Abstract
A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.
Highlights
The long-chain polyunsaturated fatty acids (LC-PUFA)1 play important roles in adult as well as infant nutrition because they serve as precursors of eicosanoids including prostaglandins and leukotrienes
In this work we describe the isolation of a desaturase-like sequence from M. alpina and demonstrate its ability to produce ⌬5-unsaturated fatty acids in bakers’ yeast and transgenic canola seeds
PCR amplification of M. alpina cDNA using degenerate primers corresponding to the sequences HHTYTN found in the second His box of cyanobacterial ⌬6-desaturases (amino acid residues 127–132 of the Synechocystis ⌬6-desaturase (Fig. 2A) [7, 8]) and HHLFP found in the 3rd His box of many desaturases (amino acid residues 306 –310 of the Synechocystis ⌬6-desaturase and 308 –312 of the Arabidopsis ⌬15-desaturase (Fig. 2A) [10] resulted in the production of a 550-base pair fragment
Summary
Strains and Growth Conditions—M. alpina ATCC32221 was grown in PD medium (Difco). Cultures were inoculated on PDA plates and grown at room temperature. For cloning in plant expression vectors, the coding region of the M. alpina desaturase gene was PCR-amplified using the following primers: 5Ј-CUACUACUACUACTCGAGCAAGATGGGAACGGACCAAGG-3Ј and 5Ј-CAUCAUCAUCAUCTCGAGCTACTCTTCCTTGGGACGGAG3Ј. These primers introduced XhoI cloning sites (underlined) upstream and downstream of the start and stop codons (in boldface), repsectively. The plasmid pCGN5530 was digested with HindIII and religated at a high DNA concentration to produce a tandem expression construct, pCGN5531 This construct contains two copies of the napin/M. alpina desaturase gene fusion oriented in the same direction with respect to transcription of the nptII selection gene. The mass spectrum of any new peak obtained was compared with that of standards (Nu Chek Prep, Elysian, MN) in the data base NBS75K.L (National Bureau of Standards)
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