L-Serine → Arachidonic Acid

Participants

Reaction Steps

A DNA fragment of about 0.95 kbp obtained by digesting plasmid pCR-MAGLELO with restriction endonucleases XbaI and SacI was ligated to a DNA fragment of about 7.7 kbp obtained by digesting the vector pESC-LEU (STRATAGENE) with restriction endonucleases XbaI and SacI to generate plasmid pESC-L-GLELO. A DNA fragment of about 1.3 kbp obtained by digesting plasmid pCR-MAΔ5DS with restriction endonuclease XbaI followed by blunt-ending and further digesting it with restriction endonuclease HindIII was ligated to a DNA fragment of about 8.7 kbp obtained by digesting plasmid pESC-L-GLELO with restriction endonuclease ApaI followed by blunt-ending and further digesting it with restriction endonuclease HindIII to generate plasmid pESC-L-GLELO:Δ5. A fragment of about 3.2 kb obtained by digesting this with restriction endonuclease PvuII was inserted into the SmaI site of pUC-LEU2 to generate plasmid pUC-LEU-GLELO:Δ5. S. cerevisiae strain YPH499 (STRATAGENE) was co-transformed with plasmid pUC-URA-Δ12:Δ6 and plasmid pUC-LEU-GLELO:Δ5. Transformed strains were selected by viability on SC-Leu, Ura agar medium (2% agar) containing, per liter, 6.7 g Yeast nitrogen base w/o amino acids (DIFCO), 20 g glucose and 1.3 g amino acid powder (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 g threonine and 1.2 g tryptophan). Random one of the selected strains was designated as ARA3-1 strain. This strain can produce arachidonic acid by expressing the Δ12 fatty acid desaturase gene, Δ6 fatty acid desaturase gene, GLELO fatty acid elongase gene, and Δ5 fatty acid desaturase gene from the GAL1/10 promoter upon cultivation in a galactose-containing medium.

Provenance & Evidence