scholarly journals Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120

2008 ◽  
Vol 190 (18) ◽  
pp. 6084-6096 ◽  
Author(s):  
Sean A. Agger ◽  
Fernando Lopez-Gallego ◽  
Thomas R. Hoye ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Functional Expression ◽  
Terpene Synthase ◽  
Putative Hybrid ◽  
Nostoc Punctiforme ◽  
Terpene Synthases ◽  
E Coli ◽  
Reductase Gene ◽  
Pcc 7120 ◽  
Defense Compound

ABSTRACT Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli

Toxicon ◽  
2003 ◽  
Vol 41 (6) ◽  
pp. 713-721 ◽  
Author(s):  
Wen-Li Yang ◽  
Li-Sheng Peng ◽  
Xiao-Fen Zhong ◽  
Jian-Wen Wei ◽  
Xiao-Yu Jiang ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Soluble Protein ◽  
Functional Expression ◽  
Sea Snake ◽  
E Coli

Isolation and characterization of β-cadinene synthase cDNA from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2012 ◽  
Vol 66 (5) ◽  
pp. 569-576 ◽  
Author(s):  
Pei-Min Kuo ◽  
Keng-Hao Hsu ◽  
Yi-Ru Lee ◽  
Fang-Hua Chu ◽  
Sheng-Yang Wang
Keyword(s):  
Functional Expression ◽  
Major Product ◽  
Gas Chromatography Mass Spectrometry ◽  
Synthesis Mechanism ◽  
E Coli ◽  
Isolation And Characterization ◽  
Ms Analysis ◽  
Chamaecyparis Formosensis ◽  
Rapid Amplification

Abstract Chamaecyparis formosensis is a precious conifer endemic in Taiwan. To understand the sesquiterpene synthesis mechanism in this tree, full-length cDNA of a putative sesquiterpene synthase (sesqui-TPS), designated Cf-Cad, was obtained by rapid amplification of complementary DNA ends-polymerase chain reaction. Cf-Cad is 1812 bp in length. To identify its function, recombinant protein from Escherichia coli was incubated with farnesyl diphosphate, which produced one major product, the structure of which was elucidated by gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS analysis, GC retention time and MS matching with authentic standards revealed that the major product was β-cadinene. This is the first report of the cloning, functional expression in E. coli and identification of a sesqui-TPS from a Cupressaceae conifer.

scholarly journals Cloning and Characterization of the Gene EncodingPasteurella haemolytica FnrP, a Regulator of theEscherichia coli Silent Hemolysin SheA

1999 ◽  
Vol 181 (12) ◽  
pp. 3845-3848 ◽  
Author(s):  
Gaylen A. Uhlich ◽  
Peter J. McNamara ◽  
John J. Iandolo ◽  
Derek A. Mosier
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Transcriptional Regulator ◽  
Pasteurella Haemolytica ◽  
Anaerobic Growth ◽  
Null Mutant ◽  
E Coli ◽  
Sequence Identity ◽  
Reductase Gene

ABSTRACT A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in hostEscherichia coli cells. The gene, designatedfnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of afnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene,frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli ΔsheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.

Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pestHelicoverpa armigera

2017 ◽  
Vol 108 (1) ◽  
pp. 77-83 ◽  
Author(s):  
D. Liu ◽  
K. Tian ◽  
Y. Yuan ◽  
M. Li ◽  
M. Zheng ◽  
...  
Keyword(s):  
Pyrethroid Resistance ◽  
Functional Characterization ◽  
Kinetic Studies ◽  
Functional Expression ◽  
Cytochrome P450 Reductase ◽  
E Coli ◽  
Important Pest ◽  
Activity Comparison

AbstractCytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12andCYP9A17) from the polyphagous pestHelicoverpa armigerain the facileEscherichia coli. The functionality ofE. coliproduced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed inE. coliwith a content of about 1.0–1.5 nmol mg−1protein in membrane preparations.In vitroassays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatestkcat/Kmvalue for MROD, and CYP9A17 for EROD, while the lowestkcat/Kmvalues for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

scholarly journals Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

2015 ◽  
Vol 112 (18) ◽  
pp. 5661-5666 ◽  
Author(s):  
Jeng-Yeong Chow ◽  
Bo-Xue Tian ◽  
Gurusankar Ramamoorthy ◽  
Brandan S. Hillerich ◽  
Ronald D. Seidel ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Model ◽  
Streptomyces Clavuligerus ◽  
Major Product ◽  
Terpene Synthase ◽  
Terpene Synthases ◽  
Guided Discovery ◽  
Functional Assignment ◽  
Reaction Channels

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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