Fig. 1. Reaction sequences leading to formation of lcPUFAs.

Fig. 2. GC/FID analysis of fatty acid methyl esters isolated from P. megasperma mycelia cultivated for 2 days on V8-oleate media. The lipids were extracted from lyophilized mycelia, esterified fatty acids were transmethylated and analyzed by GC/FID as described under Materials and methods. All fatty acids were characterized by authentic standards.

Fig. 3. GC/FID analysis of fatty acid methyl esters isolated from yeast cells supplemented simultaneously with 20:3^{Δ 11Z,14Z,17Z}, 20:3^{Δ 8Z,11Z,14Z} (di-homo-γ-linolenic acid) and 20:4^{Δ 8Z,11Z,14Z,17Z} (ω3-arachidonic acid). The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and analyzed by GC/FID as described under Materials and methods. Panel A shows the chromatogram of fatty acid methyl esters from yeast cell transformed with the empty vector pYES2 as a control. Panel B shows the GC/FID analysis of fatty acid methyl esters from yeast cells transformed with the plasmid pYES2-PmFAD5.

Fig. 4. Extent of desaturation in the lipid extract, within the different lipid classes and within the acyl-CoA-pool in yeast cell cultures expressing PmFAD5. A culture of the yeast cells transformed with pYES2-PmFAD5 was cultivated for 3 days at 30 °C in the presence of 0.01% di-homo-γ-linolenic acid. Fatty acid composition of lipid extract, the different lipid fractions and acyl-CoA-pool were determined as indicated under Materials and methods. Desaturation (%) was calculated as (product×100)/(substrate+product) using values corresponding to percent of total esterified fatty acids.

Substrate specificity of PmFAD5

Yeast cells transformed with pYES-PmFAD5 were supplemented with different fatty acids. The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and GC/FID analysis of fatty acid methyl esters isolated from these yeast cultures was performed as described under Materials and methods. All fatty acids were characterized by co-elution of authentic standards.

Conversion rates of exogenous fatty acids in yeast transformed with PmFAD5

Yeast cells transformed with pYES-PmFAD5 were supplemented with different fatty acids. The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and GC/FID analysis of fatty acid methyl esters isolated from these yeast cultures was performed as described under Materials and methods. All fatty acids were characterized by co-elution of authentic standards. Conversion was calculated as (product×100)/(substrate+product) using values corresponding to the peak area of the corresponding signals.

Fatty acid composition of different lipid fractions from yeast expressing the Δ5-desaturase from P. megasperma (PmFAD5)

Yeast cells transformed with pYES-PmFAD5 were supplemented with 20:3^Δ8Z,11Z,14Z and used to isolate different lipid fractions as described under Materials and methods. All fatty acids were characterized by co-elution of authentic standards. Desaturation (%) was calculated as (product×100)/(substrate+product) using values corresponding to percent of total fatty acids in each lipid fractions. Each value is the mean±S.D. from three to four experiments.