scholarly journals Production of 7-O-Methyl Aromadendrin, a Medicinally Valuable Flavonoid, in Escherichia coli

2011 ◽  
Vol 78 (3) ◽  
pp. 684-694 ◽  
Author(s):  
Sailesh Malla ◽  
Mattheos A. G. Koffas ◽  
Romas J. Kazlauskas ◽  
Byung-Gee Kim
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Avermitilis ◽  
Flavonoid Glycosides ◽  
Petroselinum Crispum ◽  
Cell Factory ◽  
Coumaric Acid ◽  
Content Type ◽  
E Coli ◽  
Coa Ligase ◽  
Malonyl Coa

ABSTRACT7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such asPopulus albaandEucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, anEscherichia colicell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor,p-coumaric acid, inE. colifor the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feedingp-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate–coenzyme A (CoA) ligase fromPetroselinum crispum, chalcone synthase fromPetunia hybrida, and chalcone isomerase fromMedicago sativa.In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890andnfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) fromNocardia farcinicawere also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase fromArabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase fromStreptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction inE. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μMp-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.

scholarly journals Efficient Synthesis of Eriodictyol from l-Tyrosine in Escherichia coli

2014 ◽  
Vol 80 (10) ◽  
pp. 3072-3080 ◽  
Author(s):  
Saijie Zhu ◽  
Junjun Wu ◽  
Guocheng Du ◽  
Jingwen Zhou ◽  
Jian Chen
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Antioxidant Activities ◽  
Efficient Synthesis ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
Coa Ligase ◽  
Malonyl Coa ◽  
Two Factors

ABSTRACTThe health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration inEscherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3′-hydroxylase (tF3′H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) inE. coli. This allowed the engineeredE. colito produce eriodictyol froml-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol froml-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids froml-tyrosine or even glucose inE. coli.

scholarly journals Biosynthesis of fraxetin from three different substrates using engineered Escherichia coli

2020 ◽  
Vol 63 (1) ◽  
Author(s):  
Seung Hoon An ◽  
Gyu-Sik Choi ◽  
Joong-Hoon Ahn
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Iron Homeostasis ◽  
Antibacterial Activities ◽  
Enzymatic Reactions ◽  
Biosynthesis Pathway ◽  
Coumaric Acid ◽  
E Coli ◽  
Coa Ligase ◽  
Key Enzyme

Abstract Fraxetin, which is a simple coumarin, is a phytochemical present in medicinal plants, such as Fraxinus rhynchophylla, and Cortex Fraxini. In plants, it serves as a controller of iron homeostasis. The health-enhancing activities of fraxetin, such as anticancer, neuroprotective and antibacterial activities, are known. Scopoletin 8-hydroxylase (S8H) is a key enzyme involved in the synthesis of fraxetin from scopoletin. Scopoletin can be synthesized either from esculetin by O-methylation or from ferulic acid by feruloyl CoA 6′-hydroxylase (F6′H) and 4-coumaric acid CoA ligase (4CL). To enable fraxetin synthesis, the fraxetin biosynthesis pathway was introduced into Escherichia coli. Three distinct routes, from ferulic acid, esculetin, and scopoletin, were designed for the synthesis of fraxetin. In the first approach, E. coli strain harboring S8H was used and found to synthesize 84.8 μM fraxetin from 100 μM scopoletin. Two E. coli strains were used for the other two approaches because these approaches required at least two enzymatic reactions. Through this approach, 41.4 μM fraxetin was synthesized from 100 μM esculetin, while 33.3 μM fraxetin was synthesized from 100 μM ferulic acid.

scholarly journals Boosting Secretion of Extracellular Protein byEscherichia colivia Cell Wall Perturbation

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Haiquan Yang ◽  
Xiao Lu ◽  
Jinyuan Hu ◽  
Yuan Chen ◽  
Wei Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Protein Secretion ◽  
Extracellular Protein ◽  
Deletion Mutants ◽  
Cell Factory ◽  
Content Type ◽  
E Coli ◽  
Cell Wall Peptidoglycan ◽  
Extracellular Secretion

