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The Escherichia coli highly expressed entD gene complements the pfaE deficiency in a pfa gene clone responsible for the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

Sugihara S, Orikasa Y, Okuyama H - FEMS Microbiol. Lett. (2010)

Bottom Line: In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738.Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures.Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo, Japan.

ABSTRACT
The Escherichia coli entD gene, which encodes an Sfp-type phosphopantetheinyl transferase (PPTase) that is involved in the biosynthesis of siderophore, is available as a high-expression ASKA clone (pCA24N::entD) constructed from the E. coli K-12 strain AG1. In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738. Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures. Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

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Related in: MedlinePlus

Gas chromatograms of total fatty-acid methyl esters prepared from Escherichia coli DH5α recombinants, and electron-impact MS of the unknown fatty-acid peak. Escherichia coli carrying pEPAΔ1,2,3 plus pCA24N::entD (a) and E. coli carrying pEPAΔ1,2,3 (b). Mass spectrum of the unknown peak with a retention time of 30.2 min detected in a (c). Peaks A and B are those of cis-vaccenic and 3-hydroxyl tetradecanoic acids, respectively. Heneicosanoic acid (21 : 0) was used as an internal standard. Escherichia coli DH5α cells carrying pEPAΔ1,2,3 and cells carrying pEPAΔ1,2,3 plus pCA24N::entD were cultivated at 20°C for 72 h in LB medium containing ampicillin at 50 μg mL−1 and ampicillin at 50 μg mL−1 and chloramphenicol at 30 μg mL−1, respectively.
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fig01: Gas chromatograms of total fatty-acid methyl esters prepared from Escherichia coli DH5α recombinants, and electron-impact MS of the unknown fatty-acid peak. Escherichia coli carrying pEPAΔ1,2,3 plus pCA24N::entD (a) and E. coli carrying pEPAΔ1,2,3 (b). Mass spectrum of the unknown peak with a retention time of 30.2 min detected in a (c). Peaks A and B are those of cis-vaccenic and 3-hydroxyl tetradecanoic acids, respectively. Heneicosanoic acid (21 : 0) was used as an internal standard. Escherichia coli DH5α cells carrying pEPAΔ1,2,3 and cells carrying pEPAΔ1,2,3 plus pCA24N::entD were cultivated at 20°C for 72 h in LB medium containing ampicillin at 50 μg mL−1 and ampicillin at 50 μg mL−1 and chloramphenicol at 30 μg mL−1, respectively.

Mentions: pCA24N::entD was used to transform E. coli DH5α cells carrying pEPAΔ1,2,3. GC/MS analysis of fatty-acid methyl esters prepared from E. coli DH5α cells that carried pCA24N::entD plus pEPAΔ1,2,3 revealed the presence of an unknown peak with a retention time of 30.2 min (Fig. 1a), which was not detected in E. coli DH5α host cells carrying only pEPAΔ1,2,3 (Fig. 1b). The retention time of the unknown peak was the same as that of the methyl ester of authentic EPA (data not shown). The GC/MS profile of the unknown peak shown in Fig. 1c was typical of methylene-interrupted PUFAs, and analysis of the fragmentation profile using a program from the National Institute of Standard and Technology Databases (http://www.nist.gov./srd/htm) indicated that the profile of this unknown component was closest to that of EPA. Based on these results, this compound was identified as EPA methyl ester. The content of EPA was 9.2±0.2% of the total fatty acids from cells grown at 20 °C for 72 h. PUFAs other than EPA were not detected.


The Escherichia coli highly expressed entD gene complements the pfaE deficiency in a pfa gene clone responsible for the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

Sugihara S, Orikasa Y, Okuyama H - FEMS Microbiol. Lett. (2010)

Gas chromatograms of total fatty-acid methyl esters prepared from Escherichia coli DH5α recombinants, and electron-impact MS of the unknown fatty-acid peak. Escherichia coli carrying pEPAΔ1,2,3 plus pCA24N::entD (a) and E. coli carrying pEPAΔ1,2,3 (b). Mass spectrum of the unknown peak with a retention time of 30.2 min detected in a (c). Peaks A and B are those of cis-vaccenic and 3-hydroxyl tetradecanoic acids, respectively. Heneicosanoic acid (21 : 0) was used as an internal standard. Escherichia coli DH5α cells carrying pEPAΔ1,2,3 and cells carrying pEPAΔ1,2,3 plus pCA24N::entD were cultivated at 20°C for 72 h in LB medium containing ampicillin at 50 μg mL−1 and ampicillin at 50 μg mL−1 and chloramphenicol at 30 μg mL−1, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2901515&req=5

fig01: Gas chromatograms of total fatty-acid methyl esters prepared from Escherichia coli DH5α recombinants, and electron-impact MS of the unknown fatty-acid peak. Escherichia coli carrying pEPAΔ1,2,3 plus pCA24N::entD (a) and E. coli carrying pEPAΔ1,2,3 (b). Mass spectrum of the unknown peak with a retention time of 30.2 min detected in a (c). Peaks A and B are those of cis-vaccenic and 3-hydroxyl tetradecanoic acids, respectively. Heneicosanoic acid (21 : 0) was used as an internal standard. Escherichia coli DH5α cells carrying pEPAΔ1,2,3 and cells carrying pEPAΔ1,2,3 plus pCA24N::entD were cultivated at 20°C for 72 h in LB medium containing ampicillin at 50 μg mL−1 and ampicillin at 50 μg mL−1 and chloramphenicol at 30 μg mL−1, respectively.
Mentions: pCA24N::entD was used to transform E. coli DH5α cells carrying pEPAΔ1,2,3. GC/MS analysis of fatty-acid methyl esters prepared from E. coli DH5α cells that carried pCA24N::entD plus pEPAΔ1,2,3 revealed the presence of an unknown peak with a retention time of 30.2 min (Fig. 1a), which was not detected in E. coli DH5α host cells carrying only pEPAΔ1,2,3 (Fig. 1b). The retention time of the unknown peak was the same as that of the methyl ester of authentic EPA (data not shown). The GC/MS profile of the unknown peak shown in Fig. 1c was typical of methylene-interrupted PUFAs, and analysis of the fragmentation profile using a program from the National Institute of Standard and Technology Databases (http://www.nist.gov./srd/htm) indicated that the profile of this unknown component was closest to that of EPA. Based on these results, this compound was identified as EPA methyl ester. The content of EPA was 9.2±0.2% of the total fatty acids from cells grown at 20 °C for 72 h. PUFAs other than EPA were not detected.

Bottom Line: In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738.Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures.Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo, Japan.

ABSTRACT
The Escherichia coli entD gene, which encodes an Sfp-type phosphopantetheinyl transferase (PPTase) that is involved in the biosynthesis of siderophore, is available as a high-expression ASKA clone (pCA24N::entD) constructed from the E. coli K-12 strain AG1. In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738. Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures. Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

Show MeSH
Related in: MedlinePlus