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Laijun Xing - One of the best experts on this subject based on the ideXlab platform.
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Effects of low temperature and exogenous unsaturated Fatty Acids on Fatty Acid Desaturase gene expression of Mortierella alpina ATCC 16266
Wei sheng wu xue bao = Acta microbiologica Sinica, 2012Co-Authors: Tonglei Shi, Biao Zhang, Laijun XingAbstract:OBJECTIVE In order to investigate the regulatory mechanisms of Mortierella alpina Desaturase genes by low temperature and exogenous unsaturated Fatty Acids (UFAs) at the transcriptional level. METHODS We performed time-course studies of Fatty Acid Desaturase gene expression by real-time PCR and determined Fatty Acid Desaturase gene promoter activity using promoter-reporter constructs. RESULTS Relative expression results in real-time PCR showed that the mRNA levels of three Fatty Acid Desaturase genes (Fad6, Fad12 and Fad3) were rapidly and transiently enhanced after 1 h of shifting to low temperature, in contrast, high concentration of exogenous oleic Acid (OA) which was a monounsaturated Fatty Acid containing a D9 double bond suppressed the transcription of these genes and the transcriptional response appeared to be rapid and transient. Also, there was no absolute correlation between mRNA abundance and production of corresponding Fatty Acids. The pFAD6 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by addition of UFAs to the media, and alpha-linolenic Acid (ALA) containing three double bonds appeared to have a more effective inhibition than linoleic Acid (LA) and OA. CONCLUSION These results indicate that there may be post-transcriptional control and other modes of regulation of UFAs synthesis in M. alpina when facing different stimuli such as low temperature and exogenous unsaturated Fatty Acids besides the regulation in the transcription of Fatty Acid Desaturase genes at the initial stage. Also, there may be an unknown end-product (changes in Fatty Acid compositions) feedback regulation in the transcription of Fatty Acid Desaturase genes to maintain cellular UFAs' homeostasis. In a word, we assessed mechanisms of transcriptional regulation in M. alpina Fatty Acid Desaturase gene expression for the first time and we wish to make it possible to obtain a better understanding of the mechanisms and get some theoretical knowledge to offer some guidance to the industrial production of UFAs by transgenic technology and microbial fermentation technology.
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Cloning of delta8-Fatty Acid Desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2010Co-Authors: Dongsheng Wei, Xiangdong Yang, Dongquan Guo, Xueyan Yian, Laijun XingAbstract:Delta8 Desaturase pathway, different from common delta6 Desaturase pathway, is an alternate pathway of polyunsaturated Fatty Acids biosynthesis. Delta8-Fatty Acid Desaturase is one of the key enzymes in delta8 Desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-Fatty Acid Desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino Acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-Fatty Acid Desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic Acid and omega3-eicosatrienoic Acid for the expression of delta8-Fatty Acid Desaturase gene. The results indicated that high level expressed As-Fatty Acid Desaturase could convert omega6-eicosadienoic Acid and omega3-eicosatrienoic Acid to dihomo-gamma-linolenic Acid and eicosatetraenoic Acid with substrate conversion ratio 31.2% and 46.3%, respectively.
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Cloning and expression in Saccharomyces cerevisiae of delta5-Fatty Acid Desaturase gene from Phaeodactylum tricornutum
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2009Co-Authors: Zhe Yang, Dongsheng Wei, Laijun XingAbstract:Delta5-Fatty Acid Desaturase is the key enzyme in synthesis of arachidonic Acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-Fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic Acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic Acid. The results indicated that high level expression of delta5-Fatty Acid Desaturase, and the substrate conversion reached 45.9%.
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Evolution-related amino Acids play important role in determining regioselectivity of Fatty Acid Desaturase from Pichia pastoris.
Molecular biology reports, 2008Co-Authors: Xinxin Zhang, Dongsheng Wei, Laijun XingAbstract:ω3-Fatty Acid Desaturase and Δ12-Fatty Acid Desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω3-Fatty Acid Desaturase were replaced with corresponding region of Δ12-Fatty Acid Desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino Acids of ω3-Fatty Acid Desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of Fatty Acid Desaturases may be better understood by investigating the evolutionary relationships of different Fatty Acid Desaturases.
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Identification and characterization of a novel yeast omega3-Fatty Acid Desaturase acting on long-chain n-6 Fatty Acid substrates from Pichia pastoris.
