scholarly journals Cloning and expression of the acyl thioesterase 2 (ALT2) gene in Escherichia coli for producing short- and medium-chain 2-methylketones

2015 ◽  
Vol 18 (2) ◽  
pp. 87-98
Author(s):  
Vy Le Uyen Khuat ◽  
Thao Phuong Do ◽  
Thuong Thi Hong Nguyen
Keyword(s):  
Escherichia Coli ◽  
Food Industry ◽  
Biosynthetic Pathway ◽  
Biodiesel Production ◽  
Cloning And Expression ◽  
Melting Points ◽  
E Coli ◽  
Flame Ionization ◽  
Fatty Acid Biosynthetic Pathway ◽  
Natural Insecticides

2-Methylketones are organic compounds that are more widely used in the food industry and cosmetics. Some of methylketones found in plants act as pheromones and natural insecticides. Recently, methylketones have been recognized as strong candidates for the production of renewable energy as they possess not only favourable cetane numbers but also lower hydrophilicity and melting points than fatty acids which have been used in biodiesel production. In addition, short chain methylketones have much lower melting points than long chain methylketones. In this study, we cloned and expressed in Escherichia coli C41(DE3) cells a gene for acyl thioesterase 2 (ALT2) isolated from Arabidopsis thaliana. The ALT2 enzyme could hydrolyze 3-ketoacyl-ACP (also called β-ketoacyl-ACP) intermediates in the fatty acid biosynthetic pathway into the corresponding 3-ketoacid. The resulting 3- ketoacids are unstable compounds that will be decarboxylated to produce n-1 methylketones. Methylketones released in the growth medium of E. coli expressing ALT2 were extracted by hexane and analyzed by gas chromatography with the flame ionization detector (GC-FID). The results showed that the E. coli C41(DE3) cells expressing ALT2 recombinantly produced 2-nonanone (9C), 2- undecanone (11C) and 2-tridecanone (13C) in the spent medium when were induced with 0.25 mM IPTG at 37 oC for 3.5 hours.

scholarly journals Engineering Escherichia coli FAB system using synthetic plant genes for the production of long chain fatty acids

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Elias Kassab ◽  
Monika Fuchs ◽  
Martina Haack ◽  
Norbert Mehlmer ◽  
Thomas B. Brueck
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Production System ◽  
Biosynthetic Pathway ◽  
Long Chain Fatty Acids ◽  
E Coli ◽  
Fatty Acid Biosynthetic Pathway ◽  
Long Chain ◽  
Chain Fatty Acids

Abstract Background Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. Results In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. Conclusion The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.

scholarly journals Cloning and expression of \(\textit{ pigI}\) gene in \(\textit{Escherichia coli}\) BL21 (DE3)

2021 ◽  
Vol 43 (3) ◽  
pp. 59-67
Author(s):  
Do Minh Trung ◽  
Do Hai Quynh ◽  
Nguyen Thuy Duong
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Biosynthetic Pathway ◽  
Condensation Reaction ◽  
Potential Method ◽  
Antimicrobial Activities ◽  
Cloning And Expression ◽  
E Coli ◽  
Recombinant Vector ◽  
His Tag

Prodigiosin (Pg), a secondary metabolite with anticancer and antimicrobial activities, can be produced in Serratia marcescens bacteria through the condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Among these, the MBC synthetic pathway is started by the conversion of L-proline to L-proline-AMP before this complex is covalently attached to PigG. This reaction is catalyzed by an L-prolyl-AMP ligase named PigI. Therefore, PigI protein plays an important role in the prodigiosin biosynthetic pathway. However, studies related to PigI protein have not been carried out in Vietnam yet. In this work, the pigI gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Sequence alignment results revealed that the obtained pigI gene is 99.7% identical to the four strains, CP027798, CP027796, CP021984 and CP003959. This recombinant vector pJET1.2/pigI was used to reamplify pigI, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing the recombinant vector pET22b/pigI was expressed in an auto-induced medium. The presence of PigI protein in the lysate was identified due to a 53 kDa band through Western Blot analysis using an anti-his-tag antibody. The results of our study provide a potential method for producing prodigiosin from recombinant protein in Vietnam.

scholarly journals Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5391-5397 ◽  
Author(s):  
Si Jae Park ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Biosynthetic Pathway ◽  
Pha Synthase ◽  
Enzymatic Analysis ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Enoyl Coa Hydratase

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

scholarly journals Escherichia coli as a genetic tool

1985 ◽  
Vol 95 (3) ◽  
pp. 611-618
Author(s):  
Naomi Datta
Keyword(s):  
Escherichia Coli ◽  
Academic Research ◽  
Genetically Engineered ◽  
Cloning And Expression ◽  
Biological Research ◽  
New Techniques ◽  
E Coli ◽  
Genetic Tool ◽  
The Past ◽  
Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

scholarly journals Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

2019 ◽  
Vol 476 (21) ◽  
pp. 3125-3139 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Jeannine Hess ◽  
Elen Shaw ◽  
Christina Spry ◽  
Robert Starley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Structural Biology ◽  
Ion Mobility ◽  
Biosynthetic Pathway ◽  
Screening Tool ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Structural Insights

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

scholarly journals Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryo Yoshida ◽  
Hisashi Hemmi
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Membrane Lipids ◽  
Extreme Environments ◽  
Halophilic Archaea ◽  
Coli Strain ◽  
Glycerol Moiety ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix

Abstract Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli

1991 ◽  
Vol 69 (9) ◽  
pp. 670-673
Author(s):  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Cloning And Expression ◽  
Dependent Manner ◽  
Bacteriophage Λ ◽  
Expression Library ◽  
Expression Plasmid ◽  
E Coli ◽  
Dose Dependent

A rat liver bacteriophage λ expression library was probed using polyclonal antibodies raised to purified rat liver D-β-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression.

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

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