Isolation and characterization of a delta5 FA desaturase from Pythium irregulare by heterologous expression in Saccharomyces cerevisiae and oilseed crops.
By using the polymerase chain reaction approach with two degenerate primers targeting the heme-binding and the third histidine-rich motifs in microsomal carboxyl-directed desaturases, we identified a cDNA PiD5 from Pythium irregulare encoding a delta5 desaturase. The substrate specificity of the enzyme was studied in detail by expressing PiD5 in a yeast (Saccharomyces cerevisiae) mutant strain, AMY-2alpha, where ole1, a delta9 desaturase gene, is disrupted. The result revealed that the encoded enzyme could desaturate unsaturated FA from 16 to 20 carbons beginning with delta9 and delta11 as well as delta8 ethylenic double bonds. Introduction of PiD5 into Brassica juncea under the control of a CaMV 35S constitutive promoter resulted in accumulation of several delta5-unsaturated polymethylene-interrupted FA (delta5-UPIFA) including 18:2-5,9, 18:2-5,11, 18:3-5,9,12, and 18:4-5,9,12,15 in vegetative tissues. The transgenic enzyme could also desaturate the exogenously supplied homo-gamma-linolenic acid (20:3-8,11,14) to arachidonic acid (20:4-5,8,11,14). Introduction of PiD5 into B. juncea and flax under the control of seed-specific promoters resulted in production of delta5-UPIFA, representing more than 10% of the total FA in the seeds.
Hong
Haiping
H
Research & Development, Bioriginal Food and Science Corporation, Saskatoon, Saskatchewan, Canada S7J OR1.
Datla
Nagamani
N
MacKenzie
Samuel L
SL
Qiu
Xiao
X
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D018514:Plant Structures / Q000737:chemistry / Q000235:genetics; D010944:Plants / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified; D011775:Pythium / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D018076:DNA, Complementary; D005227:Fatty Acids; D044943:Fatty Acid Desaturases