scholarly journals Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

2018 ◽  
Author(s):  
Jong-won Lee ◽  
Narayan P. Niraula ◽  
Cong T. Trinh
Keyword(s):  
Fatty Acid ◽  
Docking Simulation ◽  
Redox System ◽  
Acid Decarboxylase ◽  
Microbial Conversion ◽  
E Coli ◽  
Terminal Alkene ◽  
Terminal Alkenes ◽  
Alkene Production

ABSTRACTAlkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased ∼2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

scholarly journals Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

2015 ◽  
Author(s):  
Binbin Chen ◽  
Dong-Yup Lee ◽  
Matthew Wook Chang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Industrial Applications ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Alkene ◽  
Terminal Alkenes ◽  
One Step ◽  
Combinatorial Engineering ◽  
Culturing Conditions ◽  
Alkene Production

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

scholarly journals Regioselective synthesis, characterization and antimicrobial evaluation of amide-ether linked 1,4-disubstituted 1,2,3-triazoles

2017 ◽  
Vol 82 (9) ◽  
pp. 995-1007 ◽  
Author(s):  
Chander Kaushik ◽  
Krishan Kumar ◽  
Devinder Kumar ◽  
Satbir Mor ◽  
Ashwani Kumar ◽  
...  
Keyword(s):  
Click Reaction ◽  
Docking Simulation ◽  
Regioselective Synthesis ◽  
Spectral Techniques ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Aspergillus Brasiliensis ◽  
Antimicrobial Evaluation ◽  
Ft Ir

Regioselective synthesis of some amide?ether-linked 1,4-disubstituted 1,2,3-triazoles was realized via the copper(I)-catalyzed click reaction of 1-(prop-2-ynyloxy)naphthalene, 2-(prop-2-ynyloxy)naphthalene and 1,4-bis-(prop-2-ynyloxy)benzene with 2-azido-N-substituted acetamides. The synthesized compounds were characterized by spectral techniques viz. FT-IR, 1H- -NMR, 13C-NMR, HRMS and evaluated for their in vitro antimicrobial activity against Bacillus subtilis, Staphylococcus aureus (Gram-positive bacteria), Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria), Candida albicans and Aspergillus brasiliensis (fungi). Among the synthesized 1,4-disubstituted 1,2,3-triazoles, compound 13d displayed excellent antibacterial potential, while, compounds 7d and 13d appeared as potent fungicidal agents against the tested microbial strains. The docking simulation of the broad spectrum microbicidal disubstituted 1,2,3-triazole 13d into the active site of E. coli type II topoisomerase, DNA gyrase B enzyme was also investigated.

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 Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis

2016 ◽  
Vol 29 (6) ◽  
pp. 458-467 ◽  
Author(s):  
Ya-Hui Mao ◽  
Feng Li ◽  
Jin-Cheng Ma ◽  
Zhe Hu ◽  
Hai-Hong Wang
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Sinorhizobium Meliloti ◽  
Acyl Carrier Protein ◽  
Sequence Similarity ◽  
Acid Synthesis ◽  
In Vitro Assays ◽  
Temperature Sensitive ◽  
E Coli

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

scholarly journals The commercial antimicrobial triclosan induces high levels of antibiotic tolerance in vitro and reduces antibiotic efficacy up to 100-fold in vivo

2016 ◽  
Author(s):  
Corey Westfall ◽  
Ana Lidia Flores-Mireles ◽  
John Isaac Robinson ◽  
Aaron J.L. Lynch ◽  
Scott Hultgren ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Consumer Products ◽  
Infection Model ◽  
Antibiotic Tolerance ◽  
E Coli ◽  
Wide Range ◽  
Prophylactic Use

