scholarly journals Prototyping a valinomycin biosynthesis pathway within a cell-free transcription-translation (TX-TL) system

2016 ◽  
Author(s):  
Tiffany Zhou
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Model Organism ◽  
New Drugs ◽  
Test Case ◽  
Biosynthesis Pathway ◽  
Genetic Pathways ◽  
Anticancer Effects ◽  
A Cell

1AbstractMany natural metabolites have antibacterial, antiviral, or anticancer effects and can be developed into new drugs. However, working with the microorganisms that produce these products can be challenging since they are not as well characterized as a model organism like Escherichia coli. In this paper, we investigate the potential for a cell-free transcription-translation (TX-TL) system to provide a rapid prototyping platform for characterizing new genetic pathways. We use the valinomycin biosynthesis pathway as a test case, and we show successful heterologous expression of the heterodimeric valinomycin synthetase (VlmSyn, Vlm1: 374 kDa and Vlm2: 284 kDa) from Strep-tomyces tsusimaensis within the TX-TL system. Using LC-MS analysis, we find that valinomycin is produced at low but detectable levels, even when only one out of the three basic precursors is fed into the system. Our work represents another step towards applying cell-free biosynthesis to the discovery and characterization of new natural products.

scholarly journals Characterization of Polysaccharide Accumulations in a Cell Division Defective Mutant of Escherichia coli 15T-

Microbiology ◽  
1981 ◽  
Vol 123 (2) ◽  
pp. 323-333 ◽  
Author(s):  
J. M. SCHOEMAKER ◽  
J. M. CLARK ◽  
J. J. SAUKKONEN
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Defective Mutant ◽  
A Cell

scholarly journals Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in Streptomyces caniferus GUA-06-05-006A

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 259 ◽  
Author(s):  
Raúl García-Salcedo ◽  
Rubén Álvarez-Álvarez ◽  
Carlos Olano ◽  
Librada Cañedo ◽  
Alfredo Braña ◽  
...  
Keyword(s):  
Natural Products ◽  
Streptomyces Coelicolor ◽  
Transcriptional Unit ◽  
Constitutive Promoter ◽  
Biosynthesis Pathway ◽  
Marine Actinobacteria ◽  
Peptide Synthetase ◽  
Streptomyces Albus ◽  
Streptomyces Albus J1074

Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurally, JAs are formed by a modified β-methylphenylalanine residue, whose biosynthesis involves a methyltransferase that in Streptomyces hygroscopicus has been identified as MppJ. Up to date, three JA members (A–C) and a few other natural products containing β-methylphenylalanine have been discovered from soil-derived microorganisms. Herein, we report the identification of a gene (jomM) coding for a putative methyltransferase highly identical to MppJ in the chromosome of the marine actinobacteria Streptomyces caniferus GUA-06-05-006A. In its 5’ region, jomM clusters with two polyketide synthases (PKS) (jomP1, jomP2), a nonribosomal peptide synthetase (NRPS) (jomN) and a thioesterase gene (jomT), possibly conforming a single transcriptional unit. Insertion of a strong constitutive promoter upstream of jomP1 led to the detection of JA A, along with at least two novel JA family members (D and E). Independent inactivation of jomP1, jomN and jomM abolished production of JA A, JA D and JA E, indicating the involvement of these genes in JA biosynthesis. Heterologous expression of the JA biosynthesis cluster in Streptomyces coelicolor M1152 and in Streptomyces albus J1074 led to the production of JA A, B, C and F. We propose a pathway for JAs biosynthesis based on the findings here described.

scholarly journals Susceptibility of virulent and resistant Escherichia coli strains to non-polar and polar compounds identified in Microplumeria anomala

2020 ◽  
Vol 13 (7) ◽  
pp. 1376-1387
Author(s):  
Livia Roberta Piedade Camargo ◽  
Vania Maria de Carvalho ◽  
Ingrit Elida Collantes Díaz ◽  
Mateus Luís Barradas Paciencia ◽  
Sergio Alexandre Frana ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Protocatechuic Acid ◽  
Polar Compounds ◽  
New Drugs ◽  
Economic Losses ◽  
Isolation And Identification ◽  
E Coli ◽  
Nuclear Magnetic Resonance Spectrometry ◽  
Human Infections