Yeast (Chichester England), 2008Co-Authors: Xinxin Zhang, Dongsheng Wei, Laijun XingAbstract:A cDNA sequence putatively encoding a omega(3)-Fatty Acid Desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino Acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-Fatty Acid Desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino Acid sequence exhibited omega(3)-Fatty Acid Desaturase activity. The newly identified omega(3)-Fatty Acid Desaturase, named Pp-FAD3, is novel because it showed broad n-6 Fatty Acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 Fatty Acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-Fatty Acid Desaturase to act on long-chain n-6 Fatty Acid substrates. Heterologous expression of the newly identified omega(3) Desaturase in different hosts will be an alternative method to increase the flow of n-6 Fatty Acid intermediates into their n-3 derivatives.
Dongsheng Wei - One of the best experts on this subject based on the ideXlab platform.
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Cloning of delta8-Fatty Acid Desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2010Co-Authors: Dongsheng Wei, Xiangdong Yang, Dongquan Guo, Xueyan Yian, Laijun XingAbstract:Delta8 Desaturase pathway, different from common delta6 Desaturase pathway, is an alternate pathway of polyunsaturated Fatty Acids biosynthesis. Delta8-Fatty Acid Desaturase is one of the key enzymes in delta8 Desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-Fatty Acid Desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino Acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-Fatty Acid Desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic Acid and omega3-eicosatrienoic Acid for the expression of delta8-Fatty Acid Desaturase gene. The results indicated that high level expressed As-Fatty Acid Desaturase could convert omega6-eicosadienoic Acid and omega3-eicosatrienoic Acid to dihomo-gamma-linolenic Acid and eicosatetraenoic Acid with substrate conversion ratio 31.2% and 46.3%, respectively.
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Cloning and expression in Saccharomyces cerevisiae of delta5-Fatty Acid Desaturase gene from Phaeodactylum tricornutum
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2009Co-Authors: Zhe Yang, Dongsheng Wei, Laijun XingAbstract:Delta5-Fatty Acid Desaturase is the key enzyme in synthesis of arachidonic Acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-Fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic Acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic Acid. The results indicated that high level expression of delta5-Fatty Acid Desaturase, and the substrate conversion reached 45.9%.
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Evolution-related amino Acids play important role in determining regioselectivity of Fatty Acid Desaturase from Pichia pastoris.
Molecular biology reports, 2008Co-Authors: Xinxin Zhang, Dongsheng Wei, Laijun XingAbstract:ω3-Fatty Acid Desaturase and Δ12-Fatty Acid Desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω3-Fatty Acid Desaturase were replaced with corresponding region of Δ12-Fatty Acid Desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino Acids of ω3-Fatty Acid Desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of Fatty Acid Desaturases may be better understood by investigating the evolutionary relationships of different Fatty Acid Desaturases.
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Identification and characterization of a novel yeast omega3-Fatty Acid Desaturase acting on long-chain n-6 Fatty Acid substrates from Pichia pastoris.
Yeast (Chichester England), 2008Co-Authors: Xinxin Zhang, Dongsheng Wei, Laijun XingAbstract:A cDNA sequence putatively encoding a omega(3)-Fatty Acid Desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino Acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-Fatty Acid Desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino Acid sequence exhibited omega(3)-Fatty Acid Desaturase activity. The newly identified omega(3)-Fatty Acid Desaturase, named Pp-FAD3, is novel because it showed broad n-6 Fatty Acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 Fatty Acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-Fatty Acid Desaturase to act on long-chain n-6 Fatty Acid substrates. Heterologous expression of the newly identified omega(3) Desaturase in different hosts will be an alternative method to increase the flow of n-6 Fatty Acid intermediates into their n-3 derivatives.
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Identification and functional characterization of the delta 6-Fatty Acid Desaturase gene from Thamnidium elegans.
The Journal of eukaryotic microbiology, 2007Co-Authors: Depei Wang, Yinghui Zhang, Dongsheng Wei, Yi Cai, Laijun XingAbstract:A cDNA sequence was cloned from the filamentous fungus Thamnidium elegans As3.2806 using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends method (RACE). Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,380 bp, which encodes a 52.4 kDa peptide of 459 amino Acids. The designated amino Acid sequence has high similarity with that found in fungal delta 6-Fatty Acid Desaturases: it shows three conserved histidine-rich motifs and two hydrophobic domains. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative Desaturase, the open reading frame was cloned into the intracellular expression vector pPIC3.5K and the gene was expressed heterologously in Pichia pastoris. Accumulation of gamma-linolenic Acid to the level of 6.83% in total Fatty Acid demonstrated that the deduced amino Acid sequence possesses of delta 6-Fatty Acid Desaturase activity.
