Co-expression studies of different microalgal acyltransferase sequences

The microalgal enzymes were further investigated by co-expressing different combina-tions in yeast to find out whether the acyltransferases possibly interact with each other or even are only active within a complex of different acyltransferases. This was tested by analyzing total amounts and fatty acid composition of different lipid classes and by comparison of the obtained data with those from cultures expressing one acyltrans-ferase individually. Co-localization could be shown for example for the desaturase SCD1p and the acyltransferase DGAT2p from mouse (Man et al., 2006a). LPCAT in turn might potentially interact with both DGAT and PDAT enzymes because it mediates the transfer between acyl-CoA pool on the one hand and PC pool on the other hand.

Therefore, combinations of LPCATp with PDATp or DGATp were chosen for the follow-ing experiments. For co-expression with OtLPCAT and OlLPCAT, the sequence encod-ing the most active DGAT protein from O. tauri, OtDGAT2B, was chosen. In addition, OtPDAT from O. tauri was utilized. The nucleotide sequence was isolated by Dr. M.

Wagner after searching through the genome of O. tauri with known PDAT sequences from yeast and Arabidopsis. Expression of a partly codon-optimized version of OtPDAT resulted in solid protein amounts in yeast. The enzyme was shown to complement the BY4741 lro1Δ dga1Δ mutant only after addition of (n-3)- and (n-6)-polyunsaturated fatty acids with 18 or 20 carbon atoms (Wagner, 2008).

The yeast mutant BY4741 lro1Δ dga1Δ ale1Δ was created to avoid interference of the endogenous yeast acyltransferases with those of microalgal origin. The completely codon-optimized OlLPCAT and the partly codon-optimized OtLPCAT were cloned into pESC-LEU downstream of the GAL1 promoter. Subsequently, pESC-LEU, OlLPCAT-pESC-LEU or OtLPCAT-OlLPCAT-pESC-LEU were co-transformed with OtDGAT2B-pYES2/CT, OtPDAT-pYES2/CT or pYES2/CT into the newly created yeast mutant strain. As wild type control, BY4741 was co-transformed with pESC-LEU and pYES2/CT. Expression

cultures were grown for two days at 23 °C and subsequently harvested. Half of each sample was used for acyl-CoA extraction and measurement whereas the other half was utilized for total lipid extraction and separation of TAG from PC. Internal standards were used for quantification. Results of the performed experiment are depicted in Figure 10, Figure 11 and Figure 12. Acyl-CoA amounts were higher in the mutant com-pared to the wild type control, but this effect was not statistically significant (Figure 10 A). In all mutant expression cultures, 16:1(n-9)-fractions were elevated two to three times and percentages of 18:0 and 18:1(n-9) were lowered in comparison to wild type (Figure 10 B). TAGs were detectable only in those mutant cultures expressing OtDGAT2B (Figure 11 A), but the fatty acid composition of the TAG fraction from com-plemented mutant yeast cultures did not differ from wild type (Figure 11 B). In case of OtPDAT expression, TAG levels were not significantly higher than in the negative con-trol, the yeast mutant strain co-expressing pESC-LEU and pYES2/CT (Figure 11 A).

Low TAG levels in these cases did not lead to reliable data concerning fatty acid com-position because of detection limitations in GC measurements. PC amounts were higher in the mutant yeast strain but this effect again was not statistically significant, as can be seen in Figure 12 A. Fatty acid composition of PC was nearly the same in all tested expression cultures (Figure 12 B).

Generally it should be noted, that strong differences in total amounts as well as in fatty acid composition could be observed already between cultures co-expressing the same combination of acyltransferase nucleotide sequences. Therefore, it might be problema-tic to come to a conclusion based on the presented data.

Results

Figure 10. Acyl-CoAs after co-expression of microalgal acyltransferases in BY4741 lro1Δ dga1Δ ale1Δ

pESC-LEU, OlLPCAT-pESC-LEU or OtLPCAT-pESC-LEU were co-expressed with pYES2/CT, OtDGAT2B-pYES2/CT or OtPDAT-pYES2/CT as indicated between the diagrams for two days at 23 °C in the yeast mutant strain or in BY4741 (wt). Yeast cells were harvested, acyl-CoAs were extracted and measured. (A) total amounts and (B) mol% of different acyl chains. Acyl-CoA, acyl-Coenzyme A. Bars represent the mean + standard deviation (SD) of three to four independent clones.

Figure 11. TAG after co-expression of microalgal acyltransferases in BY4741 lro1Δ dga1Δ ale1Δ

pESC-LEU, OlLPCAT-pESC-LEU or OtLPCAT-pESC-LEU were co-expressed with pYES2/CT, OtDGAT2B-pYES2/CT or OtPDAT-pYES2/CT as indicated between the diagrams for two days at 23 °C in the yeast mutant strain or in BY4741 (wt). Yeast cells were harvested, total lipids were extracted and TAGs were analyzed. (A) total amounts and (B) mol% of different acyl chains. TAG, triacylglycerol. Bars represent the mean + SD of three to four independent clones.

Results

Figure 12. PC after co-expression of microalgal acyltransferases in BY4741 lro1Δ dga1Δ ale1Δ

pESC-LEU, OlLPCAT-pESC-LEU or OtLPCAT-pESC-LEU were co-expressed with pYES2/CT, OtDGAT2B-pYES2/CT or OtPDAT-pYES2/CT as indicated between the diagrams for two days at 23 °C in the yeast mutant strain or in BY4741 (wt). Yeast cells were harvested, total lipids were extracted and PC was analyzed. (A) total amounts and (B) mol% of different acyl chains.

PC, phosphatidylcholine. Bars represent the mean + SD of three to four independent clones.

3.2 Characterization of the microalgal desaturase Old4p in