New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases.
In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.
Domergue
Frédéric
F
Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, Germany. fredDo@botanik.uni-hamburg.de
Spiekermann
Patricia
P
Lerchl
Jens
J
Beckmann
Christoph
C
Kilian
Oliver
O
Kroth
Peter G
PG
Boland
Wilhem
W
Zähringer
Ulrich
U
Heinz
Ernst
E
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D015870:Gene Expression; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D030781:Organisms, Genetically Modified; D010802:Phylogeny; D018087:Plastids / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D015003:Yeasts / Q000235:genetics
D005227:Fatty Acids; D044943:Fatty Acid Desaturases