Lawal Garba - One of the best experts on this subject based on the ideXlab platform.
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Homology modeling and docking studies of a Δ9-Fatty Acid Desaturase from a Cold-tolerant Pseudomonas sp. AMS8.
PeerJ, 2018Co-Authors: Lawal Garba, Mohd Shukuri Mohamad Ali, Siti Nurbaya Oslan, Mohamad Ariff Mohamad Yussoff, Khairul Bariyyah Abd Halim, Siti Nor Hasmah Ishak, Raja Noor Zaliha Raja Abd RahmanAbstract:Membrane-bound Fatty Acid Desaturases perform oxygenated desaturation reactions to insert double bonds within Fatty acyl chains in regioselective and stereoselective manners. The Δ9-Fatty Acid Desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane Fatty Acid Desaturases are not available, relevant information about the enzymes are derived from their amino Acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound Desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated Fatty Acids at low temperature. Subsequently, an active Δ9-Fatty Acid Desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-Fatty Acid Desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound Desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic Acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic Acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with the in vivo activity of the Δ9-Fatty Acid Desaturase on the membrane phospholipids.
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Heterologous Expression of PA8FAD9 and Functional Characterization of a Δ9-Fatty Acid Desaturase from a Cold-Tolerant Pseudomonas sp. A8.
Molecular biotechnology, 2016Co-Authors: Lawal Garba, Mohd Shukuri Mohamad Ali, Siti Nurbaya Oslan, Raja Noor Zaliha Raja Abdul RahmanAbstract:Fatty Acid Desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated Fatty acyl-chains to produce unsaturated Fatty Acids. A gene coding for a putative Δ9-Fatty Acid Desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino Acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-Fatty Acid Desaturase-like protein activity and increased overall levels of cellular unsaturated Fatty Acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic Acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic Acid to oleic Acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-Fatty Acid Desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
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Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3.
PloS one, 2016Co-Authors: Lawal Garba, Mohd Shukuri Mohamad Ali, Siti Nurbaya Oslan, Raja Noor Zaliha Raja Abdul RahmanAbstract:Fatty Acid Desaturase enzymes play an essential role in the synthesis of unsaturated Fatty Acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic Acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- Fatty Acid Desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino Acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-Fatty Acid Desaturase capable of increasing the total amount of cellular unsaturated Fatty Acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic Acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-Fatty Acid Desaturase-like protein which was actively expressed in E. coli.
Johnathan A. Napier - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis and heterologous expression in yeast of a plant Δ6‐Fatty Acid Desaturase
Journal of experimental botany, 2001Co-Authors: Olga Sayanova, Peter R. Shewry, Frédéric Beaudoin, Balázs Libisch, Aude Castel, Johnathan A. NapierAbstract:Membrane-bound microsomal Fatty Acid Desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' Desaturases. These enzymes are also characterized by the presence of a cytochrome b 5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino Acid residues which comprise the third histidine box of a 'front end' Desaturase, the borage Δ 6 -Fatty Acid Desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.
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Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity
Plant physiology, 1999Co-Authors: Olga Sayanova, Peter R. Shewry, Johnathan A. NapierAbstract:Unlike most other plant microsomal Desaturases, the Δ 6 -Fatty Acid Desaturase from borage ( Borago officinalis ) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b 5 . To determine if this domain serves as a functional electron donor for the Δ 6 -Fatty Acid Desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ 6 -Fatty Acid Desaturase resulted in the synthesis and accumulation of Δ 6 -unsaturated Fatty Acids, this was not observed in plants transformed with N-terminally deleted forms of the Desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b 5 domain; expression of this mutant form of the Δ 6 -Desaturase in transgenic plants failed to produce Δ 6 -unsaturated Fatty Acids. These data indicate that the Cyt b 5 domain of the borage Δ 6 -Fatty Acid Desaturase is essential for enzymatic activity.
