A novel fungal ?3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4

Abstract

A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20 degrees C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel omega3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 Delta12-desaturase and Saccharomyces kluyveri omega3-desaturase, the omega3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 Delta12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri omega3-desaturase. The cloned cDNA was confirmed to encode the omega3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 omega3-desaturase differs from those of the known fungal omega3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri omega3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina omega3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans omega3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 omega3-desaturase is rather similar to that of C. elegans omega3-desaturase, but the M. alpina omega3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 omega3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.

Molecular Switch That Controls the Flux of Linoleic Acid Into N-6 or N-3 Polyunsaturated Fatty Acids in Microorganisms

“…Although it contains a complete set of desaturase genes for the biosynthesis of both ARA (n-6) and EPA (n-3), at physiological growth temperatures, it produces ARA as the only major VL-PUFA. This regulation of PUFA metabolic flux is partly due to the substrate specificity of the ∆6 Des Table 2, Sakuradani and Shimizu, 2003;Sakuradani et al, 2005;Zhu et al, 2002) which prefers n-6 fatty acid LA to n-3 ALA as substrate and its ω-3 Des must be repressed at physiological growth temperature (Shimizu et al, 1998). Although the ω-3 Des have been cloned and biochemically studied in a few microorganisms Table 5, (Gellerman and Schlenk, 1979;Pereira et al, 2004;Sakamoto et al, 1994;Wada and Murata, 1990), its expression level in oleaginous microorganisms has not been determined so far.…”

“…Main PUFAs (%) n-3 FAs/n-6 FAs LA ALA Reference ARA: 48 * (Shimizu et al, 1988 GLA: 4 DGLA: 3 AA: 9 0 51 0 *Strain was cultivated at 28°C †Strain was cultivated at 12°C §Substrate conversion yield was calculated as described previously (Sakuradani et al, 2005), Conversion yield (%) = 100× ([product]/ [product + substrate]. ∆6 Desaturase gene from M. alpina 1S-4 was expressed in A. oryzae.…”

“…∆6 Des gene from M. alpina W15 was expressed in P. pastoris. The substrate preference was calculated based on its endogenous fatty acids production (Sakuradani et al, 2005), Conversion yield (%) = 100× ([product]/ [product + substrate]. NA: Not Available.…”

“…68 80 Tonon et al, 2005) *Grown at 22°C, †Grown at 14°C, §Substrate conversion yield was calculated as described previously (Sakuradani et al, 2005), Conversion yield (%) = 100× ([product]/ [product + substrate]. ∆6 Des gene from P. irregular was expressed in Brassica Juncea, substrate preference was calculated based on its endogenous fatty acids production.…”

“…∆6 Des from all other organisms were expressed in S. cerevisiae and the substrate preference was studied by feeding the yeast with exogenous precursor fatty acids as substrates (Wada and Murata, 1990;SDA: 8 <1 25 ? Sakamoto et al, 1994) *Strain was cultivated at 28°C †Strain was cultivated at 12°C §Yield of substrate conversion was calculated as described previously (Sakuradani et al, 2005), Conversion yield (%) = 100× ([product]/ [product + substrate]. ω-3 Des gene from Synechocystis sp.…”