ABSTRACTEscherichia coliis one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced inE. coliby deleting twod,d-carboxypeptidase genes (dacAanddacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion ofdacAanddacBenhanced the accumulation of intracellular soluble peptidoglycan inE. coliand affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion ofdacAanddacBappears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacApETDuet-amykand BL21 ΔdacBpETDuet-amykwas increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion ofdacAanddacBincreased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCECell surface structure stabilization is important for extracellular secretion of proteins inEscherichia coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deletingdacAanddacBgenes encodingd,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity ofE. colias a microbial cell factory for extracellular secretion of proteins and chemicals.

scholarly journals Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin

2014 ◽  
Vol 81 (3) ◽  
pp. 840-849 ◽  
Author(s):  
Philana Veronica van Summeren-Wesenhagen ◽  
Jan Marienhagen
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Heart Diseases ◽  
Stilbene Synthase ◽  
Content Type ◽  
E Coli ◽  
Malonyl Coa ◽  
Pine Trees ◽  
Health Promoting

ABSTRACTPlant polyphenols are of great interest for drug discovery and drug development since many of these compounds have health-promoting activities as treatments against various diseases, such as diabetes, cancer, or heart diseases. However, the limited availability of polyphenols represents a major obstacle to clinical applications that must be overcome. In comparison to the quantities of these compounds obtained by isolation from natural sources or costly chemical synthesis, the microbial production of these compounds could provide sufficient quantities from inexpensive substrates. In this work, we describe the development of anEscherichia coliplatform strain for the production of pinosylvin, a stilbene found in the heartwood of pine trees which could aid in the treatment of various cancers and cardiovascular diseases. Initially, several configurations of the three-step biosynthetic pathway to pinosylvin were constructed from a set of two different enzymes for each enzymatic step. After optimization of gene expression and evaluation of different construct environments, low pinosylvin concentrations up to 3 mg/liter could be detected. Analysis of the precursor supply and a comparative analysis of the intracellular pools of pathway intermediates and product identified the limited malonyl coenzyme A (malonyl-CoA) availability and low stilbene synthase activity in the heterologous host to be the main bottlenecks during pinosylvin production. Addition of cerulenin for increasing intracellular malonyl-CoA pools and thein vivoevolution of the stilbene synthase fromPinus strobusfor improved activity inE. coliproved to be the keys to elevated product titers. These measures allowed product titers of 70 mg/liter pinosylvin from glucose, which could be further increased to 91 mg/liter by the addition ofl-phenylalanine.

scholarly journals Production of a Novel Quercetin Glycoside through Metabolic Engineering of Escherichia coli

2012 ◽  
Vol 78 (12) ◽  
pp. 4256-4262 ◽  
Author(s):  
Jeong-A Yoon ◽  
Bong-Gyu Kim ◽  
Woo Ju Lee ◽  
Yoongho Lim ◽  
Youhoon Chong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Flavonoid Glycoside ◽  
Flavonoid Glycosides ◽  
Nucleotide Sugar ◽  
Content Type ◽  
E Coli ◽  
Naturally Occurring ◽  
Quercetin Glycoside ◽  
By Products

ABSTRACTMost flavonoids exist as sugar conjugates. Naturally occurring flavonoid sugar conjugates include glucose, galactose, glucuronide, rhamnose, xylose, and arabinose. These flavonoid glycosides have diverse physiological activities, depending on the type of sugar attached. To synthesize an unnatural flavonoid glycoside,Actinobacillus actinomycetemcomitansgenetll(encoding dTDP-6-deoxy-l-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-l-mannose to dTDP-6-deoxytalose) was introduced intoEscherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases (UGTs) were screened to find a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain ofE. coliwith quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-deoxytalose) by increasing the supplement of dTDP-6-deoxytalose inE. coli, we engineered nucleotide biosynthetic genes ofE. coli, such asgalU(UTP-glucose 1-phosphate uridyltransferase),rffA(dTDP-4-oxo-6-deoxy-d-glucose transaminase), and/orrfbD(dTDP-4-dehydrorahmnose reductase). The engineeredE. colistrain produced approximately 98 mg of quercetin 3-O-(6-deoxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose and quercetin 3-O-rhamnose, were also significantly reduced.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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