Background and Aim: Escherichia coli is one of the main pathogens responsible for veterinary and human infections, and it is associated with significant economic losses in the livestock, as it causes severe diseases to humans, particularly in children. For that reason, there is a need for introducing new drugs to treat E. coli diseases. The Brazilian species richness is a source of potential new antibacterial natural products. The study aimed at the biological and chemical investigation of the organic extract obtained from the stem of Microplumeria anomala (Apocynaceae), EB127, as it was identified as a potential source of new antibacterial compounds to be used in Veterinary. Materials and Methods: The antibacterial activity was evaluated by disk diffusion and microdilution assays; chromatography, nuclear magnetic resonance spectrometry, and mass spectrometry were used in the isolation and identification of compounds. Results: EB127 showed activity against E. coli ATCC25922, and against three E. coli strains that were isolated from frigarte's cloaca, named 31/1A, 35A, and 51A. Lupeol, 3-acetyl-11-oxo-β-amyrin, 3-acetyl-11-oxo-α-amyrin, sitosterol, stigmasterol, 3β,7α-dihydroxy-cholest-5-ene, 3β-hydroxy-cholest-5-en-7-one, and 3β-hydroxy-cholest-5,22-dien-7-one were identified in fraction Hex/CHCl3, while loganin, loganic acid, methylanomaline, and anomaline were all identified in EB127 and protocatechuic acid hexoside, ferulic acid, secoxyloganin, feruloylquinic acid, vanillic acid hexoside, protocatechuic acid-4-O-β-hexoside, and rosmarinic acid were tentatively identified in fraction 10%ACN/H2O. E. coli 51A (virulent/non-resistant) showed sensitivity to the antibacterial action of fraction Hex/CHCl3 which contains alkaloids, triterpenes, and steroids, while E. coli 35A (resistant/non-virulent) were more susceptible to 10%ACN/H2O, which contains iridoids as loganin and loganic acid, and glycosylated and non-glycosylated caffeic acids. Conclusion: Fraction 10%ACN/H2O is of interest in pursuing new drugs to treat resistant E. coli, in veterinary. All compounds were isolated from the plant for the first time and have shown potential as new antibacterial natural products from Amazon plants to be used in veterinary and human diseases.

scholarly journals Characterization of SafC, a Catechol 4-O-Methyltransferase Involved in Saframycin Biosynthesis

2007 ◽  
Vol 73 (11) ◽  
pp. 3575-3580 ◽  
Author(s):  
James T. Nelson ◽  
Jaeheon Lee ◽  
James W. Sims ◽  
Eric W. Schmidt
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Caffeic Acid ◽  
Antitumor Agents ◽  
Myxococcus Xanthus ◽  
Clinical Development ◽  
Nonribosomal Peptide Synthetase ◽  
Biotechnological Potential ◽  
Peptide Synthetase

ABSTRACT Members of the saframycin/safracin/ecteinascidin family of peptide natural products are potent antitumor agents currently under clinical development. Saframycin MX1, from Myxococcus xanthus, is synthesized by a nonribosomal peptide synthetase, SafAB, and an O-methyltransferase, SafC, although other proteins are likely involved in the pathway. SafC was overexpressed in Escherichia coli, purified to homogeneity, and assayed for its ability to methylate a variety of substrates. SafC was able to catalyze the O-methylation of catechol derivatives but not phenols. Among the substrates tested, the best substrate for SafC was l-dihydroxyphenylalanine (l-dopa), which was methylated specifically in the 4′-O position (k cat/Km = 5.5 × 103 M−1 s−1). SafC displayed less activity on other catechol derivatives, including catechol, dopamine, and caffeic acid. The more labile l-5′-methyldopa was an extremely poor substrate for SafC (k cat/Km = ∼2.8 × 10−5 M−1 s−1). l-Dopa thioester derivatives were also much less reactive than l-dopa. These results indicate that SafC-catalyzed 4′-O-methylation of l-dopa occurs prior to 5′-C-methylation, suggesting that 4′-O-methylation is likely the first committed step in the biosynthesis of saframycin MX1. SafC has biotechnological potential as a methyltransferase with unique regioselectivity.

Characterization of Escherichia coli men Mutants Defective in Conversion of o -Succinylbenzoate to 1,4-Dihydroxy-2-Naphthoate

1982 ◽  
Vol 152 (3) ◽  
pp. 1132-1137
Author(s):  
Duncan J. Shaw ◽  
John R. Guest ◽  
Rangaswamy Meganathan ◽  
Ronald Bentley
Keyword(s):  
Escherichia Coli ◽  
Vitamin K ◽  
Gene Order ◽  
Cell Free Extract ◽  
E Coli ◽  
Mycobacterium Phlei ◽  
Form Part ◽  
A Cell

