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

Author(s):  
Zhenya Chen ◽  
Tongtong Chen ◽  
Shengzhu Yu ◽  
Yi-Xin Huo

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.

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.


2021 ◽  
Author(s):  
◽  
Madeleine Huber

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.


Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2766 ◽  
Author(s):  
Heba E. Hashem ◽  
Abd El-Galil E. Amr ◽  
Eman S. Nossier ◽  
Elsayed A. Elsayed ◽  
Eman M. Azmy

To develop new antimicrobial agents, a series of novel thiourea derivatives incorporated with different moieties 2–13 was designed and synthesized and their biological activities were evaluated. Compounds 7a, 7b and 8 exhibited excellent antimicrobial activity against all Gram-positive and Gram-negative bacteria, and the fungal Aspergillus flavus with minimum inhibitory concentration (MIC) values ranged from 0.95 ± 0.22 to 3.25 ± 1.00 μg/mL. Furthermore, cytotoxicity studies against MCF-7 cells revealed that compounds 7a and 7b were the most potent with IC50 values of 10.17 ± 0.65 and 11.59 ± 0.59 μM, respectively. On the other hand, the tested compounds were less toxic against normal kidney epithelial cell lines (Vero cells). The in vitro enzyme inhibition assay of 8 displayed excellent inhibitory activity against Escherichia coli DNA B gyrase and moderate one against E. coli Topoisomerase IV (IC50 = 0.33 ± 1.25 and 19.72 ± 1.00 µM, respectively) in comparison with novobiocin (IC50 values 0.28 ± 1.45 and 10.65 ± 1.02 µM, respectively). Finally, the molecular docking was done to position compound 8 into the E. coli DNA B and Topoisomerase IV active pockets to explore the probable binding conformation. In summary, compound 8 may serve as a potential dual E. coli DNA B and Topoisomerase IV inhibitor.


1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
J Vuopio-Varkila ◽  
G K Schoolnik

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.


2016 ◽  
Vol 87 (11) ◽  
pp. 1377-1386 ◽  
Author(s):  
Xihui Zhao ◽  
Qun Li ◽  
Xiaowen Li ◽  
Yanzhi Xia ◽  
Bing Wang ◽  
...  

Biopolymer nanocomposites containing metal nanoparticles have attracted much attention due to their excellent properties and broad applications. In this work, alginate fibers embedded with silver nanoparticles (AgNPs) were prepared. The as-obtained alginate-AgNP fibers exhibited antibacterial activity against both Gram microorganisms of model microbes Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). A growth kinetic study with S. aureus and E. coli displayed the inhibition of bacterial growth at the logarithmic phase. The cytotoxic effect of the fibers in human cervical cancer (HeLa) cells was assessed by cell counting kit-8 (CCK-8) assay and flow cytometry. The as-prepared alginate-AgNP fibers, particularly with high amount and long treatment time, showed high cell-killing efficiency. These findings emphasize that such alginate-AgNP fibers with multifaceted biological activities are a promising material for applications in the textile or biomedical fields.


2006 ◽  
Vol 188 (3) ◽  
pp. 909-918 ◽  
Author(s):  
Jianmin Zhong ◽  
Stephane Skouloubris ◽  
Qiyuan Dai ◽  
Hannu Myllykallio ◽  
Alan G. Barbour

ABSTRACT The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.


2021 ◽  
Vol 12 ◽  
Author(s):  
Kyung Taek Heo ◽  
Byeongsan Lee ◽  
Jae-Hyuk Jang ◽  
Jung-Oh Ahn ◽  
Young-Soo Hong

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.


2021 ◽  
Author(s):  
Yidi Liu ◽  
Xinlei Mao ◽  
Baoqi Zhang ◽  
Jinping Lin ◽  
Dongzhi Wei

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.


2020 ◽  
Vol 10 (5) ◽  
pp. 274-292
Author(s):  
Rohit Kumar ◽  
Sushil Kumar ◽  
Mohammad Asif Khan

Recently a series of Schiff bases of diphenylamine derivatives have been synthesized and evaluated in vitro for their antibacterial activity against pathogenic both Gram-positive bacteria B. subtitles and Gram-negative bacteria E. coli using ciprofloxacin as standard drug at conc. of 50 μg/ml and 100 μg/ml. Literature review revels that chalcones possesses various biological activities like antimicrobial, antiviral, anti-inflammatory, anticancer and sedative etc. Therefore the present study was designed on synthesis and pharmacological evaluation of 2-(4-(3 (Substituted Phenyl) Acryloyl) Phenoxy)-N, N Diphenylacetamides. Target compound was synthesized by reaction of chloroacetylchloride with diphenylamine to afford 2-chloro-N, N-diphenylacetamide which further by reaction with substituted Chalcones and characterized following recrystallization and evaluated for anti-microbial potential through cup-diffusion method. In results, the target compounds were tested for activity against B. Subtilis, E.Coli and C. albicans. The chalcones having the lipophilic 4-chloro group (RKCT2) showed the greatest antimicrobial activity (zone of inhibition 20 & 22 mm against. B. subtilis, E. Coli, C. Albicans respectively. It suggests further researchers to go through anti-microbial evaluations against a more varieties of bacteria and fungi. Keywords: Schiff bases of diphenylamine derivatives, antibacterial activity, Gram-positive bacteria, 2-(4-(3 (Substituted Phenyl) Acryloyl) Phenoxy)-N, N Diphenylacetamides


Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 730
Author(s):  
Nicolás Gómez-Sequeda ◽  
Marlon Cáceres ◽  
Elena E. Stashenko ◽  
William Hidalgo ◽  
Claudia Ortiz

The emergence of multidrug resistant microorganisms represents a global challenge due to the lack of new effective antimicrobial agents. In this sense, essential oils (EOs) are an alternative to be considered because of their anti-inflammatory, antiviral, antibacterial, and antibiofilm biological activities. Therefore, multiple efforts have been made to consider the potential use of EOs in the treatment of infections which are caused by resistant microorganisms. In this study, 15 EOs of both Colombian and introduced aromatic plants were evaluated against pathogenic strains of E. coli O157:H7 and methicillin resistant Staphylococcus aureus (MRSA) in planktonic and sessile states in order to identify relevant and promising alternatives for the treatment of microbial infections. Forty different compounds were identified in the 15 EO with nine of them constituted mainly by oxygenated monoterpenes (OM). EOs from Lippia origanoides, chemotypes thymol, and carvacrol, displayed the highest antibacterial activity against E. coli O157:H7 (MIC50 = 0.9 and 0.3 mg/mL, respectively) and MRSA (MIC50 = 1.2 and 0.6 mg/mL, respectively). These compounds from EOs had also the highest antibiofilm activity (inhibition percentage > 70.3%). Using scanning electron microscopy (SEM), changes in the size and morphology of both bacteria were observed when they were exposed to sub-inhibitory concentrations of L. origanoides EO carvacrol chemotype. EOs from L. origanoides, thymol, and carvacrol chemotypes represented a viable alternative for the treatment of microbial infections; however, the Selectivity Index (SI ≤ 3) indicated that it was necessary to study alternatives to reduce its in vitro cytotoxicity.


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