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Characterization and expression of a Fatty Acid Desaturase from Borago officinalis
Journal of Experimental Botany, 1999Co-Authors: Olga Sayanova, Peter R. Shewry, Johnathan A. NapierAbstract:accumulated in the leaves, indicating that the cDNA encoded A homologue of the FAD2 microsomal D12-Fatty Acid desatur- a microsomal D6-Fatty Acid Desaturase (Sayanova et al., ase was isolated from borage (Borago officinalis), a species 1997). This D6-Fatty Acid Desaturase was unlike any previnoted for the synthesis of unusual Fatty Acids. Expression of ously characterized higher plant microsomal Desaturases, due the borage FAD2 transcript was correlated with the levels of to the presence of an N-terminal extension of 100 amino linoleic Acid, and expression of this FAD2 homologue was Acids, which showed homology to the electron transport highest in non-photosynthetic tissues. chain protein cytochrome b 5 (Napier et al., 1997). The presence of this N-terminal extension has subsequently been
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Isolation of a Δ5-Fatty Acid Desaturase gene from Mortierella alpina
The Journal of biological chemistry, 1998Co-Authors: Louise V. Michaelson, Colin M. Lazarus, Gareth Griffiths, Johnathan A. Napier, A. K. StobartAbstract:Arachidonic Acid (C20:4 Delta5,8,11,14) is a polyunsaturated Fatty Acid synthesized by the Delta5-Fatty Acid desaturation of di-homo-gamma-linolenic Acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-Fatty Acid Desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal Desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic Acid. The M. alpina Delta5-Desaturase is the first example of a cloned Delta5-Desaturase, and differs from other fungal Desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.
Qi Zhang - One of the best experts on this subject based on the ideXlab platform.
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Heteroexpression of Rhizopus arrhizus delta6-Fatty Acid Desaturase gene in Pichia pastoris
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2005Co-Authors: Qi Zhang, Ying Sun, You-wei Chen, Biao Zhang, Laijun XingAbstract:Delta6-Fatty Acid Desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated Fatty Acids. A cDNA sequence putatively encoding a delta6-Fatty Acid Desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-Fatty Acid Desaturase gene which had an open reading frame of 1377bp coding 458 amino Acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-Fatty Acid Desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total Fatty Acids were extracted from the induced cells and methylated. The resultant Fatty Acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty Acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate Fatty Acid substrates including C16:1, C17:1, C18:1, linoleic Acid and alpha-linolenic Acid without chain length specificity of Fatty Acids. Furthermore, modification of sequence flanking AUG codon of this delta6-Fatty Acid Desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-Fatty Acid Desaturase gene.
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Expression of delta6-Fatty Acid Desaturase gene from Mortierella alpina in Pichia pastoris
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2004Co-Authors: Ying Sun, Qi Zhang, Laijun XingAbstract:Gamma-linolenic Acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated Fatty Acid, plays an important role in hormone regulation and Fatty Acid metabolization. Delta6-Fatty Acid Desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic Acid (C18:2delta9,12) in the production of gamma-linolenic Acid. A deficiency of GLA may have occurred when delta6-Fatty Acid Desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-Fatty Acid Desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-Fatty Acid Desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular Fatty Acid composition by GC. The resultant chromatograms of Fatty Acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the Fatty Acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-Fatty Acid Desaturase was expressed intracellularly in P. pastoris and gamma-linolenic Acid reached 16.26% of the total Fatty Acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.
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Identification and characterization of a novel Δ6-Fatty Acid Desaturase gene from Rhizopus arrhizus
FEBS Letters, 2003Co-Authors: Qi Zhang, Ying Sun, Laijun XingAbstract:Abstract A cDNA sequence putatively encoding a Δ6-Fatty Acid Desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino Acids of 52 kDa. The deduced amino Acid sequence showed high similarity to those of fungal Δ6-Fatty Acid Desaturases which comprised the characteristics of membrane-bound Desaturases, including three conserved histidine-rich motifs and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative Desaturase, the coding sequence was expressed heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the coding product of the sequence exhibited Δ6-Fatty Acid Desaturase activity by the accumulation of γ-linolenic Acid.
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Identification and characterization of a novel Delta6-Fatty Acid Desaturase gene from Rhizopus arrhizus.
FEBS letters, 2003Co-Authors: Qi Zhang, Ying Sun, Laijun XingAbstract:A cDNA sequence putatively encoding a Delta(6)-Fatty Acid Desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino Acids of 52 kDa. The deduced amino Acid sequence showed high similarity to those of fungal Delta(6)-Fatty Acid Desaturases which comprised the characteristics of membrane-bound Desaturases, including three conserved histidine-rich motifs and hydropathy profile. A cytochrome b(5)-like domain was observed at the N-terminus. To elucidate the function of this novel putative Desaturase, the coding sequence was expressed heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the coding product of the sequence exhibited Delta(6)-Fatty Acid Desaturase activity by the accumulation of gamma-linolenic Acid.