Four independent menaquinone (vitamin K 2 )-deficient mutants of Escherichia coli , blocked in the conversion of o -succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from Mycobacterium phlei . Mutants lacking DHNA synthase (and therefore complementing with M. phlei DHNA synthase) were designated menB , and the mutant lacking OSB-CoA synthetase (and therefore complementing with M. phlei OSB-CoA synthetase) was designated menE . The menB mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3- 14 C 2 ]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the menE mutant under these conditions. Experiments with strains lysogenized by a λ men transducing phage (λG68) and transduction studies with phage P1 indicated that the menB and menE genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the E. coli linkage map. Evidence was obtained for the clockwise gene order gyrA ....-B-D, where the asterisk denotes the uncertain position of menE relative to menC and menB . The transducing phage (λG68) contained functional menB, menC , and menE genes, but only part of the menD gene, and it was designated λ menC B(D) .

scholarly journals Isolation of PDX2, a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria

2001 ◽  
Vol 183 (11) ◽  
pp. 3383-3390 ◽  
Author(s):  
Marilyn Ehrenshaft ◽  
Margaret E. Daub
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Degenerate Primer ◽  
Biosynthesis Pathway ◽  
Protein Motifs ◽  
E Coli ◽  
Biosynthesis Gene ◽  
Targeted Gene Replacement

ABSTRACT In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to theE. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that containPDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

scholarly journals Medicinal plants - Therapeutic effective compounds

2018 ◽  
pp. 21-28
Author(s):  
Odontuya G
Keyword(s):  
Biological Sciences ◽  
Natural Products ◽  
Medicinal Plants ◽  
Raw Materials ◽  
Interdisciplinary Approach ◽  
New Drugs ◽  
Secondary Products ◽  
Active Compounds ◽  
Chemical Structures

In plants, substances starting with a simple skeleton to highly complex chemical structures having specific activities are synthesized. Plants are still considered a resource of valuable raw materials for producing different therapeutic and health supporting products, as well as a pool of significant pharmacological active compounds. According to the analysis of the recent PubMed research trends, it was observed that the number of scientific research interest targeting plant-derived natural products had increased. New approaches for discovery of medicinal plants, isolation, identification and characterization of secondary metabolites, and resupply of plant-derived natural products are being developed. Meanwhile, the usage of medicinal plants and plant-derived natural products for any disease conditions is being increased worldwide. There is a limited possibility to isolate therapeutically active compounds from natural-originated plants and resupply them in the market. Therefore, alternative resupply approaches are being developed that they rely on biotechnological products and chemical synthesis. Currently, the research on medicinal plants and plant-derived secondary products is going with success using a broad interdisciplinary approach of chemical and biological sciences involving advanced technologies. Medicinal plants are a source of therapeutically active compounds and new drugs, and other health supporting products. Эмийн ургамал - Эмчилгээний идэвхт нэгдэл Хураангуй: Ургамалд хамгийн энгийнээс эхлээд маш нарийн нийлмэл бүтэц байгууламжтай эмчилгээний идэвхт бодис нийлэгжиж байдаг ба ургамал нь эрүүл мэндийн бүтээгдэхүүний үнэт түүхий эд бөгөөд фармакологийн идэвхт нэгдлийн сан болж байна. Сүүлийн үеийн PubMed-д хэвлэгдсэн бүтээлүүдэд дүн шинжилгээ хийхэд ургамлын гаралтай байгалийн нэгдлийг шинжлэх ухааны үндэслэлтэйгээр сонирхон судлах явдал өссөн байна. Эмийн ургамлыг олж илрүүлэх, түүнээс хоѐрдогч метаболитыг ялгах, шинж чанарыг тодорхойлох, таньж илрүүлэх, ханган нийлүүлэх арга зүй, аргачлал боловсронгуй болж, аливаа өвчний эмчилгээнд ургамлыг хэрэглэх хандлага өсөж байна. Эмчилгээний идэвхтэй бодисыг зөвхөн байгалийн ургамлаас ялган зах зээлд ханган нийлүүлэх боломжгүй тул биотехнологийн арга болон химийн нийлэгжүүлэлт гэсэн өөр бусад аргыг хэрэглэж байна. Ийм чиглэлийн судалгааг хими, биологийн шинжлэх ухааны олон салбарын мэдлэг, мэдээллийн уялдаа холбоонд тулгуурлан орчин үеийн ололт, дэвшилтэт технологийг ашиглан хийж байна. Ургамал бол эмчилгээний идэвхт бодис ба шинэ эм, эрүүл мэндийн бүтээгдэхүүний эх сурвалж юм. Түлхүүр үгс: ургамал, хоёрдогч метаболит, эмчилгээний идэвх, дэвшилтэт технологи

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