumi ◽  
Takukji Ichino ◽  
Natsumi Isaka ◽  
Akifumi Sugiyama ◽  
Yozo Okazaki ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Cell Death ◽  
Membrane Lipids ◽  
Saturated Fatty Acids ◽  
Membrane Lipid ◽  
Lithospermum Erythrorhizon ◽  
Cell Secretion ◽  
Oil Droplets

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. The mechanism of secretion remains largely unknown, because hydrophobic metabolites, which may form oil droplets or crystals in cytosol, inducing cell death, cannot be directly secreted by transporters. Moreover, some secondary metabolic lipids react with cytosolic components leading to their decomposition. Lipophilic metabolites should thus be solubilized by matrix lipids and compartmentalized by membrane lipids. The mechanism of lipophilic metabolite secretion was assessed using shikonin, a red naphthoquinone lipid, in Lithospermum erythrorhizon. Cell secretion of shikonin also involved the secretion of about 30% of triacylglycerol (TAG), composed predominantly of saturated fatty acids. Shikonin production was associated with the induction of large amounts of the membrane lipid phosphatidylcholine. Together with in vitro reconstitution, these findings suggest a novel role for TAG as a matrix lipid for the secretion of lipophilic metabolites.

scholarly journals The Antibacterial and Anti-biofilm Activities of Two Thiazolidione Derivatives (H2-60 and H2-81) Against Clinical Enterococcus Faecium Strains

2021 ◽  
Author(s):  
Zhong Chen ◽  
Yanpeng Xiong ◽  
Yuanyuan Tang ◽  
Yuxi Zhao ◽  
Junwen Chen ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Histidine Kinase ◽  
Enterococcus Faecium ◽  
Biofilm Formation ◽  
Confocal Laser ◽  
Competent Cell ◽  
E Coli ◽  
Viable Cells ◽  
Mic Value

Abstract Background: Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase (HK) YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the YycG recombinant proteins containing HisKA and HATPase_c domain of E. faecium DO were in vitro expressed in E. coli competent cell BL21 (DE3) and then purified for the autophosphorylation test, indicating these two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium.Results: The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium was in the range from 3.125mg/L to 25mg/L. Moreover, either H2-60 or H2-81showed the excellent bactericidal activity against E. faecium with the single dose or its combination with daptomycin (4 × MIC) by time-killing assay. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by two thiazolidione derivatives (H2-60 and H2-81) within 1/4×MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with its monotherapy (78.17% and 74.48%vs.41.59%, respectively, P< 0.01). Conclusion: Two thiazolidione derivatives (H2-60 and H2-81) exhibit the robust antibacterial and anti-biofilm activity against E. faecium by targeting the histidine kinase (HK) YycG.

scholarly journals IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways in Escherichia coli under aerobic growth conditions

2009 ◽  
Vol 420 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Guoqiang Tan ◽  
Jianxin Lu ◽  
Jacob P. Bitoun ◽  
Hao Huang ◽  
Huangen Ding
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Biosynthetic Pathway ◽  
Excision Repair ◽  
Growth Conditions ◽  
Cluster Assembly ◽  
Aerobic Growth ◽  
Aerobic Conditions ◽  
E Coli

IscA/SufA paralogues are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. Whereas deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogues blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthetic pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in enzyme ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.

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