AbstractThe antimicrobial triclosan is used in a wide range of consumer products ranging from toothpaste, cleansers, socks, and baby toys. A bacteriostatic inhibitor of fatty acid synthesis, triclosan is extremely stable and accumulates in the environment. Approximately 75% of adults in the US have detectable levels of the compound in their urine, with a sizeable fraction of individuals (>10%) having urine concentrations equal to or greater than the minimal inhibitory concentration for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Previous work has identified connections between defects in fatty acid synthesis and accumulation of the alarmone guanosine tetraphosphate (ppGpp), which has been repeatedly associated with antibiotic tolerance and persistence. Based on these data, we hypothesized that triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics. Here we report that clinically relevant concentrations of triclosan increased E. coli and MRSA tolerance to bactericidal antibiotics as much as 10,000 fold in vitro and reduced antibiotic efficacy up to 100-fold in a mouse urinary tract infection model. Genetic analysis indicated that triclosan-mediated antibiotic tolerance requires ppGpp synthesis, but is independent of growth. These data highlight an unexpected and certainly unintended consequence of adding high concentrations of antimicrobials in consumer products, supporting an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.ImportanceAdded as a prophylactic to a wide range of consumer products, the fatty acid synthesis inhibitor triclosan accumulates to high levels in humans and the environment. Based on links between defects in fatty acid synthesis and accumulation of the alarmone ppGpp, we hypothesized that triclosan would render cells tolerant to bactericidal compounds due to ppGpp-mediated inhibition of biosynthetic capacity. Our data indicate that clinically relevant concentrations of triclosan induces higher tolerance of E. coli and methicillin resistant S. aureus (MRSA) to a panel of bactericidal antibiotics up to 10,000-fold. In a urinary tract infection model, mice exposed to triclosan exhibited bacterial loads ~100-fold higher in the bladder than control animals following ciprofloxacin challenge. These findings highlight an unexpected consequence of antimicrobials in consumer products and support an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.

Photobiocatalytic decarboxylation for olefin synthesis

2015 ◽  
Vol 51 (10) ◽  
pp. 1918-1921 ◽  
Author(s):  
Ioannis Zachos ◽  
Sarah Katharina Gaßmeyer ◽  
Daniel Bauer ◽  
Volker Sieber ◽  
Frank Hollmann ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Fatty Acids ◽  
Fatty Acid ◽  
High Selectivity ◽  
Acid Decarboxylase ◽  
Oxidative Decarboxylation ◽  
Efficient System ◽  
Olefin Synthesis ◽  
Terminal Alkenes

The oxidative decarboxylation of fatty acids to terminal alkenes was accomplished with high selectivity by combining a fatty acid decarboxylase OleTJE with the light-catalyzed generation of the cosubstrate hydrogen peroxide, resulting in a simple and efficient system for the light-driven cleavage of C–C bonds.

scholarly journals Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Prabhakar Singh ◽  
Rajesh Kumar Kesharwani ◽  
Krishna Misra ◽  
Syed Ibrahim Rizvi
Keyword(s):  
Plasma Membrane ◽  
Curcuma Longa ◽  
Docking Simulation ◽  
Redox System ◽  
Redox Status ◽  
Dependent Manner ◽  
Plasma Membrane Redox System ◽  
Plasma Membrane Redox

Plasma membrane redox system (PMRS) is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated fromCurcuma longa,has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar ratsin vitroandin vivoand validated through anin silicodocking simulation study using Molegro Virtual Docker (MVD). Effects of curcumin were also evaluated on level of glutathione (GSH) and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP). Results show that curcumin significantly (p<0.01) downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochromeb5reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP) of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects.

scholarly journals Protective Effect of Theaflavin on Erythrocytes Subjected toIn VitroOxidative Stress

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Mahejabeen Fatima ◽  
Rajesh Kumar Kesharwani ◽  
Krishna Misra ◽  
Syed Ibrahim Rizvi
Keyword(s):  
Oxidative Stress ◽  
Radical Scavenging ◽  
Docking Simulation ◽  
Black Tea ◽  
Redox System ◽  
Epidemiological Studies ◽  
Protective Role ◽  
Protective Mechanism ◽  
Molegro Virtual Docker

Antioxidant and free radical scavenging effect of black tea theaflavins has been shown in many epidemiological studies. In the present work we report the protective mechanism of tea theaflavins on biomarkers of oxidative stress, which are elevated during stress conditions. We hereby report thein vitroeffect of theaflavins on erythrocyte malondialdehyde (MDA), intracellular reduced glutathione (GSH), and plasma membrane redox system (PMRS) of rats. The effect of theaflavin on PMRS has also been validated through anin silicodocking simulation study using Molegro Virtual Docker (MVD). We report that theaflavins show significant protection to erythrocyte against oxidative stress induced bytert-butyl hydroperoxide (t-BHP). The findings suggest a possible protective role of theaflavins as antioxidant.

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